text:
Agrobacterium tumefaciens -mediated transformation of Rhipsalidopsis gaertneri
E. A. Al-Ramamneh; S. Sriskandarajah; M. Serek
A protocol for Agrobacterium tumefaciens-mediated genetic transformation of Rhipsalidopsis cv. CB5 was developed. Calluses derived from phylloclade explants and sub-cultured onto fresh callus induction medium over a period of 9–12 months were co-cultivated with A. tumefaciens LBA4404. Plasmid constructs carrying the nptII gene, as a selectable marker, and the reporter uidA gene were used. Transformed Rhipsalidopsis calluses with a vigorous
Agrobacterium tumefaciens -mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.)
A. J. E. Bettany; S. Dalton; E. Timms; B. Manderyck; M. Dhanoa; P. Morris
Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and #-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and
Agrobacterium tumefaciens -Mediated Transformation of Rosa hybrida using the Green Fluorescent Protein (GFP) Gene
C. K. Kim; J. D. Chung; S. H. Park; A. M. Burrell; K. K. Kamo; D. H. Byrne
Embryogenic calluses of Rosa hybrida cultivar Tineke were transformed with Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBIN m-gfp5-ER into which the virE\\/virG genes had been inserted. Visualization of GFP-expressing cells enabled visual selection of dividing, embryogenic cell clusters that were transgenic. When the Agrobacterium strain with the bifunctional fusion marker containing additional virE\\/virG genes was used, the number
An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens
Ariel D. Arencibia; Elva R. Carmona; Pilar Tellez; Ming-Tsair Chan; Su-May Yu; Luis E. Trujillo; Pedro Oramas
This is the first successful report of the recovery of morphologically normal transgenic sugarcane plants from co-cultivation of calluses with Agrobacterium tumefaciens. Transformation frequencies (total of transgenic plants\\/number of cell clusters) were between 9.4 × 10-3 and 1.15 × 10-2. In our experiments, both LBA4404 (pTOK233) and EHA101 (pMTCA3IG), carrying a super-binary vector or supervirulent strain, respectively, were successful for
Agrobacterium tumefaciens -mediated transformation of Campanula carpatica : factors affecting transformation and regeneration of transgenic shoots
Sridevy Sriskandarajah; Stefan Frello; Kirsten Jřrgensen; Margrethe Serek
An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and
Developing an Agrobacterium tumefaciens -mediated genetic transformation for a selenium-hyperaccumulator Astragalus racemosus
Diane E. Darlington; Chiu-Yueh Hung; Jiahua Xie
Agrobacterium\\u000a tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The\\u000a optimal concentration of kanamycin that could effectively inhibit cell growth and division in
Agrobacterium tumefaciens -mediated creeping bentgrass ( Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration
H. Luo; Q. Hu; K. Nelson; C. Longo; A. P. Kausch; J. M. Chandlee; J. K. Wipff; C. R. Fricker
Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation
Salttolerant transgenic perennial ryegrass ( Lolium perenne L.) obtained by Agrobacterium tumefaciens-mediated transformation of the vacuolar Na +\\/H + antiporter gene
Yu-Ye Wu; Qi-Jun Chen; Min Chen; Jia Chen; Xue-Chen Wang
The objective of this study was to obtain a salt-tolerant perennial ryegrass (Lolium perenne L.) by transforming it with a rice vacuolar membrane Na+\\/H+ antiporter gene via the Agrobacterium-mediated method. To optimize the transformation conditions, two Agrobacterium tumefaciens strains, LBA4404 and EHA105, carrying plasmid pCAMBIA3301, were used to transform embryogenic calli of perennial ryegrass; two factors affecting transformation efficiency, acetosyringone
[Agrobacterium-mediated sunflower transformation (Helianthus annuus L.) in vitro and in Planta using strain of LBA4404 harboring binary vector pBi2E with dsRNA-suppressor proline dehydrogenase gene].
Tishchenko, E N; Komisarenko, A G; Mikhal'skaia, S I; Sergeeva, L E; Adamenko, N I; Morgun, B V; Kochetov, A V
To estimate the efficiency of proline dehydrogenase gene suppression towards increasing of sunflower (Helianthus annuus L.) tolerance level to water deficit and salinity, we employed strain LBA4404 harboring pBi2E with double-stranded RNA-suppressor, which were prepared on basis arabidopsis ProDH1 gene. The techniques of Agrobacterium-mediated transformation in vitro and in planta during fertilization sunflower have been proposed. There was shown the genotype-depended integration of T-DNA in sunflower genome. PCR-analysis showed that ProDH1 presents in genome of inbred lines transformed in planta, as well as in T1- and T2-generations. In trans-genic regenerants the essential accumulation of free L-proline during early stages of in vitro cultivation under normal conditions was shown. There was established the essential accumulation of free proline in transgenic regenerants during cultivation under lethal stress pressure (0.4 M mannitol and 2.0% sea water salts) and its decline upon the recovery period. These data are declared about effectiveness of suppression of sunflower ProDH and gene participation in processes connected with osmotolerance. PMID:25184200
Agrobacterium -mediated transformation of Campanula glomerata
Y. Joung; M. Roh; K. Kamo; J. Song
A transformation system for Campanula glomerata 'Acaulis' based on the co-cultivation of leaf explants with Agrobacterium tumefaciens LBA4404 or EHA105 was developed. A. tumefaciens was eliminated when the explants were cultured on medium containing 400 mg\\/l vancomycin and 100 mg\\/l cefotaxime. Transgenic plants containing the uidA gene that codes for #-glucuronidase (gus) were obtained following co-cultivation with either strain of
Agrobacterium -mediated genetic transformation of safflower ( Carthamus tinctorius L.)
K. Sri Shilpa; V. Dinesh Kumar; M. Sujatha
Reproducible and highly efficient protocols for shoot regeneration and genetic transformation mediated by Agrobacterium have been established for safflower (Carthamus tinctorius L.). Agrobacterium tumefaciens strain LBA 4404 with gus reporter gene and hygromycin (hpt gene) as plant selection marker was used as the plant transformation vector. Genetic transformation experiments were carried\\u000a out to evaluate the efficacy of various parameters such as genotype,
Transient expression of ? ? ? ?-glucuronidase reporter gene in Agrobacterium-inoculated shoots of various teak clones
Sri Nanan Widiyanto; Arfri Sukmawan; Nancy Haro; Heni Rahmania
Agrobacterium tumefaciens strain LBA4404 carrying the pBI.121 binary plasmid was used in transformation to introduce the gus (ß-glucuronidase\\/GUS) gene into teak shoot-tissues. In vitro regenerated shoots from various teak clones, i.e. the ITB, GT, P97, P96, P75, P20, and P108 clones were vacuum-infiltrated for 5 min in the suspension culture of A. tumefaciens. Seven days after selection period, the evidence
Transgenic regal pelargoniums that express the rol C gene from Agrobacterium rhizogenes exhibit a dwarf floral and vegetative phenotype
M. R. Boase; C. S. Winefield; T. A. Lill; M. J. Bendall
Summary The regal pelargonium, ev. Dubonnet, was transformed using the disarmed Agrobacterium tumefaciens strains LBA4404 or EHA105 containing the binary vector pLN70. This plasmid carries on its T-DNA the rolC gene from Agrobacterium rhizogenes under control of the CaMV 35S promoter and the npt II selectable marker gene under a NOS promoter. Six independent transformants were produced and grouped according to
Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots.
Crane, Cynthia; Wright, Elane; Dixon, Richard A; Wang, Zeng-Yu
Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes. PMID:16575594
Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots.
Sriskandarajah, Sridevy; Frello, Stefan; Jřrgensen, Kirsten; Serek, Margrethe
An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar "Blue Uniform" was more responsive than "White Uniform". Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by beta-glucuronidase and ELISA analyses, respectively. PMID:15114492
Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration.
Luo, H; Hu, Q; Nelson, K; Longo, C; Kausch, A P; Chandlee, J M; Wipff, J K; Fricker, C R
Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns. PMID:14615907
Transformation of rice mediated by Agrobacterium tumefaciens
Yukoh Hiei; Toshihiko Komari; Tomoaki Kubo
Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient
Exogenous phytohormone-independent growth and regeneration of tobacco plants transgenic for the 6b gene of Agrobacterium tumefaciens AKE10.
Wabiko, H; Minemura, M
The 6b gene of Agrobacterium tumefaciens AKE10 (AK-6b) induces crown gall tumors on certain plants but so far there have been no reports of the gene being able to induce tumors on culture medium. We cloned T-DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes from A. tumefaciens AKE10. Tobacco (Nicotiana tabacum) leaf discs infected with A. tumefaciens LBA4404 carrying the clones produced shooty calli on hormone-free Murashige-Skoog medium. The relevant T-DNA segment was integrated into plant DNA as determined by Southern hybridization. Some of these immature shoots spontaneously developed into mature shoots, of which several leaves displayed morphological abnormalities. When leaf discs of these mature plants were placed onto the same medium numerous shoots developed from the wounding sites, indicating that the transgenic plants possessed a high regenerative potential. Northern blot and reverse transcriptase-polymerase chain reaction analyses showed a large accumulation of the AK-6b transcripts in the shooty calli, but only a limited degree in mature plants, demonstrating that AK-6b expression is regulated in plants and essential for the early stages of regeneration. Cytokinin levels in the shooty calli were comparable to those in normal shoots, suggesting that shoot regeneration is not mediated by the modulation of cytokinin content. PMID:8938404
The Oncogenes of Agrobacterium Tumefaciens and Agrobacterium Rhizogenes
Monica T. Britton; Matthew A. Escobar; Abhaya M. Dandekar
The common soil bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes are unique genetic pathogens capable of fundamentally redirecting plant metabolism in order to generate macroscopic tissue\\u000a masses (crown galls and hairy roots, respectively) which support the growth of large populations of Agrobacteria. Central to pathogenesis is the horizontal transfer of a suite of oncogenes from the tumor-inducing (Ti) plasmids of A.
Agrobacterium tumefaciens -mediated transformation of Robinia pseudoacacia
T. Igasaki; T. Mohri; H. Ichikawa; K. Shinohara
Robinia pseudoacacia (black locust) plants were regenerated after co-cultivation of stem and leaf segments with Agrobacterium tumefaciens strain GV3101 (pMP90) that harbored a binary vector that included genes for ?-glucuronidase (GUS) and hygromycin phosphotransferase. Successful transformation was confirmed by the ability of stem and\\u000a leaf segments to produce calli in the presence of hygromycin, by histochemical and fluorometric assays of
Cellulose Synthesis in Agrobacterium tumefaciens
Alan R. White; Ann G. Matthysse
We have cloned the celC gene and its homologue from E. coli, yhjM, in an expression vector and expressed the both genes in E. coli; we have determined that the YhjM protein is able to complement in vitro cellulose synthesis by extracts of A. tumefaciens celC mutants, we have purified the YhjM protein product and are currently examining its enzymatic activity; we have examined whole cell extracts of CelC and various other cellulose mutants and wild type bacteria for the presence of cellulose oligomers and cellulose; we have examined the ability of extracts of wild type and cellulose mutants including CelC to incorporate UDP-14C-glucose into cellulose and into water-soluble, ethanol-insoluble oligosaccharides; we have made mutants which synthesize greater amounts of cellulose than the wild type; and we have examined the role of cellulose in the formation of biofilms by A. tumefaciens. In addition we have examined the ability of a putative cellulose synthase gene from the tunicate Ciona savignyi to complement an A. tumefaciens celA mutant. The greatest difference between our knowledge of bacterial cellulose synthesis when we started this project and current knowledge is that in 1999 when we wrote the original grant very few bacteria were known to synthesize cellulose and genes involved in this synthesis were sequenced only from Acetobacter species, A. tumefaciens and Rhizobium leguminosarum. Currently many bacteria are known to synthesize cellulose and genes that may be involved have been sequenced from more than 10 species of bacteria. This additional information has raised the possibility of attempting to use genes from one bacterium to complement mutants in another bacterium. This will enable us to examine the question of which genes are responsible for the three dimensional structure of cellulose (since this differs among bacterial species) and also to examine the interactions between the various proteins required for cellulose synthesis. We have carried out one preliminary experiment of this type and have successfully complemented an A. tumefaciens CelC mutant with the homologous gene (yhjM) from E. coli.
Strain specificity in transformation of alfalfa by Agrobacterium tumefaciens
Deborah A. Samac
Production of transgenic alfalfa plants by Agrobacterium-mediated transformation requires Agrobacterium infection and regeneration from tissue culture. Variation in alfalfa (Medicago sativa L.) germplasm for resistance to oncogenic and disarmed strains of A. tumefaciens (Smith & Townsend) Conn was tested in plant populations representing the nine distinct sources of alfalfa germplasm introduced into North America and used to develop modern varieties.
Shape-dependent bactericidal activity of TiO2 for the killing of Gram-negative bacteria Agrobacterium tumefaciens under UV torch irradiation.
Aminedi, Raghavendra; Wadhwa, Gunveen; Das, Niranjan; Pal, Bonamali
This paper demonstrated the relative bactericidal activity of photoirradiated (6W-UV Torch, ??>?340 nm and intensity?=?0.64 mW/cm(2)) P25-TiO2 nanoparticles, nanorods, and nanotubes for the killing of Gram-negative bacterium Agrobacterium tumefaciens LBA4404 for the first time. TiO2 nanorod (anatase) with length of 70-100 nm and diameter of 10-12 nm, and TiO2 nanotube with length of 90-110 nm and diameter of 9-11 nm were prepared from P-25 Degussa TiO2 (size, 30-50 nm) by hydrothermal method and compared their biocidal activity both in aqueous slurry and thin films. The mode of bacterial cell decomposition was analyzed through transmission electron microscopy (TEM), Fourier transform-infrared (FT-IR), and K(+) ion leakage. The antimicrobial activity of photoirradiated TiO2 of different shapes was found to be in the order P25-TiO2?>?nanorod?>?nanotube which is reverse to their specific surface area as 54?79?176 m(2) g(-1), evidencing that the highest activity of P25-TiO2 nanoparticles is not due to surface area as their crystal structure and surface morphology are entirely different. TiO2 thin films always exhibited less photoactivity as compared to its aqueous suspension under similar conditions of cell viability test. The changes in the bacterial surface morphology by UV-irradiated P25-TiO2 nanoparticles was examined by TEM, oxidative degradation of cell components such as proteins, carbohydrates, phospholipids, nucleic acids by FT-IR spectral analysis, and K(+) ion leakage (2.5 ppm as compared to 0.4 ppm for control culture) as a measure of loss in cell membrane permeability. PMID:23608987
Complete Genome Sequence of Agrobacterium tumefaciens Ach5.
Huang, Ya-Yi; Cho, Shu-Ting; Lo, Wen-Sui; Wang, Yi-Chieh; Lai, Erh-Min; Kuo, Chih-Horng
Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease. The strain Ach5 was isolated from yarrow (Achillea ptarmica L.) and is the wild-type progenitor of other derived strains widely used for plant transformation. Here, we report the complete genome sequence of this bacterium. PMID:26044425
Virulence of Agrobacterium tumefaciens Strain A281 on Legumes 1
Hood, Elizabeth E.; Fraley, Robert T.; Chilton, Mary-Dell
This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an l,l-succinamopine strain. We tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains. PMID:16665283
Complete Genome Sequence of Agrobacterium tumefaciens Ach5
Huang, Ya-Yi; Cho, Shu-Ting; Lo, Wen-Sui; Wang, Yi-Chieh; Lai, Erh-Min
Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease. The strain Ach5 was isolated from yarrow (Achillea ptarmica L.) and is the wild-type progenitor of other derived strains widely used for plant transformation. Here, we report the complete genome sequence of this bacterium. PMID:26044425
Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens - transformed roots and Agrobacterium rhizogenes- transformed hairy roots
Cynthia Crane; Elane Wright; Richard A. Dixon; Zeng-Yu Wang
Medicago\\u000a truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1,
Agrobacterium tumefaciens and Agrobacterium rhizogenes transformed roots of the parasitic plant Triphysaria versicolor retain parasitic competence
Alexey Tomilov; Natalya Tomilova; John I. Yoder
Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria\\u000a versicolor Fischer and C. Meyer. Agrobacterium\\u000a tumefaciens and Agrobacterium\\u000a rhizogenes were both able to transiently
Transformation of forage legumes using Agrobacterium tumefaciens
K. J. Webb
Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce
Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37
Andrew Spencer; John D. Hamill; Michael J. C. Rhodes
Crown gall initiation on Mentha × piperita var. citrata (Ehrh.) Briq. (mint) was investigated using a range of wild type and mutant strains of Agrobacterium tumefaciens. Axenic transformed shoot cultures of Mentha 'citrata' were established on plant stems inoculated with the nopaline strain T37 of Agrobacterium tumefaciens. The presence of T-DNA in the transformed tissues and the absence of bacterial
Evidence of Migration and Endophytic Presence of Agrobacterium tumefaciens in Rose Plants
Rubén Martí; Jaime Cubero; Antonio Daza; Jaime Piquer; Carmen I. Salcedo; Clara Morente; María M. López
Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in
Transformation of forage legumes using Agrobacterium tumefaciens.
Webb, K J
Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce roots and both Onobrychis viciifolia and Lotus corniculatus produced teratomatous galls. The shoots elongated in the latter species only. In L. corniculatus, tissues that were infected by five bacterial strains were capable of shoot regeneration when cultured on a hormone-free medium. The transformed nature of these shoots was confirmed by their failure to root, the production of callus from leaves cultured on hormone-free medium and the presence of opines. PMID:24247771
Progress of cereal transformation technology mediated by Agrobacterium tumefaciens
Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko
Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens. PMID:25426132
Agrobacterium tumefaciens -mediated transformation of callus cells of Crataegus aronia
A. M. Al Abdallat; J. S. Sawwan; B. Al Zoubi
A genetic transformation system has been developed for callus cells of Crataegus\\u000a aronia using Agrobacterium\\u000a tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with\\u000a 5 mg l?1 Indole-3-butyric acid (IBA) and 0.5 mg l?1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different\\u000a types and concentrations
Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene
Background Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. Results Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100?mg?L-1 along with 250?mg?L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200??M acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3?mg?L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. Conclusion Rough lemon has been successfully transformed via Agrobacterium tumefaciens with ?-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated. PMID:22691292
Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58
Michelle R. Rondon; Katie S. Ballering; Michael G. Thomas
Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains
Genome Sequence of the Plant Pathogen and Biotechnology Agent Agrobacterium tumefaciens C58
Brad Goodner; Gregory Hinkle; Stacie Gattung; Nancy Miller; Mary Blanchard; Barbara Qurollo; Barry S. Goldman; Yongwei Cao; Manor Askenazi; Conrad Halling; Lori Mullin; Kathryn Houmiel; Jeffrey Gordon; Mark Vaudin; Oleg Iartchouk; Andrew Epp; Fang Liu; Clifford Wollam; Mike Allinger; Dahlia Doughty; Charlaine Scott; Courtney Lappas; Brian Markelz; Casey Flanagan; Chris Crowell; Jordan Gurson; Caroline Lomo; Carolyn Sear; Graham Strub; Chris Cielo; Steven Slater
Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the
Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions
Cangelosi, G.A.; Hung, L.; Puvanesarajah, V.; Stacey, G.; Ozga, D.A.; Leigh, J.A.; Nester, E.W.
The authors isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned, R. meliloti exo loci. They also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-..beta..-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.
Agrobacterium tumefaciens -mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium
Gento Tsuji; Satoshi Fujii; Naoki Fujihara; Chika Hirose; Seiji Tsuge; Tomonori Shiraishi; Yasuyuki Kubo
Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells
T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus.
Pardo, A G; Kemppainen, M; Valdemoros, D; Duplessis, S; Martin, F; Tagu, D
The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus. PMID:16178458
Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane.
Joyce, Priya; Kuwahata, Melissa; Turner, Nicole; Lakshmanan, Prakash
A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8-4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and beta-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1-3 transgene insertion events that were randomly integrated in the majority of the plants produced. PMID:20041254
Crystal Structure of Uronate Dehydrogenase from Agrobacterium tumefaciens*
Parkkinen, Tarja; Boer, Harry; Jänis, Janne; Andberg, Martina; Penttilä, Merja; Koivula, Anu; Rouvinen, Juha
Uronate dehydrogenase from Agrobacterium tumefaciens (AtUdh) belongs to the short-chain dehydrogenase/reductase superfamily and catalyzes the oxidation of d-galacturonic acid and d-glucuronic acid with NAD+ as a cofactor. We have determined the crystal structures of an apo-form of AtUdh, a ternary form in complex with NADH and product (substrate-soaked structure), and an inactive Y136A mutant in complex with NAD+. The crystal structures suggest AtUdh to be a homohexamer, which has also been observed to be the major form in solution. The monomer contains a Rossmann fold, essential for nucleotide binding and a common feature of the short-chain dehydrogenase/reductase family enzymes. The ternary complex structure reveals a product, d-galactaro-1,5-lactone, which is bound above the nicotinamide ring. This product rearranges in solution to d-galactaro-1,4-lactone as verified by mass spectrometry analysis, which agrees with our previous NMR study. The crystal structure of the mutant with the catalytic residue Tyr-136 substituted with alanine shows changes in the position of Ile-74 and Ser-75. This probably altered the binding of the nicotinamide end of NAD+, which was not visible in the electron density map. The structures presented provide novel insights into cofactor and substrate binding and the reaction mechanism of AtUdh. This information can be applied to the design of efficient microbial conversion of d-galacturonic acid-based waste materials. PMID:21676870
Developmental Effects of Zeatin, Ribosyl-Zeatin, and Agrobacterium tumefaciens B6 on Certain Mosses
Spiess, Luretta D.
Eight species of mosses studied were divided into two groups on the basis of their developmental responses to ribosyl-trans-zeatin and Agro-bacterium tumefaciens B6. All eight produced either gametophores or callus on the protonema in response to 6-(?,?-dimethylallylamino) purine and trans-zeatin. Three which produced normal gametophores with A. tumefaciens yielded callus or abnormal gametophores with ribosyl-trans-zeatin. Ribosyl-trans-zeatin and A. tumefaciens were relatively ineffective on five other mosses. Characteristics of protonemal growth common to each of these two groups are described. PMID:16659608
Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955
Yves Dessaux; Jacques Tempé; Stephen K. Farrand
The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 15955 was investigated. A partial HindIII digest of pTi15955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine,
Factors influencing Agrobacterium tumefaciens -mediated genetic transformation of Eleusine coracana (L.) Gaertn
Manju Sharma; Aditi Kothari-Chajer; Swati Jagga-Chugh; S. L. Kothari
Agrobacterium-mediated transformation protocol has been developed for Eleusine coracana (var. PR-202) by varying several factors which influence T-DNA delivery. Green nodular regenerative calli with meristematic\\u000a nodules of seed origin were used as the target tissue for Agrobacterium\\u000a tumefaciens-mediated gene transfer. The highest frequency of transformation (44.4%) was observed when callus was infected, co-cultivated\\u000a and incubated at 22°C. Incorporation of higher
Production of herbicide-resistant transgenic sweet potato plants through Agrobacterium tumefaciens method
Hye Jin Choi; Thummala Chandrasekhar; Hyo-Yeon Lee; Kyung-Moon Kim
Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding ?-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l?1 PPT,
Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens -mediated transformation
Donna G. Barfield; Eng-Chong Pua
An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg\\/L AgNO3 and 0.5–2 mg\\/L BA in combination
Mapping of the Interaction Between Agrobacterium tumefaciens and Vanda Kasem's Delight Orchid Protocorm-Like Bodies.
Gnasekaran, Pavallekoodi; Subramaniam, Sreeramanan
Physical contact between A. tumefaciens and the target plant cell walls is essential to transfer and integrate the transgene to introduce a novel trait. Chemotaxis response and attachment of Agrobacterium towards Vanda Kasem's Delight (VKD) protocorm-like bodies (PLBs) were studied to analyse the interaction between Agrobacterium and PLB during the transformation event. The study shows that initially A. tumefaciens reversibly attached to PLB surface via polar and lateral mode of adherence followed by the irreversible attachment which involved the production of cellulosic fibril by A. tumefaciens. Cellulosic fibril allows formation of biofilm at the tip of trichome. Contrarily, attachment mutant Escherichia coli strain DH5? was significantly deficient in the attachment process. Spectrophotometric GUS assay showed the mean value of attachment by A. tumefaciens was 8.72 % compared to the negative control E. coli strain DH5? that produced 0.16 %. A. tumefaciens swarmed with sharper and brighter edge when severe wounding was applied to the PLBs producing the highest swarming ratio of 1.46 demonstrating the positive effect of the plant exudates on bacterial movement. The study shows that VKD's PLBs are the suitable explants for Agrobacterium-mediated transformation since the bacteria expressed higher competency rate. PMID:26063938
Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots
Si-Jun Zheng; Ludmila Khrustaleva; Betty Henken; Eri Sofiari; Evert Jacobsen; Chris Kik; Frans A. Krens
This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the
Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassica juncea
Pierre J. Charest; V. N. Iyer; Brian L. Miki
Brassica napus and Brassica juncea were infected with a number of Agrobacterium tumefaciens strains. Tumourigenesis was very rapid and extremely efficient on B. juncea with all but one of the strains. Tumourigenesis on B. napus varied widely. It was very efficient with the nopaline strains, was reduced with the succinamopine strain A281 and was very weak with the octopine strains.
Agrobacterium tumefaciens -mediated transformation to alter ethylene and cytokinin biosynthesis in broccoli
Nigel E. Gapper; Marian J. McKenzie; Mary C. Christey; Robert H. Braun; Simon A. Coupe; Ross E. Lill; Paula E. Jameson
Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. Postharvest treatment of broccoli with 6-benzylaminopurine delays senescence, whilst exogenous ethylene has been shown to accelerate this process following harvest. To alter ethylene biosynthesis, broccoli was transformed, using Agrobacterium tumefaciens-mediated transformation, with an antisense ACC oxidase gene from broccoli driven by the asparagine synthetase promoter from asparagus. In addition, broccoli was
Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis
Aktas, Meriyem; Danne, Linna; Möller, Philip; Narberhaus, Franz
Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation. PMID:24723930
Technology Transfer Automated Retrieval System (TEKTRAN)
Anthuriums exhibit limited resistance to bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae and to the nematodes Radopholus simile and Meloidogyne javanica. Agrobacterium tumefaciens transformation of embryogenic calli with strains LBA4404, EHA105, and AGLO resulted in transgenic p...
Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens
Muller, A.; Manzara, T.; Lurquin, P.F.
Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype. In vitro crown gall transformation of dicotyledonous plant cells has been demonstrated after cocultivation of cell-wall regenerating mesophyll protoplasts with Agrobacterium tumefaciens cells. In addition, it has been shown that protoplasts freshly isolated from suspension cultures, when treated with A. tumefaciens spheroplasts and a fusogen, also generated cells displaying a typical crown gall phenotype, i.e., phytohormone-independent growth and opine synthesis. Subsequently, both techniques were used to transfer and express foreign genes in plant cells via A. tumefaciens T-DNA integration. For practical purposes, it would be advantageous to be able to perform crown gall transformation of plant cells in tissue culture. The authors report here for the first time the production of Nicotiana tabacum crown gall cells after cocultivation of callus tissue with A. tumefaciens A136 cells. 11 references, 1 figure, 1 table.
Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens.
Wang, Ling Ling; Wang, En Tao; Liu, Jie; Li, Ying; Chen, Wen Xin
Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129(T) and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules. PMID:16897296
Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens.
Bidney, D; Scelonge, C; Martich, J; Burrus, M; Sims, L; Huffman, G
Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol. PMID:1310058
Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants
Ebrahim Firoozabady; David L. DeBoer; Donald J. Merlo; Edward L. Halk; Lorraine N. Amerson; Kay E. Rashka; Elizabeth E. Murray
Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies
Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens -mediated transformation of embryogenic tissue
Toru Taniguchi; Yasunori Ohmiya; Manabu Kurita; Miyoko Tsubomura; Teiji Kondo
A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58\\/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on\\u000a hygromycin and kanamycin medium, respectively.
Agrobacterium tumefaciens -mediated transformation as a tool for random mutagenesis of Colletotrichum trifolii
Hiroyuki Takahara; Gento Tsuji; Yasuyuki Kubo; Mikihiro Yamamoto; Kazuhiro Toyoda; Yoshishige Inagaki; Yuki Ichinose; Tomonori Shiraishi
We transformed Colletotrichum trifolii, the causal agent of alfalfa anthracnose, using Agrobacterium tumefaciens as a new tool for random insertional mutagenesis. Fungal spores of C. trifolii were transformed with T-DNA including the hygromycin phosphotransferase gene ( hph). Southern analysis showed that every randomly selected transformant had a unique hybridization pattern of T-DNA, suggesting that the T-DNA was randomly integrated into
Parameters affecting the efficiency of Agrobacterium tumefaciens -mediated transformation of Colletotrichum graminicola
Jennifer L. Flowers; Lisa J. Vaillancourt
We have developed an Agrobacterium\\u000a tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than\\u000a previously available polyethylene glycol-mediated protocols, and falcate spores can be used instead of protoplasts for transformation.\\u000a Various experimental parameters were tested for their effects on
Isolation of a strain of Agrobacterium tumefaciens ( Rhizobium radiobacter ) utilizing methylene urea (ureaformaldehyde) as nitrogen source
Marja E. Koivunen; Christophe Morisseau; William R. Horwath; Bruce D. Hammock
Methylene ureas (MU) are slow-release nitrogen fertilizers degraded in soil by microbial enzymatic activity. Improved utilization of MU in agricultural production requires more knowledge about the organisms and enzymes responsible for its degradation. A Gram-negative, MU-degrading organism was isolated from a soil in Sacramento Valley, California. The bacterium was identified as Agrobacterium tumefaciens(recently also known as Rhizobium radiobacter) using both
A Novel Non-wounding Transient Expression Assay for Cereals Mediated by Agrobacterium tumefaciens
Surendar Reddy Dhadi; Aparna Deshpande; Wusirika Ramakrishna
A novel Agrobacterium tumefaciens-mediated transient expression assay (AmTEA) was developed for young plants of different cereal species and the model dicot\\u000a Arabidopsis thaliana. AmTEA was evaluated using five promoters (six constructs) and two reporter genes, gus and egfp. The constitutive 35S promoter and the promoter of the rice glutaredoxin gene showed gus and egfp expression in the cereals analyzed in
Ornamental traits modification by Rol genes in Osteospermum ecklonis transformed with Agrobacterium tumefaciens
Annalisa Giovannini; Michela Zottini; Giacomo Morreale; Angelo Spena; Andrea Allavena
Summary Transgenic plants of Osteospermum ecklonis were produced by cocultivation of leaf fragments with Agrobacterium tumefaciens harboring rol genes from A. rhizogenes. The phenotypic alterations caused by the different transgenes were evaluated in field trials. The genetic manipulation produced\\u000a transgenic plants characterized by the following features: 1) increased number of flowers (e.g., 35SrolC and rolABC); 2) early flowering (e.g., 35SrolC); 3)
Transformation of pollen embryo-derived explants by Agrobacterium tumefaciens in Hyoscyamus niger
Shanjun Tu; R. S. Sangwan; V. Raghavan; D. P. S. Verma; B. S. Sangwan-Norreel
Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient ß-glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were
Roles of Agrobacterium tumefaciens RirA in Iron Regulation, Oxidative Stress Response, and Virulence
Patchara Ngok-Ngam; Nantaporn Ruangkiattikul; A. Mahavihakanont; S. S. Virgem; R. Sukchawalit; S. Mongkolsuk
The analysis of genetics and physiological functions of Agrobacterium tumefaciens RirA (rhizobial iron regulator) has shown that it is a transcription regulator and a repressor of iron uptake systems. The rirA mutant strain (NTLrirA) overproduced siderophores and exhibited a highly constitutive expression of genes involved in iron uptake (fhuA, irp6A, and fbpA) compared to that of the wild-type strain (NTL4).
Transcriptional Activation of Agrobacterium tumefaciens Virulence Gene Promoters in Escherichia coli Requires the A. tumefaciens rpoA Gene, Encoding the Alpha Subunit of RNA Polymerase
The two-component regulatory system, composed of virA and virG, is indispensable for transcription of virulence genes within Agrobacterium tumefaciens. However, virA and virG are insufficient to activate transcrip- tion from virulence gene promoters within Escherichia coli cells, indicating a requirement for additional A. tumefaciens genes. In a search for these additional genes, we have identified the rpoA gene, encoding the
Phosphorus limitation increases attachment in Agrobacterium tumefaciens and reveals a conditional functional redundancy in adhesin biosynthesis
Xu, Jing; Kim, Jinwoo; Danhorn, Thomas; Merritt, Peter M.; Fuqua, Clay
Bacterial responses to phosphorus limitation, commonly inorganic phosphate (Pi), are important survival mechanisms in a variety of environments. The two-component sensor kinase PhoR and its cognate response regulator PhoB are central to the Pi limitation response of many bacteria and control the large Pho regulon. Limitation for Pi significantly increased attachment and biofilm formation by the plant pathogen Agrobacterium tumefaciens, and this was driven by PhoB. Surprisingly, it was also found that both phoR and phoB were essential in A. tumefaciens. Expression of a plasmid-borne copy of the low affinity Pi transporter (pit) from Sinorhizobium meliloti in A. tumefaciens abolished the phoB and phoR essentiality in A. tumefaciens and allowed direct demonstration of the requirement for this regulatory system in the biofilm response. Increased attachment under Pi limitation required a unipolar polysaccharide (UPP) adhesin. Mutation of a polyisoprenylphosphate hexose-1-phosphate transferase (PHPT) called uppE abolished UPP production and prevented surface attachment under Pi-replete conditions, but this was rescued under Pi limitation, and this rescue required phoB. In low Pi conditions, either uppE or a paralogous gene Atu0102 is functionally redundant, but only uppE functions in UPP synthesis and attachment when Pi is replete. This conditional functional redundancy illustrates the influence of phosphorus availability on A. tumefaciens surface colonization. PMID:23103488
Coordination of Division and Development Influences Complex Multicellular Behavior in Agrobacterium tumefaciens
Fuqua, Clay
The ?-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse ?-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJ homologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ?pleC and ?divK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ?pleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions. PMID:23437210
Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris.
Cha, Thye San; Yee, Willy; Aziz, Ahmad
The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient ?-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 ?M acetosyringone, Agrobacterium density of 1.0 units (OD(600)) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L(-1)) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga. PMID:22805959
Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens
Wilms, Ina; Overlöper, Aaron; Nowrousian, Minou; Sharma, Cynthia M.; Narberhaus, Franz
Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other ?-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium. PMID:22336765
Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only
Valérie Bonhomme; Dominique Laurain-Mattar; Jérôme Lacoux; Marc-André Fliniaux; Annie Jacquin-Dubreuil
Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rolABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in
Proteomic changes in grape embryogenic callus in response to Agrobacterium tumefaciens-mediated transformation.
Zhao, Fengxia; Chen, Lihua; Perl, Avihai; Chen, Shangwu; Ma, Huiqin
Agrobacterium tumefaciens-mediated transformation is highly required for studies of grapevine gene function and of huge potential for tailored variety improvements. However, grape is recalcitrant to transformation, and the underlying mechanism is largely unknown. To better understand the overall response of grapevine to A. tumefaciens-mediated transformation, the proteomic profile of cv. Prime embryogenic callus (EC) after co-cultivation with A. tumefaciens was investigated by two-dimensional electrophoresis and MALDI-TOF-MS analysis. Over 1100 protein spots were detected in both inoculated and control EC, 69 of which showed significantly differential expression; 38 of these were successfully identified. The proteins significantly up-regulated 3 d after inoculation were PR10, resistance protein Pto, secretory peroxidase, cinnamoyl-CoA reductase and different expression regulators; down-regulated proteins were ascorbate peroxidase, tocopherol cyclase, Hsp 70 and proteins involved in the ubiquitin-associated protein-degradation pathway. A. tumefaciens transformation-induced oxidative burst and modified protein-degradation pathways were further validated with biochemical measurements. Our results reveal that agrobacterial transformation markedly inhibits the cellular ROS-removal system, mitochondrial energy metabolism and the protein-degradation machinery for misfolded proteins, while the apoptosis signaling pathway and hypersensitive response are strengthened, which might partially explain the low efficiency and severe EC necrosis in grape transformation. PMID:21889056
Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens
Möller, Philip; Overlöper, Aaron; Förstner, Konrad U.; Wen, Tuan-Nan; Sharma, Cynthia M.; Lai, Erh-Min; Narberhaus, Franz
As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens. PMID:25330313
Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids.
Martínez, E; Palacios, R; Sánchez, F
Rhizobium phaseoli CFN299 forms nitrogen-fixing nodules in Phaseolus vulgaris (bean) and in Leucaena esculenta. It has three plasmids of 185, 225, and 410 kilobases. The 410-kilobase plasmid contains the nitrogenase structural genes. We have transferred these plasmids to the plasmid-free strain Agrobacterium tumefaciens GMI9023. Transconjugants containing different combinations of the R. phaseoli plasmids were obtained, and they were exhaustively purified before nodulation was assayed. Only transconjugants harboring the 410-kilobase plasmid nodulate P. vulgaris and L. esculenta. Nodules formed by all such transconjugants are able to reduce acetylene. Transconjugants containing the whole set of plasmids from CFN299 nodulate better and fix more nitrogen than the transconjugants carrying only the Sym plasmid. Microscopic analysis of nodules induced by A. tumefaciens transconjugants reveals infected cells and vascular bundles. None of the A. tumefaciens transconjugants, not even the one with the whole set of plasmids from CFN299, behaves in symbiosis like the original R. phaseoli strain; the transconjugants produce fewer nodules and have lower acetylene reduction (25% as compared to the original R. phaseoli strain) and more amyloplasts per nodule. More than 2,000 bacterial isolates from nodules of P. vulgaris and L. esculenta formed by the transconjugants were analyzed by different criteria. Not a single rhizobium could be detected. Our results show that R. phaseoli plasmids may be expressed in the A. tumefaciens background and direct the formation of effective, differentiated nodules. Images PMID:3584072
Controlling the interplay between Agrobacterium tumefaciens and plants during the transient expression of proteins.
Buyel, J F
In May 2012, the first plant-derived biopharmaceutical protein received full regulatory approval for therapeutic use in humans. Although plant-based expression systems have many advantages, they can suffer from low expression levels and, depending on the species, the presence of potentially toxic secondary metabolites. Transient expression mediated by Agrobacterium tumefaciens can be used to increase product yields but may also increase the concentration of secondary metabolites generated by plant defense responses. We have recently investigated the sequence of defense responses triggered by A. tumefaciens in tobacco plants and considered how these can be modulated by the transient expression of type III effectors from Pseudomonas syringae. Here we discuss the limitations of this approach, potential solutions and additional issues concerning transient expression in plants that should be investigated in greater detail. PMID:25997443
Interaction between Meloidogyne incognita and Agrobacterium tumefaciens or Fusarium oxysporum f. sp. lycopersici on Tomato.
El-Sherif, A G; Elwakil, M A
Agrobacterium tumefaciens stimulated and Fusarium oxysporum f. sp. lycopersici inhibited development and reproduction of Meloidogyne incognita when applied to the opposite split root of tomato, Lycopersicon esculentum cv. Tropic, plants. The lowest rate of nematode reproduction occurred after 2,000 juveniles were applied and the fungus was present in the opposite split root. The effects of all three pathogens alone on the growth of roots and shoots of tomato plants were evident, but M. incognita had a greater effect alone than did either of the other pathogens. The length of split roots was reduced by the infection of M. incognita and A. tumefaciens or F. oxysporum f. sp. lycopersici. The number of galls induced by nematodes on roots was higher where the bacterium was applied and lower where the fungus was applied to the opposite split root. PMID:19283119
Production of transgenic creeping bentgrass Agrostis stolonifera var. palustris plants by Agrobacterium tumefaciens -mediated transformation using hygromycin selection
Ning Han; Dong Chen; Hong-Wu Bian; Min-Juan Deng; Mu-Yuan Zhu
A protocol was developed for Agrobacterium tumefaciens-mediated transformation of creeping bentgrass [Agrostis stolonifera L. var. palustris (Huds) Farw]. The transformation was performed using the vector pCAMBIA 1301 which contains the reporter (uidA) gene and the selectable marker hygromycin phosphotransferase (hph) gene. Embryogenic calli initiated from mature seeds were infected with A. tumefaciens strain EHA105 followed by hygromycin selection. Effects of
Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein
Barbara Schrammeijer; Eddy Risseeuw; Werner Pansegrau; William L Crosby; Paul J. J Hooykaas
The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region–encoded transport system (reviewed in [1–5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB\\/VirD4 transport system
Combined Genetic and Physical Map of the Complex Genome of Agrobacterium tumefaciens
Goodner, Brad W.; Markelz, Brian P.; Flanagan, M. Casey; Crowell, Chris B.; Racette, Jodi L.; Schilling, Brittany A.; Halfon, Leah M.; Mellors, J. Scott; Grabowski, Gregory
A combined genetic and physical map of the Agrobacterium tumefaciens A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies. PMID:10464183
Rapid induction of Agrobacterium tumefaciens -mediated transgenic roots directly from adventitious roots in Panax ginseng
Jung Yeon Han; Yong Eui Choi
Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1\\/2 MS solid medium lacking NH4NO3 and containing 3.0 mg l?1 IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious\\u000a root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring ?-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed
Agrobacterium tumefaciens -mediated transformation of Lotus tenuis and regeneration of transgenic lines
F. D. Espasandin; M. M. Collavino; C. V. Luna; R. C. Paz; J. R. Tarragó; O. A. Ruiz; L. A. Mroginski; P. A. Sansberro
A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which\\u000a carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants
[Saline tolerance white clover transformed with the betaine aldehyde dehyrogenase gene by Agrobacterium tumefaciens].
Chen, Chuan-Fang; Li, Yi-Wen; Chen, Yu; Bai, Jian-Rong; Li, Hui; Zhu, Yin-Feng; Chen, Shou-Yi; Jia, Xu
The white clover has been transformed with the Betaine Aldehyde Dehydrogenase (BADH) gene cloned from Atriplex hortensis by Agrobacterium tumefaciens. The relative electronic conductivity of the transgenic plants under 1% NaCl stress for 48 hours was about 20%, less than the control plant's relative electronic conductivity (more than 40%), these showed the cell membrane of the transgenic plants has been less injured than control plants under salt stress. The other experience showed that the transgenic plant could grow well in water culture included 0.5% NaCl for more than two weeks, but the control plants could not. PMID:15468926
Enhanced hydantoinase and N -carbamoylase activity on immobilisation of Agrobacterium tumefaciens
Ingrid M. Foster; Rosemary D. Dorrington; Stephanie G. Burton
Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C. 3.5.1) activity on reaction with N-carbamylglycine. The hydantoinase (dihydropyrimidinase E.C. 188.8.131.52) and N-carbamoylase activities remained stable over 4 weeks storage at 4?°C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase.
Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation
T. Hatanaka; Y. E. Choi; T. Kusano; H. Sano
Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5??M N6–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing ?-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase\\u000a II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100?mg\\/l).\\u000a The
Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer.
McCullen, Colleen A; Binns, Andrew N
Host recognition and macromolecular transfer of virulence-mediating effectors represent critical steps in the successful transformation of plant cells by Agrobacterium tumefaciens. This review focuses on bacterial and plant-encoded components that interact to mediate these two processes. First, we examine the means by which Agrobacterium recognizes the host, via both diffusible plant-derived chemicals and cell-cell contact, with emphasis on the mechanisms by which multiple host signals are recognized and activate the virulence process. Second, we characterize the recognition and transfer of protein and protein-DNA complexes through the bacterial and plant cell membrane and wall barriers, emphasizing the central role of a type IV secretion system-the VirB complex-in this process. PMID:16709150
Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis
Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus. PMID:22166586
Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors.
Bélanger, C; Canfield, M L; Moore, L W; Dion, P
Little is known about the effect of the host on the genetic stability of bacterial plant pathogens. Crown gall, a plant disease caused by Agrobacterium tumefaciens, may represent a useful model to study this effect. Indeed, our previous observations on the natural occurrence and origin of nonpathogenic agrobacteria suggest that the host plant might induce loss of pathogenicity in populations of A. tumefaciens. Here we report that five different A. tumefaciens strains initially isolated from apple tumors produced up to 99% nonpathogenic mutants following their reintroduction into axenic apple plants. Two of these five strains were also found to produce mutants on pear and/or blackberry plants. Generally, the mutants of the apple isolate D10B/87 were altered in the tumor-inducing plasmid, harboring either deletions in this plasmid or point mutations in the regulatory virulence gene virG. Most of the mutants originating from the same tumor appeared to be of clonal origin, implying that the host plants influenced agrobacterial populations by favoring growth of nonpathogenic mutants over that of wild-type cells. This hypothesis was confirmed by coinoculation of apple rootstocks with strain D10B/87 and a nonpathogenic mutant. PMID:7601840
Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility
Tomlinson, Amelia D.; Ramey-Hartung, Bronwyn; Day, Travis W.; Merritt, Peter M.; Fuqua, Clay
The ubiquitous plant pathogen Agrobacterium tumefaciens attaches efficiently to plant tissues and abiotic surfaces and can form complex biofilms. A genetic screen for mutants unable to form biofilms on PVC identified disruptions in a homologue of the exoR gene. ExoR is a predicted periplasmic protein, originally identified in Sinorhizobium meliloti, but widely conserved among alphaproteobacteria. Disruptions in the A. tumefaciens exoR gene result in severely compromised attachment to abiotic surfaces under static and flow conditions, and to plant tissues. These mutants are hypermucoid due to elevated production of the exopolysaccharide succinoglycan, via derepression of the exo genes that direct succinoglycan synthesis. In addition, exoR mutants have lost flagellar motility, do not synthesize detectable flagellin and are diminished in flagellar gene expression. The attachment deficiency is, however, complex and not solely attributable to succinoglycan overproduction or motility disruption. A. tumefaciens ExoR can function independently of the ChvG–ChvI two component system, implicated in ExoR-dependent regulation in S. meliloti. Mutations that suppress the exoR motility defect suggest a branched regulatory pathway controlling succinoglycan synthesis, motility and biofilm formation. PMID:20576688
Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens
V. Levée; E. Garin; K. Klimaszewska; A. Séguin
A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein
Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58.
Rondon, Michelle R; Ballering, Katie S; Thomas, Michael G
Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron. Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain. Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium. Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce. These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore. Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp. PCC7120, rather than to known siderophore biosynthetic enzymes. Given these properties, it is proposed that the siderophore produced by A. tumefaciens C58 will have a unique chemical structure. Production of the siderophore was not required for virulence of A. tumefaciens when tested with a standard stem inoculation assay. PMID:15528670
Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells.
Matthysse, A G; Holmes, K V; Gurlitz, R H
The attachment of virulent strains of Agrobacterium tumefaciens to plant cells is the first step in the bacterial induction of tumors. Binding of A. tumefaciens to carrot tissue culture cells occurred as a two-step process. The initial step was the attachment of the bacteria to the plant cell wall. Living plant cells were not required. Bacterial attachment to heat-killed or glutaraldehyde-fixed carrot cells proceeded with only slightly altered kinetics and unaltered bacterial strain specificity. After the bacteria bound to the carrot cell surface, scanning electron microscopy showed that fibrils developed, surrounded the bacteria, and anchored them to the plant cell surface. These fibrils were synthesized by the bacteria and not by the plant cell since they were also made after the attachment of A. tumefaciens to dead carrot cells and since under some conditions the bacteria synthesized fibrils in the absence of plant cells. Calcofluor staining, acid hydrolysis, enzymatic digestion studies, and infrared spectroscopy showed that the fibrils were composed of cellulose. The formation of these cellulose fibrils occurred during the attachment of virulent strains of A. tumefaciens to plant cells in vitro. The fibrils anchored the bacteria to the plant cell surface and entrapped additional bacteria. The multiplication of entrapped and attached bacteria resulted in the formation of large clusters of bacteria held close to the plant cell wall and plasma membrane by cellulose fibrils. This high concentration of bacteria may facilitate transfer of Ti plasmid deoxyribonucleic acid to the plant cell resulting in the formation of tumors. PMID:7462151
Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)
NASA Technical Reports Server (NTRS)
Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)
Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.
Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens.
Mackay, J; Séguin, A; Lalonde, M
Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and axenic cultures were established. Octopine or nopaline, respective of the strain type used for inoculation, was detected in tumorous cultures. Southern blot analyses demonstrated T-DNA integration by hybridization of DNA from tumors with tmr and nos gene probes. One clone of B. papyrifera produced tumors with a morphogenic character, unusual in calli of this species, generating viable shoots which did not synthesize opine. PMID:24241754
The structure of a putative S-formylglutathione hydrolase from Agrobacterium tumefaciens
van Straaten, Karin E; Gonzalez, Claudio F; Valladares, Ricardo B; Xu, Xiaohui; Savchenko, Alexei V; Sanders, David A R
The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 Ĺ resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S-formylglutathione hydrolase (AtuSFGH). The three-dimensional structure of AtuSFGH contains the ?/? hydrolase fold topology and exists as a homo-dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes C—O bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S-glutathionylation is also proposed. PMID:19653299
Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens
Lassalle, Florent; Campillo, Tony; Vial, Ludovic; Baude, Jessica; Costechareyre, Denis; Chapulliot, David; Shams, Malek; Abrouk, Danis; Lavire, Céline; Oger-Desfeux, Christine; Hommais, Florence; Guéguen, Laurent; Daubin, Vincent; Muller, Daniel; Nesme, Xavier
The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species. PMID:21795751
Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation
Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites. PMID:24949275
Deletion analysis of the mannopine synthase gene promoter in sunflower crown gall tumors and Agrobacterium tumefaciens.
DiRita, V J; Gelvin, S B
We have used deletion mutagenesis to analyze a TR-DNA promoter from the octopine-type Ti plasmid pTiB6806. The promoter for the gene encoding mannopine synthase (mas) was cloned upstream of the bacterial kanamycin-resistance gene neomycin phosphotransferase II (NPT II). Bal31 deletion mutagenesis was used to generate deletion derivatives of the mas/NPTII gene beginning 1353 bp upstream of the initiation of transcription and extending to 120 bp downstream from the mRNA start site. Deletions that left intact 318 bp upstream of transcription initiation had no detectable effect on the ability of tumors harboring the deletion to synthesize correctly initiated mRNA or to grow on the kanamycin analogue G418. Deletion to-138 destroyed the ability of sunflower crown gall tumors to grow on G418 although low levels of the mas/NPTII transcript were detected in one tumor line. Deletions that left only 57 bp upstream of transcription initiation allowed neither growth on G418 nor detectable mas/NPTII synthesis, even though the CCAAT and TATAA homologies were intact. The mas promoter is functional in Agrobacterium tumefaciens and we present data concerning the effects of the Bal31 deletions on the growth of A. tumefaciens on kanamycin. PMID:3039293
Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens
Voigt, Boris; Menzel, Diedrik; Baluška, František; Villanueva, Marco A.
Plant-targeted pCB302 plasmids containing sequences encoding gfp fusions with a microtubule-binding domain; gfp with the fimbrin actin-binding domain 2; and gfp with AtRACK1C from Arabidopsis thaliana, all harbored in Agrobacterium tumefaciens, were used to assay heterologous expression on three different clades of the photosynthetic dinoflagellate, Symbiodinium. Accessibility to the resistant cell wall and through the plasma membrane of these dinoflagellates was gained after brief but vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection of the cells expressing the hybrid proteins, which showed a characteristic green fluorescence, although they appeared to lose their photosynthetic pigments and did not further divide. Cell GFP expression frequency measured as green fluorescence emission yielded 839 per every 106 cells for Symbiodinium kawagutii, followed by 640 and 460 per every 106 cells for Symbiodinium microadriaticum and Symbiodinium sp. Mf11, respectively. Genomic PCR with specific primers amplified the AtRACK1C and gfp sequences after selection in all clades, thus revealing their presence in the cells. RT-PCR from RNA of S. kawagutii co-incubated with A. tumefaciens harboring each of the three vectors with their respective constructs, amplified products corresponding to the heterologous gfp sequence while no products were obtained from three distinct negative controls. The reported procedure shows that mild abrasion followed by co-incubation with A. tumefaciens harboring heterologous plasmids with CaMV35S and nos promoters can lead to expression of the encoded proteins into the Symbiodinium cells in culture. Despite the obvious drawbacks of the procedure, this is an important first step towards a stable transformation of Symbiodinium. PMID:26167858
Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens.
Loper, J E; Kado, C I
The host range of Agrobacterium tumefaciens 1D1109, known to induce crown gall only on grapevine (Vitis spp.), was extended to include many plant species by transferring a tumor-inducing plasmid (pTi) from strain 1D1, a broad-host-range pathogen. The pTi plasmid was mobilized by the conjugative plasmid pRK2, which was inserted into 1D1 by mating with Escherichia coli J53(pRK2). The resulting transconjugants were screened for their ability to induce crown gall tumors on hosts other than grapevine by inoculation into sunflower. Transconjugants that were virulent on sunflower were then tested on 36 different host plants and compared with host-limited strain 1D1109 and the donor strain. Two transconjugants induced tumors on the same 28 plant species as those of the original plasmid donor 1D1(pRK2) (pTi). These results show that pRK2 promoted transfer of the pTi plasmid and suggest that the pTi plasmid rather than the A. tumefaciens chromosome determined the host range of the pathogen. Insertion of pRK2 alone did not extend the host range of strain 1D1109. Insertion of pS-a into A. tumefaciens 1D1 by mating with E. coli J53-1 (pS-a) resulted in the concomitant loss of pTi and virulence. There appears to be incompatibility between pTi and pS-a. Images PMID:457613
Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens
Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.
ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.
Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones.
Wolterink-van Loo, Suzanne; Escamilla Ayala, Abril A; Hooykaas, Paul J J; van Heusden, G Paul H
Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 5' end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His6-tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA. PMID:25505187
Regeneration of transgenic Picea glauca, P. Mariana , and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens
Krystyna Klimaszewska; Denis Lachance; Gervais Pelletier; Marie-Anne Lelu; Armand Séguin
Summary Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58\\/pMP90\\/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (?-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation\\u000a procedure. Of the three
High-efficiency transformation of Lycium barbarum mediated by Agrobacterium tumefaciens and transgenic plant regeneration via somatic embryogenesis
Z. Hu; J. Yang; G. Guo; G. Zheng
We have developed a reliable and high-frequency system of transformation and regeneration via somatic embryogenesis (SE) of Lycium barbarum. Leaf segments were co-cultivated with Agrobacterium tumefaciens EHA101 (pIG121Hm) carrying the neomycin phosphotransferase II gene as a selectable marker and an intron-#-glucuronidase (GUS) gene as a reporter marker. On the medium for callus-induction, which contained 50 mg l-1 kanamycin (Km), approximately
High Efficiency Transgene Segregation in Co-Transformed Maize Plants using an Agrobacterium Tumefaciens 2 T-DNA Binary System
Michael Miller; Laura Tagliani; Ning Wang; Benjamin Berka; Dennis Bidney; Zuo-Yu Zhao
For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker
Role a Agrobacterium tumefaciens ChvA protein in export of. beta. -1,2-glucan
Cangelosi, G.A.; Martinetti, G.; Leigh, J.A.; Lee, Chi Chang; Theines, C. (Univ. of Washington, Seattle (USA))
Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic {beta}-1,2-glucan. Whereas chvB is required for {beta}-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral {beta}-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble {beta}-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for {beta}-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.
A Glutathione Transferase from Agrobacterium tumefaciens Reveals a Novel Class of Bacterial GST Superfamily
Skopelitou, Katholiki; Dhavala, Prathusha; Papageorgiou, Anastassios C.; Labrou, Nikolaos E.
In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Ĺ resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity. PMID:22496785
Fha Interaction with Phosphothreonine of TssL Activates Type VI Secretion in Agrobacterium tumefaciens
Lin, Jer-Sheng; Wu, Hsin-Hui; Hsu, Pang-Hung; Ma, Lay-Sun; Pang, Yin-Yuin; Tsai, Ming-Daw; Lai, Erh-Min
The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed. PMID:24626341
Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens.
Hou, Feng; Miyakawa, Takuya; Takeshita, Daijiro; Kataoka, Michihiko; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru
(R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293?K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72?Ĺ resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2(1), with unit-cell parameters a = 62.0, b = 126.4, c = 62.0?Ĺ, ? = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V(M) = 2.08?Ĺ(3)?Da(-1)). PMID:23027756
Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens
Hou, Feng; Miyakawa, Takuya; Takeshita, Daijiro; Kataoka, Michihiko; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru
(R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293?K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72?Ĺ resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P21, with unit-cell parameters a = 62.0, b = 126.4, c = 62.0?Ĺ, ? = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V M = 2.08?Ĺ3?Da?1). PMID:23027756
Agrobacterium tumefaciens mediated transient expression of plant cell wall-degrading enzymes in detached sunflower leaves.
Jung, Sang-Kyu; Lindenmuth, Benjamin E; McDonald, Karen A; Hwang, Hwang; Bui, Mai Q Nguyen; Falk, Bryce W; Uratsu, Sandra L; Phu, My L; Dandekar, Abhaya M
For biofuel applications, synthetic endoglucanase E1 and xylanase (Xyn10A) derived from Acidothermus cellulolyticus were transiently expressed in detached whole sunflower (Helianthus annuus L.) leaves using vacuum infiltration. Three different expression systems were tested, including the constitutive CaMV 35S-driven, CMVar (Cucumber mosaic virus advanced replicating), and TRBO (Tobacco mosaic virus RNA-Based Overexpression Vector) systems. For 6-day leaf incubations, codon-optimized E1 and xylanase driven by the CaMV 35S promoter were successfully expressed in sunflower leaves. The two viral expression vectors, CMVar and TRBO, were not successful although we found high expression in Nicotiana benthamiana leaves previously for other recombinant proteins. To further enhance transient expression, we demonstrated two novel methods: using the plant hormone methyl jasmonic acid in the agroinfiltration buffer and two-phase optimization of the leaf incubation temperature. When methyl jasmonic acid was added to Agrobacterium tumefaciens cell suspensions and infiltrated into plant leaves, the functional enzyme production increased 4.6-fold. Production also increased up to 4.2-fold when the leaf incubation temperature was elevated above the typical temperature, 20C, to 30C in the late incubation phase, presumably due to enhanced rate of protein synthesis in plant cells. Finally, we demonstrated co-expression of E1 and xylanase in detached sunflower leaves. To our knowledge, this is the first report of (co)expression of heterologous plant cell wall-degrading enzymes in sunflower. PMID:25180328
Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens
Liu, D.; Thomas, P.W.; Momb, J.; Hoang, Q.Q.; Petsko, G.A.; Ringe, D.; Fast, W.
N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.
Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens*
Ochiai, Akihito; Yamasaki, Masayuki; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku
Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a ?-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-l-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/?GGG) were determined at 2.10- and 2.99-? resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an ?/?-barrel + anti-parallel ?-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/?GGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His311 and Tyr365 as the catalytic base and acid, respectively. Two structural determinants, i.e. a short ?-helix in the central ?/?-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme. PMID:20507980
Preliminary crystallographic analysis of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens
Cupp-Vickery, Jill R.; Igarashi, Robert Y.; Meyer, Christopher R.
ADP-glucose pyrophosphorylase catalyzes the conversion of glucose-1-phosphate and ATP to ADP-glucose and pyrophosphate, a key regulated step in both bacterial glycogen and plant starch biosynthesis. Crystals of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens (420 amino acids, 47?kDa) have been obtained by the sitting-drop vapor-diffusion method using lithium sulfate as a precipitant. A complete native X-ray diffraction data set was collected to a resolution of 2.0?Ĺ from a single crystal at 100?K. The crystals belong to space group I222, with unit-cell parameters a = 92.03, b = 141.251, c = 423.64?Ĺ. To solve the phase problem, a complete anomalous data set was collected from a selenomethionyl derivative. These crystals display one-fifth of the unit-cell volume of the wild-type crystals, with unit-cell parameters a = 85.38, b = 93.79, c = 140.29?Ĺ and space group I222. PMID:16511013
The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization
Pan, Yi; Fiscus, Valena; Meng, Wuyi; Zheng, Zhida; Zhang, Lian-Hui; Fuqua, Clay; Chen, Lingling (IMCB-Singapore); (Indiana)
The Agrobacterium tumefaciens BlcR is a member of the emerging isocitrate lyase transcription regulators that negatively regulates metabolism of {gamma}-butyrolactone, and its repressing function is relieved by succinate semialdehyde (SSA). Our crystal structure showed that BlcR folded into the DNA- and SSA-binding domains and dimerized via the DNA-binding domains. Mutational analysis identified residues, including Phe{sup 147}, that are important for SSA association; BlcR{sup F147A} existed as tetramer. Two BlcR dimers bound to target DNA and in a cooperative manner, and the distance between the two BlcR-binding sequences in DNA was critical for BlcR-DNA association. Tetrameric BlcR{sup F147A} retained DNA binding activity, and importantly, this activity was not affected by the distance separating the BlcR-binding sequences in DNA. SSA did not dissociate tetrameric BlcR{sup F147A} or BlcR{sup F147A}-DNA. As well as in the SSA-binding site, Phe{sup 147} is located in a structurally flexible loop that may be involved in BlcR oligomerization. We propose that SSA regulates BlcR DNA-binding function via oligomerization.
Crystal structure of AGR_C_4470p from Agrobacterium tumefaciens
Vorobiev, Sergey M.; Neely, Helen; Seetharaman, Jayaraman; Ma, Li-Chung; Xiao, Rong; Acton, Thomas B.; Montelione, Gaetano T.; Tong, Liang
We report here the crystal structure at 2.0 Ĺ resolution of the AGR_C_4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR_C_4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR_C_4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR_C_4470p in E. coli, in addition to the ChuS protein. PMID:17322535
Evolution of Enzymatic Activities in the Enolase Superfamily: Galactarate Dehydratase III from Agrobacterium tumefaciens C58
The genome of Agrobacterium tumefaciens C58 encodes 12 members of the enolase superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars for high-throughput screening, one protein (UniProt entry A9CG74; locus tag Atu4196) showed activity with both m-galactarate and d-galacturonate. Two families of galactarate dehydratases had been discovered previously in the ENS, GalrD/TalrD [Yew, W. S., et al. (2007) Biochemistry46, 9564–9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry48, 11546–11558]; these have different active site acid/base catalysis and have no activity with d-galacturonate. A9CG74 dehydrates m-galactarate to form 2-keto-3-deoxy-galactarate but does not dehydrate d-galacturonate as expected. Instead, when A9CG74 is incubated with d-galacturonate, 3-deoxy-d-xylo-hexarate or 3-deoxy-d-lyxo-hexarate is formed. In this reaction, instead of abstracting the C5 proton ? to the carboxylate group, the expected reaction for a member of the ENS, the enzyme apparently abstracts the proton ? to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to the benzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarate as a carbon source under the conditions tested in this study, although it does utilize d-galacturonate, which is a likely precursor to m-galactarate. The gene encoding A9CG74 and several genome proximal genes were upregulated with d-galacturonate as the carbon source. One of these, a member of the dihydrodipicolinate synthase superfamily, catalyzes the dehydration and subsequent decarboxylation of 2-keto-3-deoxy-d-galactarate to ?-ketoglutarate semialdehyde, thereby providing a pathway for the conversion of m-galactarate to ?-ketoglutarate semialdehyde. PMID:24926996
Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.
Muniz, C R; da Silva, G F; Souza, M T; Freire, F C O; Kema, G H J; Guedes, M I F
Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation. PMID:24634294
Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants
Gulshan Chhabra; Darshna Chaudhary; Manish Sainger; Pawan K. Jaiwal
Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses 'nodular calli' developed on the BAP - pretreated daughter frond explants in B5 medium containing sucrose (1.0 %) with 2,4-D (5.0 ?M) and 2-iP (50.0 ?M) or 2,4-D (50.0 ?M) and TDZ (5.0 ?M) under light\\u000a conditions. These calli were co-cultured
Evaluation of methods for celery ( Apium Graveolens L.) transformation using Agrobacterium tumefaciens and the bar gene as selectable marker
A. V. Loskutov; G.-Q. Song; K. C. Sink
Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery.\\u000a Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for\\u000a 2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and
Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only.
Bonhomme, V; Laurain-Mattar, D; Lacoux, J; Fliniaux, M; Jacquin-Dubreuil, A
Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures. PMID:10989174
Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome.
Allardet-Servent, A; Michaux-Charachon, S; Jumas-Bilak, E; Karayan, L; Ramuz, M
Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed-field gel electrophoresis (PFGE) reveals four replicons: two large molecules of 3,000 and 2,100 kb, the 450-kb cryptic plasmid, and the 200-kb Ti plasmid. Digestion by PacI or SwaI generated 12 or 14 fragments, respectively. The two megabase-sized replicons, used as probes, hybridize with different restriction fragments, showing that these replicons are two independent genetic entities. A 16S rRNA probe and genes encoding functions essential to the metabolism of the organism were found to hybridize with both replicons, suggesting their chromosomal nature. In PFGE, megabase-sized circular DNA does not enter the gel. The 2.1-Mb chromosome always generated an intense band, while the 3-Mb band was barely visible. After linearization of the DNA by X-irradiation, the intensity of the 3-Mb band increased while that of the 2.1-Mb remained constant. This suggests that the 3-Mb chromosome is circular and that the 2.1-Mb chromosome is linear. To confirm this hypothesis, genomic DNA, trapped in an agarose plug, was first submitted to PFGE to remove any linear DNA present. The plug was then recovered, and the remaining DNA was digested with either PacI or SwaI and then separated by PFGE. The fragments corresponding to the small chromosome were found to be absent, while those corresponding to the circular replicon remained, further proof of the linear nature of the 2.1-Mb chromosome. Images PMID:8253676
The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 is functional in Agrobacterium tumefaciens.
González, Tania; Eng, Felipe; Fraga, Reinaldo; Fonseca, Jennifer; Amores, Isis
The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors ?-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high ?-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens. PMID:23873160
Genetic Analysis of Agrobacterium tumefaciens Unipolar Polysaccharide Production Reveals Complex Integrated Control of the Motile-to-Sessile Switch
Xu, Jing; Kim, Jinwoo; Koestler, Benjamin J.; Choi, Jeong-Hyeon; Waters, Christopher M.; Fuqua, Clay
Summary Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c-di-GMP) lead to surface-contact-independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c-di-GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive cyclic diguanosine monophosphate phosphodiesterase also elevate UPP production and attachment, consistent with c-di-GMP activation of surface-dependent adhesin deployment. PMID:23829710
CelR, an Ortholog of the Diguanylate Cyclase PleD of Caulobacter, Regulates Cellulose Synthesis in Agrobacterium tumefaciens
Barnhart, D. Michael; Su, Shengchang; Baccaro, Brenna E.; Banta, Lois M.
Cellulose fibrils play a role in attachment of Agrobacterium tumefaciens to its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, including A. tumefaciens. In this study, we identified two putative diguanylate cyclase genes, celR (atu1297) and atu1060, that influence production of cellulose in A. tumefaciens. Overexpression of either gene resulted in increased cellulose production, while deletion of celR, but not atu1060, resulted in decreased cellulose biosynthesis. celR overexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis of celR and ?cel mutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing both celR and celA, which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lack celA use CelR, called PleD, to regulate specific processes associated with polar localization and cell division. PMID:24038703
Cyanuric acid biodegradation by a mixed bacterial culture of Agrobacterium tumefaciens and Acinetobacter sp. in a packed bed biofilm reactor.
Galíndez-Nájera, S P; Llamas-Martínez, M A; Ruiz-Ordaz, N; Juárez-Ramírez, C; Mondragón-Parada, M E; Ahuatzi-Chacón, D; Galíndez-Mayer, J
Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L(-1)), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L(-1)) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support. PMID:19002512
Agrobacterium tumefaciens Deploys a Superfamily of Type VI Secretion DNase Effectors as Weapons for Interbacterial Competition In Planta
Ma, Lay-Sun; Hachani, Abderrahman; Lin, Jer-Sheng; Filloux, Alain; Lai, Erh-Min
Summary The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many Proteobacteria to target effectors/toxins into both eukaryotic and prokaryotic cells. We report that Agrobacterium tumefaciens, a soil bacterium that triggers tumorigenesis in plants, produces a family of type VI DNase effectors (Tde) that are distinct from previously known polymorphic toxins and nucleases. Tde exhibits an antibacterial DNase activity that relies on a conserved HxxD motif and can be counteracted by a cognate immunity protein, Tdi. In vitro, A. tumefaciens T6SS could kill Escherichia coli but triggered a lethal counterattack by Pseudomonas aeruginosa upon injection of the Tde toxins. However, in an in planta coinfection assay, A. tumefaciens used Tde effectors to attack both siblings cells and P. aeruginosa to ultimately gain a competitive advantage. Such acquired T6SS-dependent fitness in vivo and conservation of Tde-Tdi couples in bacteria highlights a widespread antibacterial weapon beneficial for niche colonization. PMID:24981331
Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass ( Panicum virgatum)
J. S. VanderGheynst; H.-Y. Guo; C. W. Simmons
Agrobacterium tumefaciens-mediated transient expression (agroinfiltration) experiments were performed in harvested switchgrass (Panicum virgatum) leaves to identify the effects of wounding by bead beating, surfactant concentration and vacuum application on in planta ?-glucuronidase expression and leaf decay. Expression was scored based on a consistent pattern of visual observations of histochemical staining over the leaf surface as might be observed in stable
Identification of pathogenicity-related genes in the vascular wilt fungus verticillium dahliae by agrobacterium tumefaciens-mediated t-DNA insertional mutagenesis.
Technology Transfer Automated Retrieval System (TEKTRAN)
Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that underpin pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transform...
Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. II. Effect of the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis
E. Storti; P. Bogani; P. Bettini; P. Bittini; M. L. Guardiola; M. G. Pellegrini; D. Inzé; M. Buiatti
We have studied the effect of a change in the endogenous hormone equilibria on the competence of tomato (Lycopersicon esculentum) cells to defend themselves against the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Calluses from cvs 'Davis' and 'Red River', respectively resistant and susceptible to Fusarium and transgenic for an auxin- or cytokinin-synthesizing gene from Agrobacterium tumefaciens, were used. The
Agrobacterium tumefaciens and A. rhizogenes use different proteins to transport bacterial DNA into the plant cell nucleus
Ream, Walt
Summary Agrobacterium tumefaciens and A. rhizogenes transport single?stranded DNA (ssDNA; T?strands) and virulence proteins into plant cells through a type IV secretion system. DNA transfer initiates when VirD2 nicks border sequences in the tumour?inducing plasmid, attaches to the 5? end, and pilots T?strands into plant cells. Agrobacterium tumefaciens translocates ssDNA?binding protein VirE2 into plant cells where it targets T?strands into the nucleus. Some A. rhizogenes strains lack VirE2 but transfer T?strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant. VirE2 and full?length GALLS (GALLS?FL) contain nuclear localization sequences that target these proteins to the plant cell nucleus. VirE2 binds cooperatively to T?strands allowing it to move ssDNA without ATP hydrolysis. Unlike VirE2, GALLS?FL contains ATP?binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. VirE2 may accumulate in the nucleus and pull T?strands into the nucleus using the force generated by cooperative DNA binding. GALLS?FL accumulates inside the nucleus where its predicted ATP?dependent strand transferase may pull T?strands into the nucleus. These different mechanisms for nuclear import of T?strands may affect the efficiency and quality of transgenic events in plant biotechnology applications. PMID:21255274
Agrobacterium tumefaciens and A. rhizogenes use different proteins to transport bacterial DNA into the plant cell nucleus.
Ream, Walt
Agrobacterium tumefaciens and A. rhizogenes transport single-stranded DNA (ssDNA; T-strands) and virulence proteins into plant cells through a type IV secretion system. DNA transfer initiates when VirD2 nicks border sequences in the tumour-inducing plasmid, attaches to the 5' end, and pilots T-strands into plant cells. Agrobacterium tumefaciens translocates ssDNA-binding protein VirE2 into plant cells where it targets T-strands into the nucleus. Some A. rhizogenes strains lack VirE2 but transfer T-strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant. VirE2 and full-length GALLS (GALLS-FL) contain nuclear localization sequences that target these proteins to the plant cell nucleus. VirE2 binds cooperatively to T-strands allowing it to move ssDNA without ATP hydrolysis. Unlike VirE2, GALLS-FL contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. VirE2 may accumulate in the nucleus and pull T-strands into the nucleus using the force generated by cooperative DNA binding. GALLS-FL accumulates inside the nucleus where its predicted ATP-dependent strand transferase may pull T-strands into the nucleus. These different mechanisms for nuclear import of T-strands may affect the efficiency and quality of transgenic events in plant biotechnology applications. PMID:21255274
Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones.
Chateau, S; Sangwan, R S; Sangwan-Norreel, B S
Many plant species and/or genotypes are highly recalcitrant to Agrobacterium-mediated genetic transformation, and yet little is known about this phenomenon. Using several Arabidopsis genotypes/ecotypes, the results of this study indicated that phytohormone pretreatment could overcome this recalcitrance by increasing the transformation rate in the known recalcitrant genotypes. Transient expression of a T-DNA encoded ss-glucuronidase (GUS) gene and stable kanamycin resistance were obtained for the ten Arabidopsis genotypes tested as well as for the mutant uvh1 (up to 69% of petioles with blue spots and up to 42% resistant calli). Cultivation of Arabidopsis tissues on phytohormones for 2-8 d before co-cultivation with Agrobacterium tumefaciens significantly increased transient GUS gene expression by 2-11-fold and stable T-DNA integration with petiole explants. Different Arabidopsis ecotypes revealed differences in their susceptibility to Agrobacterium-mediated transformation and in their type of reaction to pre-cultivation (three types of reactions were defined by gathering ecotypes into three groups). The Arabidopsis uvh1 mutant described as defective in a DNA repair system showed slightly lower competence to transformation than did its progenitor Colombia. This reduced transformation competence, however, could be overcome by 4-d pre-culture with phytohormones. The importance of pre-cultivation with phytohormones for genetic transformation is discussed. PMID:11141170
An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens
Julie Belles-Isles; Mathieu Dusabenyagasani; Francine M. Tremblay
An efficient and reproducible procedure for the transformation of white spruce (Picea glauca (Moench) Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, con- taining the neomycin phosphotransferase II (nptII) gene providing kanamycin
Endophytic Occupation of Root Nodules and Roots of Melilotus dentatus by Agrobacterium tumefaciens
Ling Ling Wang; En Tao Wang; Jie Liu; Ying Li; Wen Xin Chen
Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis
Characterization of transposon Tn5-facilitated donor strains and development of a chromosomal linkage map for Agrobacterium tumefaciens.
Pischl, D L; Farrand, S K
We have further characterized the transposon Tn5-facilitated chromosomal gene transfer system developed for Agrobacterium tumefaciens 15955. Using a strain whose chromosome contained Tn5, we compared the chromosome-mobilizing ability of plasmid pDP37 (containing Tn5) with that of its parent plasmid R68.45. For R68.45, we observed nonpolar transmission from multiple origins. For pDP37 we found polarized transmission from a single origin near ilv. When we examined the transmission gradients of a number of pDP37-containing donor strains each differing at the site of the chromosomal insertion we found just two classes. One set of donors transmitted markers with a gradient of Ilv+ greater than Rifr greater than His+ greater than Met+, whereas the second set transferred His+ greater than Rifr greater than Ilv+ greater than Met+. Using representatives from each transmission class of donor strains, we conducted matings to measure the degree of linkage between pairs of adjacent donor markers. From this information we developed a map of the A. tumefaciens 15955 chromosome. Attempts to isolate R-prime plasmids or Hfr-like donors were unsuccessful. PMID:6330023
Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site
Lamparter, Tilman; Michael, Norbert; Mittmann, Franz; Esteban, Berta
Phytochromes are photochromic photoreceptors with a bilin chromophore that are found in plants and bacteria. The soil bacterium Agrobacterium tumefaciens contains two genes that code for phytochrome-homologous proteins, termed Agrobacterium phytochrome 1 and 2 (Agp1 and Agp2). To analyze its biochemical and spectral properties, Agp1 was purified from the clone of an E. coli overexpressor. The protein was assembled with the chromophores phycocyanobilin and biliverdin, which is the putative natural chromophore, to photoactive holoprotein species. Like other bacterial phytochromes, Agp1 acts as light-regulated His kinase. The biliverdin adduct of Agp1 represents a previously uncharacterized type of phytochrome photoreceptor, because photoreversion from the far-red absorbing form to the red-absorbing form is very inefficient, a feature that is combined with a rapid dark reversion. Biliverdin bound covalently to the protein; blocking experiments and site-directed mutagenesis identified a Cys at position 20 as the binding site. This particular position is outside the region where plant and some cyanobacterial phytochromes attach their chromophore and thus represents a previously uncharacterized binding site. Sequence comparisons imply that the region around Cys-20 is a ring D binding motif in phytochromes. PMID:12186972
Technology Transfer Automated Retrieval System (TEKTRAN)
In order to study genetic factors of pathogenicity, insertional mutants of Sclerotinia sclerotiorum were generated using Agrobacterium tumefacience-mediated transformation with both mycelial fragments and ascospores. Transformants were tested for number of insertions by Southern hybridization and f...
Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system
Narender S. Nehra; Ravindra N. Chibbar; Kutty K. Kartha; Raju S. S. Datla; William L. Crosby; Cecil Stushnoff
An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II)
Global analysis of differentially expressed genes and proteins in the wheat callus infected by Agrobacterium tumefaciens.
Zhou, Xiaohong; Wang, Ke; Lv, Dongwen; Wu, Chengjun; Li, Jiarui; Zhao, Pei; Lin, Zhishan; Du, Lipu; Yan, Yueming; Ye, Xingguo
Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops. PMID:24278131
Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens
Zhou, Xiaohong; Wang, Ke; Lv, Dongwen; Wu, Chengjun; Li, Jiarui; Zhao, Pei; Lin, Zhishan; Du, Lipu; Yan, Yueming; Ye, Xingguo
Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops. PMID:24278131
The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens.
Stachel, S E; Nester, E W
The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3-lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty-four vir::Tn3-lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta-galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non-inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant-inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant-inducible loci (pin) which are non-essential for virulence. PMID:3017694
An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.
Gupta, Vijayta; Ur Rahman, Laiq
Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66 %) with an average of 5.08?±?0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5 mg/l BAP and 5 mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200 mg/l) for selection, standardization of cocultivation time (45 min) and acetosyringone concentration (150 ?M) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600 bp npt II fragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocol established during the study will open new vistas for genetic manipulation and introduction of desired genes for genetic improvement of T. erecta. PMID:25504508
Genetic and Environmental Factors Affecting T-Pilin Export and T-Pilus Biogenesis in Relation to Flagellation of Agrobacterium tumefaciens
The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells. Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role
Oncogene 6b from Agrobacterium tumefaciens Induces Abaxial Cell Division at Late Stages of Leaf Development and Modifies Vascular Development in Petioles
Shinji Terakura; Saeko Kitakura; Masaki Ishikawa; Yoshihisa Ueno; Tomomichi Fujita; Chiyoko Machida; Hiroetsu Wabiko; Yasunori Machida
; The 6b gene in the T-DNA region of the Ti plasmids of Agrobacterium tumefaciens and A. vitis is able to generate shooty calli in phytohormone-free culture of leaf sections of tobacco transformed with 6b. In the present study, we report characteristic morphological abnormalities of the leaves of transgenic tobacco and Arabidopsis that express 6b from pTiAKE10 (AK-6b), and altered
Study on the Genetic Transformation of Gentian by Gene Recombinant
Yan Yu Qing; Wang Yang; Xu Xiang Ling
Transformation recombinant vector pMHL7133-Gus linked with rol gene which be cloned from Agrobacterium Rhizogenes R1000 through Agrobacterium tumefaciens LBA4404 into explant of gentian lamina, inducing rol gene express and producing hair root. Meanwhile, using the Agrobacterium Rhizogenes R1000 infect gentian directly as a comparison, we built two sets of transform system of Agrobacterium for hairy root through researching on all
Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 has priority over Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority and Rule 23a, Note 1 as applied to the corresponding specific epithets. Opinion 94. Judicial Commission of the International Committee on Systematics of Prokaryotes.
Tindall, B J
The Judicial Commission affirms that, according to the Rules of the International Code of Nomenclature of Bacteria (including changes made to the wording), the combination Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 has priority over the combination Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority as applied to the corresponding specific epithets. The type species of the genus is Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942, even if treated as a later heterotypic synonym of Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942. Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 is typified by the strain defined on the Approved Lists of Bacterial Names and by strains known to be derived from the nomenclatural type. PMID:25288664
6-Hydroxy-3-Succinoylpyridine Hydroxylase Catalyzes a Central Step of Nicotine Degradation in Agrobacterium tumefaciens S33
Huang, Haiyan; Wang, Shuning
Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8±1.85 µmol min?1 mg protein?1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53±0.03 mM 2,5-DHP was produced from 0.76±0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP. PMID:25054198
6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.
Li, Huili; Xie, Kebo; Huang, Haiyan; Wang, Shuning
Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8 ± 1.85 µmol min-1 mg protein-1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53 ± 0.03 mM 2,5-DHP was produced from 0.76 ± 0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP. PMID:25054198
Factors affecting Agrobacterium tumefaciens -mediated genetic transformation of Lycium barbarum L
Zhong Hu; Yi-Rui Wu; Wei Li; Huan-Huan Gao
Summary Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation,\\u000a including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included
Transformation of passionfruit ( Passiflora edulis fv flavicarpa Degener.) using Agrobacterium tumefaciens
G. Manders; W. C. Otoni; F. B. d'Utra Vaz; N. W. Blackball; J. B. Power; M. R. Davey
Leaf and stem explants of passionfruit (Passiflora eadulis fv flavicarpa) were co-cultivated with a disarmed strain of Agrobacterium tunefaciens harbouring the co-integrate vector pMON200. Four plants of passionfruit were regenerated from leaf explants on agar-solidified Murashige and Skoog (1962) based medium containing 4.43 M 6-benzyl-aminopurine and supplemented with 86 M kanamycin sulphate. The four plants were rooted by transfer to
Agrobacterium tumefaciens-mediated transformation in the entomopathogenic fungus Lecanicillium lecanii and development of benzimidazole fungicide resistant strains.
Zhang, Yan-Jun; Zhao, Jin-Jin; Xie, Ming; Peng, De-Liang
Lecanicillium lecanii has been used in the biological control of several insects in agricultural practice. Since the gene manipulation tools for this entomopathogenic fungus have not been sufficiently developed, Agrobacterium tumefaciens-mediated transformation (ATMT) in L. lecanii was investigated in this study, using the wild-type isolate FZ9906 as a progenitor strain and the hygromycin B resistance (hph) gene as a selection marker. Furthermore, a field carbendazim-resistant (mrt) gene from Botrytis cinerea was expressed in L. lecanii FZ9906 via the ATMT system. The results revealed that the frequency of transformation surpassed 25transformants/10(6) conidia, most of the putative transformants contained a single copy of T-DNA, and the T-DNA inserts were stably inherited after five generations. All putative transformants had indistinguishable biological characteristics relative to the wild-type strain, excepting two transformants with altered growth habits or virulence. Moreover, the resistance of the putative transformants to carbendazim (MBC) was improved, and the highest one was 380-fold higher than the wild-type strain. In conclusion, ATMT is an effective and suitable system for L. lecanii transformation, and will be a useful tool for the basic and application research of gene functions and gene modifications of this strain. PMID:25107375
[Cotransformation of aspen and birch with three T-DNA regions from two different replicons in one Agrobacterium tumefaciens strain].
Lebedev, V G; Shestibratov, K A; Shadrina, T E; Bulatova, I V; Abramochkin, D G; Miroshnikov, A I
The cointegration rate into the aspen and birch genomes of foreign genes from a binary vector and a disarmed Ti plasmid pCBE21 carried by the same Agrobacterium tumefaciens strain was studied. The cotransformation rate for the genes within the Ti plasmid varied from 30 to 100%; while the transformation rate for the gene from T(L) region was twofold higher as compared with the T(R) region. On the average, the gene transfer from all three T-DNAs was recorded in 10.9% of the transgenic lines. For the vector pBI121, the cotransformation rates for the genes from both regions of pCBE21 T-DNA were higher as compared with the vector pGS. In addition, a concurrent transfer of the genes from the Ti plasmid T(L) and T(R) regions was recorded only after the transformation with the vector pBI121. These results can be used for constructing woody plants containing several genes. PMID:21261057
Site-directed mutagenesis in Escherichia coli of a stable R772::Ti cointegrate plasmid from Agrobacterium tumefaciens.
Hille, J; van Kan, J; Klasen, I; Schilperoort, R
The host range of an octopine Ti plasmid is limited to Rhizobiaceae. This has been extended also to Escherichia coli in the form of a stable cointegrate with the wide-host-range plasmid R772. Its structure was studied by constructing a physical map of R772 and of the R772::pTiB6 cointegrate. An insertion sequence present in R772, called IS70, turned out to be involved in cointegrate formation. We found one intact copy of IS70 and a small segment of IS70, respectively, at the junctions of R772 and Ti DNA. The absence of a complete second copy of IS70 is a likely explanation for the stability of the cointegrate plasmid. A procedure for site-directed mutagenesis of this cointegrate plasmid in E. coli is described. The effect of mutations in the Ti plasmid part can be studied subsequently by transferring the cointegrate into Agrobacterium tumefaciens. The advantage of this procedure for Ti plasmids over other methods used at present is discussed. Images PMID:6302080
Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens.
Perry, K L; Kado, C I
Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA. PMID:7085561
Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens.
Perry, K L; Kado, C I
Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA. Images PMID:7085561
Cell-autonomous cytokinin-independent growth of tobacco cells transformed by Agrobacterium tumefaciens strains lacking the cytokinin biosynthesis gene.
Black, R C; Binns, A N; Chang, C F; Lynn, D G
Mutations at the cytokinin biosynthesis locus (tmr) of Agrobacterium tumefaciens usually result in strains that induce tumors exhibiting the rooty phenotype associated with high auxin-to-cytokinin ratios. However, tobacco (Nicotiana tabacum cv Havana 425) leaf disc explants responded to tmr- mutant strain A356 by producing rapidly growing, unorganized tumors, indicating that these lines can grow in a cytokinin-independent fashion despite the absence of a functional tmr gene. Several methods have been used to characterize the physiological and cellular basis of this phenotype. The results indicate that tmr- tumors have a physiologically distinct mechanism for cytokinin-independent growth in comparison to tumors induced by wild-type bacteria. The cytokinin-independent phenotype of the tmr- transformants appears to be cell autonomous in nature: only the transformed cells and their progeny were capable of cytokinin-independent growth. Specifically, the tmr- tumors did not accumulate cytokinin, and clonal analysis indicated the tmr- transformed cells were not capable of stimulating the growth of neighboring nontransformed cells. Finally, the cytokinin-independent phenotype of the tmr- transformants was shown to be cold sensitive, whereas the wild-type tumors exhibited a cold-resistant cytokinin-independent phenotype. Potential mechanisms for this novel form of cytokinin-independent growth, including the role of the dehydrodiconiferyl alcohol glucosides found in both tumor types, are discussed. PMID:8058843
Protein encoded by oncogene 6b from Agrobacterium tumefaciens has a reprogramming potential and histone chaperone-like activity
Ishibashi, Nanako; Kitakura, Saeko; Terakura, Shinji; Machida, Chiyoko; Machida, Yasunori
Crown gall tumors are formed mainly by actions of a group of genes in the T-DNA that is transferred from Agrobacterium tumefaciens and integrated into the nuclear DNA of host plants. These genes encode enzymes for biosynthesis of auxin and cytokinin in plant cells. Gene 6b in the T-DNA affects tumor morphology and this gene alone is able to induce small tumors on certain plant species. In addition, unorganized calli are induced from leaf disks of tobacco that are incubated on phytohormone-free media; shooty teratomas, and morphologically abnormal plants, which might be due to enhanced competence of cell division and meristematic states, are regenerated from the calli. Thus, the 6b gene appears to stimulate a reprogramming process in plants. To uncover mechanisms behind this process, various approaches including the yeast-two-hybrid system have been exploited and histone H3 was identified as one of the proteins that interact with 6b. It has been also demonstrated that 6b acts as a histone H3 chaperon in vitro and affects the expression of various genes related to cell division competence and the maintenance of meristematic states. We discuss current views on a role of 6b protein in tumorigenesis and reprogramming in plants. PMID:25389429
The Brucella suis Homologue of the Agrobacterium tumefaciens Chromosomal Virulence Operon chvE Is Essential for Sugar Utilization but Not for Survival in Macrophages
Alvarez-Martinez, Maria-Teresa; Machold, Jan; Weise, Christoph; Schmidt-Eisenlohr, Heike; Baron, Christian; Rouot, Bruno
Brucella strains possess an operon encoding type IV secretion machinery very similar to that coded by the Agrobacterium tumefaciens virB operon. Here we describe cloning of the Brucella suis homologue of the chvE-gguA-gguB operon of A. tumefaciens and characterize the sugar binding protein ChvE (78% identity), which in A. tumefaciens is involved in virulence gene expression. B. suis chvE is upstream of the putative sugar transporter-encoding genes gguA and gguB, also present in A. tumefaciens, but not adjacent to that of a LysR-type transcription regulator. Although results of Southern hybridization experiments suggested that the gene is present in all Brucella strains, the ChvE protein was detected only in B. suis and Brucella canis with A. tumefaciens ChvE-specific antisera, suggesting that chvE genes are differently expressed in different Brucella species. Analysis of cell growth of B. suis and of its chvE or gguA mutants in different media revealed that ChvE exhibited a sugar specificity similar to that of its A. tumefaciens homologue and that both ChvE and GguA were necessary for utilization of these sugars. Murine or human macrophage infections with B. suis chvE and gguA mutants resulted in multiplication similar to that of the wild-type strain, suggesting that virB expression was unaffected. These data indicate that the ChvE and GguA homologous proteins of B. suis are essential for the utilization of certain sugars but are not necessary for survival and replication inside macrophages. PMID:11514518
Ti plasmid-specified chemotaxis of Agrobacterium tumefaciens C58C1 toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants.
Ashby, A M; Watson, M D; Loake, G J; Shaw, C H
Twelve phenolic compounds with related structures were analyzed for their ability to act as chemoattractants for Agrobacterium tumefaciens C58C1 and as inducers of the Ti plasmid virulence operons. The results divided the phenolic compounds into three groups: compounds that act as strong vir inducers and are chemoattractants for A. tumefaciens C58C1 harboring the nopaline Ti plasmid pDUB1003 delta 31, but not the isogenic cured strain; compounds that are at best weak vir inducers and are weak chemoattractants for Ti plasmid-harboring and cured A. tumefaciens C58C1; and compounds that are vir noninducers and are also nonattractants. A strong correlation between vir-inducing ability and Ti plasmid requirement for chemotaxis is thus established. In addition, chemical structure rules for vir induction and chemotaxis are outlined. Positive chemotaxis toward root and shoot homogenates from monocotyledonous and dicotyledonous plants was observed. At low extract concentrations, chemotaxis was enhanced by the presence of Ti plasmid. The chemoattractants do not derive from intact cell walls. Lack of attraction is not responsible for the apparent block to monocot transformation by A. tumefaciens. PMID:3410827
Agrobacterium radiobacter strains K84, K1026 and K84 Agr produce an antibiotic?like substance, active in vitro against A. tumefaciens and phytopathogenic Erwinia and Pseudomonas spp
R. Peńalver; B. Vicedo; C. I. Salcedo; M. M. López
Agrobacterium radiobacter strains K84, K1026 and K84 Agr produced in vitro an antibiotic?like substance (ALS 84), different from agrocin 84 and observed in mannitol?glutamate medium. Twenty five out of 39 A. tumefaciens strains of biovars 1, 2 and 3 were sensitive to ALS 84 regardless of their sensitivity to agrocin 84. Sensitivity in A. tumefaciens strain C58 was not encoded
Stimulation of Agrobacterium tumefaciens T-DNA transfer by overdrive depends on a flanking sequence but not on helical position with respect to the border repeat.
Shurvinton, C E; Ream, W
T-DNA transfer by Agrobacterium tumefaciens depends on the right border repeat of the T-DNA and is greatly stimulated by overdrive, an adjacent sequence. We report that the function of overdrive does not depend on helical position with respect to the border repeat. A synthetic 24-bp overdrive and a 12-bp region containing a fully conserved 8-bp core overdrive sequence stimulated virulence equally, but full function required additional bases to the left of the 24-bp sequence. Images PMID:1885533
Genome sequence and mutational analysis of plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a zinc-lead mine tailing.
Hao, Xiuli; Xie, Pin; Johnstone, Laurel; Miller, Susan J; Rensing, Christopher; Wei, Gehong
The plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286, isolated from the nodules of Robinia pseudoacacia growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of Robinia plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of A. tumefaciens CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies, R. pseudoacacia inoculated with A. tumefaciens CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among A. tumefaciens CCNWGS0286, A. tumefaciens C58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil. PMID:22636006
Genome Sequence and Mutational Analysis of Plant-Growth-Promoting Bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a Zinc-Lead Mine Tailing
Hao, Xiuli; Xie, Pin; Johnstone, Laurel; Miller, Susan J.
The plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286, isolated from the nodules of Robinia pseudoacacia growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of Robinia plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of A. tumefaciens CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies, R. pseudoacacia inoculated with A. tumefaciens CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among A. tumefaciens CCNWGS0286, A. tumefaciens C58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil. PMID:22636006
Genetic transformation of Begonia tuberhybrida by Ri rol genes
Shigeto Kiyokawa; Yasuhiro Kikuchi; Hiroshi Kamada; Hiroshi Harada
We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar
A Genome-Wide Survey of Highly Expressed Non-Coding RNAs and Biological Validation of Selected Candidates in Agrobacterium tumefaciens
Lee, Keunsub; Huang, Xiaoqiu; Yang, Chichun; Lee, Danny; Ho, Vincent; Nobuta, Kan; Fan, Jian-Bing; Wang, Kan
Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs) have been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-Kingdom gene transfer. Using whole transcriptome sequencing and an ncRNA search algorithm developed for this work, we identified 475 highly expressed candidate ncRNAs from A. tumefaciens C58, including 101 trans-encoded small RNAs (sRNAs), 354 antisense RNAs (asRNAs), 20 5? untranslated region (UTR) leaders including a RNA thermosensor and 6 riboswitches. Moreover, transcription start site (TSS) mapping analysis revealed that about 51% of the mapped mRNAs have 5? UTRs longer than 60 nt, suggesting that numerous cis-acting regulatory elements might be encoded in the A. tumefaciens genome. Eighteen asRNAs were found on the complementary strands of virA, virB, virC, virD, and virE operons. Fifteen ncRNAs were induced and 7 were suppressed by the Agrobacterium virulence (vir) gene inducer acetosyringone (AS), a phenolic compound secreted by the plants. Interestingly, fourteen of the AS-induced ncRNAs have putative vir box sequences in the upstream regions. We experimentally validated expression of 36 ncRNAs using Northern blot and Rapid Amplification of cDNA Ends analyses. We show functional relevance of two 5? UTR elements: a RNA thermonsensor (C1_109596F) that may regulate translation of the major cold shock protein cspA, and a thi-box riboswitch (C1_2541934R) that may transcriptionally regulate a thiamine biosynthesis operon, thiCOGG. Further studies on ncRNAs functions in this bacterium may provide insights and strategies that can be used to better manage pathogenic bacteria for plants and to improve Agrobacterum-mediated plant transformation. PMID:23950988
The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants
Resmi, Thulasi Raveendrannair; Hohn, Thomas; Hohn, Barbara; Veluthambi, Karuppannan
Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases. PMID:26008704
The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants.
Resmi, Thulasi Raveendrannair; Hohn, Thomas; Hohn, Barbara; Veluthambi, Karuppannan
Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases. PMID:26008704
A Signaling Pathway Involving the Diguanylate Cyclase CelR and the Response Regulator DivK Controls Cellulose Synthesis in Agrobacterium tumefaciens
Barnhart, D. Michael; Su, Shengchang
The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR, is required for synthesis of this polymer in A. tumefaciens. In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as the conserved aspartate residue of a CheY-like receiver domain, for stimulating cellulose production. Moreover, a site-directed mutation within the PilZ domain of CelA, the catalytic subunit of the cellulose synthase complex, greatly reduced cellulose production. In addition, deletion of divK, the first gene of the divK-celR operon, also reduced cellulose production. This requirement for divK was alleviated by expression of a constitutively active form of CelR, suggesting that DivK acts upstream of CelR activation. Based on bacterial two-hybrid assays, CelR homodimerizes but does not interact with DivK. The mutation in divK additionally affected cell morphology, and this effect was complementable by a wild-type copy of the gene, but not by the constitutively active allele of celR. These results support the hypothesis that CelR is a bona fide c-di-GMP synthase and that the nucleotide signal produced by this enzyme activates CelA via the PilZ domain. Our studies also suggest that the DivK/CelR signaling pathway in Agrobacterium regulates cellulose production independent of cell cycle checkpoint systems that are controlled by divK. PMID:24443526
Characterization of a novel Agrobacterium tumefaciens Galactarolactone Cycloisomerase Enzyme for Direct Conversion of d-Galactarolactone to 3-Deoxy-2-keto-l-threo-hexarate*
Andberg, Martina; Maaheimo, Hannu; Boer, Harry; Penttilä, Merja; Koivula, Anu; Richard, Peter
Microorganisms use different pathways for d-galacturonate catabolism. In the known microbial oxidative pathway, d-galacturonate is oxidized to d-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and ?-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes d-galacturonic acid to d-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the d-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on d-galactarolactone and d-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from d-galactaro-1,4-lactone and d-glucaro-1,4-lactone conversion is in both cases the l-threo form of 3-deoxy-2-keto-hexarate. PMID:22493433
Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. II. Effect of the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis.
Storti, E; Bogani, P; Bettini, P; Bittini, P; Guardiola, M L; Pellegrini, M G; Inzé, D; Buiatti, M
We have studied the effect of a change in the endogenous hormone equilibria on the competence of tomato (Lycopersicon esculentum) cells to defend themselves against the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Calluses from cvs 'Davis' and 'Red River', respectively resistant and susceptible to Fusarium and transgenic for an auxin- or cytokinin-synthesizing gene from Agrobacterium tumefaciens, were used. The integration of Agrobacterium hormone-related genes into susceptible cv 'Red River' can bring the activation of defense processes to a stable competence as assessed by the inhibition of mycelial growth in dual culture and gem-tube elongation of Fusarium conidia, the determination of callose contents, peroxidase induction and ion leakage in the presence of fusaric acid. This is particularly true when the transformation results in a change of phytohormone equilibria towards an higher cytokin in concentration. On the contrary, in resistant cv 'Davis' the inhibition of both fungal growth in dual culture and conidia germination is higher when the hormone balance is modified in favour of the auxins. No significant effect was observed for ion leakage and peroxidase induction, probably because of a constitutive overproduction of cytokinins in 'Davis' cells. PMID:24185887
Cloning and analysis of Agrobacterium tumefaciens C58 loci involved in glutamine biosynthesis: Neither the glnA (GSI) nor the glnII (GSII) gene plays a special role in virulence
S. Rossbach; J. Schell; F. J. Bruijn
Using heterologous complementation of a glutamine synthetase deficient (glnA; GS-) Escherichia coli mutant strain and heterologous DNA hybridization probes from Rhizobium meliloti and Bradyrhizobium japonicum, three distinct Agrobacterium tumefaciens loci involved in glutamine biosynthesis were identified. These loci correspond to the glnA (GSI), glnII (GSII) and a third previously unidentified locus, which is capable of complementing an E. coli glnA
Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus
S. Krastanova; M. Perrin; P. Barbier; G. Demangeat; P. Cornuet; N. Bardonnet; L. Otten; L. Pinck; B. Walter
Summary Control of fanleaf disease induced by the Grapevine Fanleaf Nepovirus (GFLV) today is based on sanitary selection and soil disinfection with nematicides. This way of control is not always efficient and nematicides can be dangerous pollutants. Coat protein (CP) mediated protection could be an attractive alternative. We have transferred a chimeric CP gene of GFLV-F13 via Agrobacterium tumefaciens LBA4404 into
Transgenic indica rice cv. ADT 43 expressing a ? 1 -pyrroline-5-carboxylate synthetase ( P5CS ) gene from Vigna aconitifolia demonstrates salt tolerance
Alagarsamy Karthikeyan; Shumugiah Karutha Pandian; Manikandan Ramesh
To develop salt tolerant rice, the P5CS gene of Vigna aconitifolia, encoding for proline synthesis, was introduced into the popular indica rice cultivar ADT 43. Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1301\\/P5CS, carrying the proline synthesis encoding gene P5CS, was co-cultivated with embryogenic callus of rice. Adding 100 ?M acetosyringone to the Linsmaier and Skoog (LS) liquid
Production of pineapple transgenic plants assisted by temporary immersion bioreactors
P. Espinosa; J. Lorenzo; A. Iglesias; L. Yabor; E. Menéndez; J. Borroto; L. Hernández; A. Arencibia
A procedure for producing pineapple [Ananas comosus (L.) Merr.] transgenic plants was developed that involved selection by micropropagation in temporary immersion bioreactors (TIBs). Pineapple calluses ranging in size from 1.5 mm to 2.0 mm that were co-cultivated with Agrobacterium tumefaciens strains AT2260 (pIG121Hm) and LBA4404 (pTOK233) for 24 h produced the highest percentage (40%) of GUS+ calluses. Phosphinothricin and hygromycin,
Modification of plant architecture in Limonium spp. induced by rol genes
Antonio Mercuri; Simona Bruna; Laura De Benedetti; Gianluca Burchi; Tito Schiva
An Agrobacterium-mediated transformation system for Limonium has been developed. The leaf explants of the sterile hybrid L116 (Limonium otolepis, Kuntze × Limonium latifolium, Kuntze) were inoculated with A. tumefaciens LBA4404 harboring the binary vector pBin19 containing a T-DNA fragment encompassing rol A,B and C genes of A. rhizogenes Ri plasmid (pRi1855). Transgenic shoots, regenerated on selection medium, were micropropagated, rooted,
Overexpression of rice TLPD34 enhances dollar-spot resistance in transgenic bentgrass
Daolin Fu; Ned A. Tisserat; Yanmei Xiao; Derek Settle; Subbaratnam Muthukrishnan; George H. Liang
Creeping bentgrass (Agrostis palustris Huds.) is an important cool-season turfgrass and has been extensively used in golf course putting greens. To improve host resistance to fungal diseases, the rice thaumatin-like protein (TLPD34) gene was introduced into creeping bentgrass cv. 'Crenshaw' by using Agrobacterium-mediated transformation. Plant transformation was performed on mature seed-derived embryogenic calli by using A. tumefaciens strain LBA4404 in
Genetic transformation of flax ( Linum usitatissimum ) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase
Nazir Basiran; Philip Armitage; Roderick John Scott; John Draper
Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Kmr callus developing on hypocotyl sections. The totipotency of the Kmr callus was dependent upon its origin. T-DNA was visualised
In vivo analysis of DNA binding and ligand interaction of BlcR, an IclR-type repressor from Agrobacterium tumefaciens
Pan, Yi; Wang, Yi; Fuqua, Clay
Agrobacterium tumefaciens BlcR represses transcription of the blcABC operon, which is involved in metabolism of ?-butyrolactone, and this repression is alleviated by succinate semialdehyde (SSA). BlcR exists as a homodimer, and the blcABC promoter DNA contains two BlcR-binding sites (IR1 and IR2) that correspond to two BlcR dimers. In this study, we established an in vivo system to examine the SSA-responsive control of BlcR transcriptional regulation. The endogenous blcR, encoded in the pAtC58 plasmid of A. tumefaciens C58, was not optimal for investigating the effect of SSA on BlcR repression, probably due to the SSA degradation mediated by the pAt-encoded blcABC. We therefore introduced blcR (and the blcABC promoter DNA, separately) exogenously into a strain of C58 cured of pAtC58 (and pTiC58). We applied this system to interrogate BlcR–DNA interactions and to test predictions from our prior structural and biochemical studies. This in vivo analysis confirmed the previously mapped SSA-binding site and supported a model by which DNA coordinates formation of a BlcR tetramer. In addition, we identified a specific lysine residue (K59) as an important determinant for DNA binding. Moreover, based on isothermal titration calorimetry analysis, we found IR1 to play the dominant role in binding to BlcR, relative to IR2. Together, these in vivo results expand the biochemical findings and provide new mechanistic insights into BlcR–DNA interactions. PMID:23449918
A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens
Feirer, Nathan; Xu, Jing; Allen, Kylie D.; Koestler, Benjamin J.; Bruger, Eric L.; Waters, Christopher M.; White, Robert H.
ABSTRACT The motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogen Agrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP in A. tumefaciens are controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins. PMID:26126849
Biodegradation of All Stereoisomers of the EDTA Substitute Iminodisuccinate by Agrobacterium tumefaciens BY6 Requires an Epimerase and a Stereoselective C-N Lyase
Cokesa, Z?eljko; Knackmuss, Hans-Joachim; Rieger, Paul-Gerhard
Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe2+-, Mg2+-, and Ca2+-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn2+-IDS were rather scant and very slow on Cu2+-IDS. Growth and turnover experiments with A. tumefaciens BY6 indicated that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of R,S-IDS, generating d-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all stereoisomers into a catabolic pathway initiated by a stereoselective lyase. PMID:15240267
Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection.
Pantazis, Christopher J; Fisk, Sarah; Mills, Kerri; Flinn, Barry S; Shulaev, Vladimir; Veilleux, Richard E; Dan, Yinghui
KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying. PMID:23160638
Development of Efficient Plant Regeneration and Transformation System for Impatiens Using Agrobacterium tumefaciens and Multiple Bud Cultures as Explants
Background Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. Results In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. Conclusion We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation (with no need for sterilization or a greenhouse to grow stock plants), 2) flexibility (available all the time) for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using Agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of Agrobacterium-inoculated tissues. PMID:20696066
Delineation of the interaction domains of Agrobacterium tumefaciens VirB7 and VirB9 by use of the yeast two-hybrid assay.
Das, A; Anderson, L B; Xie, Y H
The Agrobacterium tumefaciens VirB proteins are postulated to form a transport pore for the transfer of T-DNA. Formation of the transport pore will involve interactions among the VirB proteins. A powerful genetic method to study protein-protein interaction is the yeast two-hybrid assay. To test whether this method can be used to study interactions among the VirB membrane proteins, we studied the interaction of VirB7 and VirB9 in yeast. We recently demonstrated that VirB7 and VirB9 form a protein complex linked by a disulfide bond between cysteine 24 of VirB7 and cysteine 262 of VirB9 (L. Anderson, A. Hertzel, and A. Das, Proc. Natl. Acad. Sci. USA 93:8889-8894, 1996). We now demonstrate that VirB7 and VirB9 interact in yeast, and this interaction does not require the cysteine residues essential for the disulfide linkage. By using defined segments in fusion constructions, we mapped the VirB7 interaction domain of VirB9 to residues 173 to 275. In tumor formation assays, both virB7C24S and virB9C262S expressed from a multicopy plasmid complemented the respective deletion mutation, indicating that the cysteine residues may not be essential for DNA transfer. PMID:9171381
The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA.
Hood, E E; Helmer, G L; Fraley, R T; Chilton, M D
We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid. Images PMID:3782037
Agrobacterium tumefaciens T-DNA gene 6b stimulates rol-induced root formation, permits growth at high auxin concentrations and increases root size.
Tinland, B; Rohfritsch, O; Michler, P; Otten, L
All Agrobacterium tumefaciens strains studied up to now transfer an active 6b gene to plant cells. However, the role of this gene in natural tumour induction is unknown. Various effects of 6b on plant cell growth have been described, but the precise mechanism by which 6b causes these effects has not been elucidated. Earlier experiments indicated that the 6b gene might increase auxin sensitivity as do the A. rhizogenes rol genes. The 6b gene from Tm4 (T-6b) was therefore compared with the rolB and rolABC genes. Although T-6b was unable to induce root formation, it strongly interfered with root induction and root elongation. In rolABC/T-6b coinfection experiments on carrots, T-6b-transformed cells stimulated root outgrowth of rolABC-transformed cells, indicating that the biologically active T-6b product is diffusible. Carrot rolABC roots containing the T-6b gene rapidly developed into unorganized calli. Nicotiana rustica roots with rolABC and T-6b continued their development, but became very large. Fragments of such roots formed callus at alpha-naphthaleneacetic acid concentrations which inhibited growth of rolABC and normal root fragments, suggesting that the role of 6b genes in natural tumour induction may be to reduce the inhibitory effects of high auxin levels and to keep cells in an undifferentiated state. PMID:2259331
Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens.
Krasutsky, Sergiy G; Urbansky, Marek; Davis, Chad E; Lherbet, Christian; Coates, Robert M; Poulter, C Dale
The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes. PMID:25184438
Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile.
Khan, Shamshad Ahmad; Ur Rahman, Laiq; Shanker, Karuna; Singh, Manju
Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS?+?1 mg/l BAP?+?1 mg/l NAA, while indirect regeneration from callus was obtained on MS?+?1 mg/l BAP?+?2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling. PMID:24154495
Genetic analysis of the agrocinopine catabolic region of Agrobacterium tumefaciens Ti plasmid pTiC58, which encodes genes required for opine and agrocin 84 transport.
Hayman, G T; Beck von Bodman, S; Kim, H; Jiang, P; Farrand, S K
The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacterium tumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy. acc is approximately 6 kb in size. It consists of at least five genes, accA through accE, as defined by complementation analysis using subcloned fragments and transposon insertion mutations of acc carried on different plasmids within the same cell. All five regions are required for agrocin 84 sensitivity, and at least four are required for agrocinopine and agrocin 84 uptake. The complementation results are consistent with the hypothesis that each of the five regions is separately transcribed. Maxicell experiments showed that the first of these genes, accA, encodes a 60-kDa protein. Analysis of osmotic shock fractions showed this protein to be located in the periplasm. The DNA sequence of the accA region revealed an open reading frame encoding a predicted polypeptide of 59,147 Da. The amino acid sequence encoded by this open reading frame is similar to the periplasmic binding proteins OppA and DppA of Escherichia coli and Salmonella typhimurium and OppA of Bacillus subtilis. Images PMID:8366042
The thuEFGKAB Operon of Rhizobia and Agrobacterium tumefaciens Codes for Transport of Trehalose, Maltitol, and Isomers of Sucrose and Their Assimilation through the Formation of Their 3-Keto Derivatives
Ampomah, Osei Yaw; Avetisyan, Anna; Hansen, Espen; Svenson, Johan; Huser, Thomas; Bhuvaneswari, T. V.
The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide-forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism. PMID:23772075
The thuEFGKAB operon of rhizobia and agrobacterium tumefaciens codes for transport of trehalose, maltitol, and isomers of sucrose and their assimilation through the formation of their 3-keto derivatives.
Ampomah, Osei Yaw; Avetisyan, Anna; Hansen, Espen; Svenson, Johan; Huser, Thomas; Jensen, John Beck; Bhuvaneswari, T V
The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide-forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism. PMID:23772075
In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation in asakura-sanshoo (Zanthoxylum piperitum (L.) DC. F. inerme Makino) an important medicinal plant
Zeng, Xiaofang; Zhao, Degang
Context: Asakura-sanshoo (Zanthoxylum piperitum [L.] DC. f. inerme Makino) is an important medicinal plant in East Asia. Transgenic technique could be applied to improve plant traits and analyze gene function. However, there is no report on regeneration and genetic transformation in Asakura-sanshoo. Aims: To establish a regeneration and Agrobacterium tumefaciens-mediated genetic transformation system in Asakura-sanshoo, which could be used for cultivar improvement and gene function analysis. Settings and Design: The various combinations of indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and naphthalene acetic acid (NAA) were explored for the optimal plant regeneration from petiole and stem of Asakura-sanshoo. The half-strength woody plant medium (WPM) with different concentrations of NAA and IBA was used to induce root. For genetic transformation, A. tumefaciens strain EHA-105 harboring the plasmid pBin-Ex-H-ipt which carries the isopentenyl transferase (ipt) gene, ?-glucuronidase (GUS) gene and kanamycin resistance gene neomycin phosphotransferase II (NPTII) were used. The transformation efficiency was detected by the kanamycin resistant frequency. Materials and Methods: Petioles and stems were obtained from the in vitro cultured Asakura-sanshoo. The petiole and stem segments were precultured for 3 days, and then inflected using the bacterium at the concentration of OD600 0.5–0.8 for 10 min, followed by 3 days co-cultivation. Selection of the transgenic plants was carried out after 7 days the regeneration using gradient kanamycin at 30 mg/L and 50 mg/L, respectively. Successful transformed plants were confirmed by GUS histochemical assays, polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), and Southern blotting analysis. Results: The highest shoots regeneration was obtained on WPM supplement with 0.5 mg/L BA and 0.2 mg/L NAA. The optimal rooting medium was half strength macro-element WPM. The kanamycin resistant frequency of petiole and stem was 24.66% and 25.93%, respectively. Thirty-five shoots in thousands adventitious buds were confirmed through GUS histochemical assays, PCR, RT-PCR, and Southern blotting. The regeneration shoot per explants elevated 5.85 fold compared with the wild-type plants. Conclusions: Individual transgenic Asakura-sanshoo lines were obtained. In this paper, it first revealed the expression of ipt gene significantly promoted the adventitious buds induction in Asakura-sanshoo as the same action as in other plants. PMID:25829778
Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system
Background The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. Results The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ?FaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A. Conclusion The new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this. PMID:25048842
Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker.
Fang, Weiguo; Zhang, Yongjun; Yang, Xingyong; Zheng, Xuelian; Duan, Hui; Li, Yi; Pei, Yan
Beauveria bassiana has been investigated for use in the biological control of several insects in agricultural practice. To understand the molecular basis of virulence and host specificity and to improve the entomopathogenicity of B. bassiana, we have developed a simple, highly efficient and reliable Agrobacterium-mediated transformation method for B. bassiana using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. Most transformants contained single copies of T-DNA and the T-DNA inserts were stably inherited after five generations. With this highly efficient transformation method for B. bassiana, we also obtained two putative T-DNA-tagged mutants that may have altered growth habits or virulence. Thus, the described protocol could provide a useful tool to manipulate the genetic make-up and to tag genes that may be important for virulence or growth and development of B. bassiana. PMID:14992856
Agrobacterium tumefaciens VirB9, an Outer-Membrane-Associated Component of a Type IV Secretion System, Regulates Substrate Selection and T-Pilus Biogenesis
Jakubowski, Simon J.; Cascales, Eric; Krishnamoorthy, Vidhya; Christie, Peter J.
Agrobacterium tumefaciens translocates DNA and protein substrates between cells via a type IV secretion system (T4SS) whose channel subunits include the VirD4 coupling protein, VirB11 ATPase, VirB6, VirB8, VirB2, and VirB9. In this study, we used linker insertion mutagenesis to characterize the contribution of the outer-membrane-associated VirB9 to assembly and function of the VirB/D4 T4SS. Twenty-five dipeptide insertion mutations were classified as permissive for intercellular substrate transfer (Tra+), completely transfer defective (Tra?), or substrate discriminating, e.g., selectively permissive for transfer only of the oncogenic transfer DNA and the VirE2 protein substrates or of a mobilizable IncQ plasmid substrate. Mutations inhibiting transfer of DNA substrates did not affect formation of close contacts of the substrate with inner membrane channel subunits but blocked formation of contacts with the VirB2 and VirB9 channel subunits, which is indicative of a defect in assembly or function of the distal portion of the secretion channel. Several mutations in the N- and C-terminal regions disrupted VirB9 complex formation with the outer-membrane-associated lipoprotein VirB7 or the inner membrane energy sensor VirB10. Several VirB9.i2-producing Tra+ strains failed to elaborate T pilus at detectable levels (Pil?), and three such Tra+ Pil? mutant strains were rendered Tra? upon deletion of virB2, indicating that the cellular form of pilin protein is essential for substrate translocation. Our findings, together with computer-based analyses, support a model in which distinct domains of VirB9 contribute to substrate selection and translocation, establishment of channel subunit contacts, and T-pilus biogenesis. PMID:15866936
Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes.
Hong, Seung-Beom; Peebles, Christie A M; Shanks, Jacqueline V; San, Ka-Yiu; Gibson, Susan I
We have established Catharanthus roseus hairy root cultures transgenic for the rol ABC genes from T(L)-DNA of the agropine-type Agrobacterium rhizogenes strain A4. The rol ABC hairy root lines exhibit a wild-type hairy root syndrome in terms of growth and morphology on solid medium. However, they differ from wild-type hairy root lines in that they more frequently have excellent adaptability to liquid medium and do not appear to form calli during cultivation. Moreover, they do not produce detectable levels of mannopine and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root lines. The absence of these opines does not appear to cause the rol ABC lines to have higher levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines, rol ABC lines produce very similar levels of total alkaloids despite wide variations in individual alkaloid contents. This work demonstrates that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharanthus roseus. PMID:16261632
Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds.
Manickavasagam, M; Ganapathi, A; Anbazhagan, V R; Sudhakar, B; Selvaraj, N; Vasudevan, A; Kasthurirengan, S
Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing beta-glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l(-1)) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l(-1) 6-benzyladenine (BA) and 5.0 mg l(-1) PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l(-1) BA, 1.0 mg l(-1) kinetin (Kin), 0.5 mg l(-1) alpha-napthaleneacetic acid (NAA) and 5.0 mg l(-1) PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l(-1) NAA and 5.0 mg l(-1) PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical beta-glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 micro M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50-60% of the transgenic plants sprayed with BASTA (60 g l(-1) glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%. PMID:15133712
Centrifugation Assisted Agrobacterium tumefaciens- mediated Transformation (CAAT) of embryogenic cell suspensions of banana ( Musa spp. Cavendish AAA and Lady finger AAB)
Harjeet Khanna; Doug Becker; Jennifer Kleidon; James Dale
Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants\\/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two
Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana.
Shamloul, Moneim; Trusa, Jason; Mett, Vadim; Yusibov, Vidadi
Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin). PMID:24796351
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Shamloul, Moneim; Trusa, Jason; Mett, Vadim; Yusibov, Vidadi
Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin). PMID:24796351
Agrobacterium and Tumor Induction: A Model System.
ERIC Educational Resources Information Center
Lennox, John E.
The author offers laboratory procedures for experiments using the bacterium, Agrobacterium tumefaciens, which causes crown gall disease in a large number of plants. Three different approaches to growing a culture are given. (SA)
Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic ?(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants
Ińón de Iannino, Nora; Briones, Gabriel; Tolmasky, Marcelo; Ugalde, Rodolfo A.
The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic ?(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic ?(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic ?(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic ?(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic ?(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic ?(1-2) glucan may be a virulence factor in Brucella infection. PMID:9721274
Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble).
Khurana, Jigyasa; Chugh, Archana; Khurana, Paramjit
The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum. Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1). Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration. At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively). Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12%. Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009. Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations. Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1). PMID:13677634
Engineering cotton ( Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences
Jamil A. HashmiYusuf; Yusuf Zafar; Muhammad Arshad; Shahid Mansoor; Shaheen Asad
Several important biological processes are performed by distinct functional domains found on replication-associated protein\\u000a (Rep) encoded by AC1 of geminiviruses. Two truncated forms of replicase (tAC1) gene, capable of expressing only the N-terminal\\u000a 669 bp (5?AC1) and C-terminal 783 bp (3?AC1) nucleotides cloned under transcriptional control of the CaMV35S were introduced\\u000a into cotton (Gossypium hirsutum L.) using LBA4404 strain of Agrobacterium tumefaciens
Resistance of transgenic tobacco seedlings expressing the Agrobacterium tumefaciens C58-6b gene, to growth-inhibitory levels of cytokinin is associated with elevated IAA levels and activation of phenylpropanoid metabolism.
Gális, Ivan; Simek, Petr; Van Onckelen, Henri A; Kakiuchi, Yasutaka; Wabiko, Hiroetsu
We previously reported that the Agrobacterium tumefaciens C58-6b gene confers resistance to growth-inhibitory levels of exogenously applied N(6)-benzyladenine (BA, cytokinin) in transgenic tobacco (Nicotiana tabacum) seedlings. Here, we found that intracellular levels of indoleacetic acid (IAA, auxin) increased in transgenics but declined in wild-type seedlings upon BA treatment. Since exogenously supplied 1-naphthalene acetic acid (NAA), a stable synthetic auxin, counteracted the growth inhibition of wild-type seedlings by BA, we suggest that BA-induced growth inhibition in wild-type seedlings occurs, at least in part, as a result of intracellular IAA deficiency. Further HPLC analysis of cell extracts from BA-treated seedlings revealed that a fluorescent compound, later identified as the phenylpropanoid, scopolin, and the major phenolic compound, chlorogenic acid, accumulated earlier in transgenics than in wild-type seedlings. Gene transcripts encoding phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and 4-coumarate:CoA ligase, which are responsible for the early steps of phenylpropanoid biosynthesis, accumulated earlier and to higher levels in transgenics than in wild-type seedlings as determined by Northern hybridization analysis, thus accounting for the early accumulation of scopolin and chlorogenic acid in transgenics. As some phenolic compounds, including chlorogenic acid and scopoletin (aglycon of scopolin) are suggested to inhibit IAA catabolism, we further propose that C58-6b gene expression protects IAA from degradation by inducing the early phenylpropanoid pathway. PMID:12198197
High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium-mediated genetic transformation of tobacco.
Pathi, Krishna Mohan; Tula, Suresh; Tuteja, Narendra
A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87-96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis. PMID:23518589
Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14
Nyaboga, Evans N.; Njiru, Joshua M.; Tripathi, Leena
Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1–2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70–80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava. PMID:26113851
Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens-mediated plant transformation.
Boyko, Alex; Matsuoka, Aki; Kovalchuk, Igor
Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors. Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases the frequency of both homologous recombination and plant transgenesis. Here we tested the influence of KCl and salts of rare earth elements, Ce and La on the efficiency of Agrobacterium-mediated plant transformation. We found that exposure to KCl, CeCl(3) and LaCl(3) leads to an increase in recombination frequency in Arabidopsis and tobacco. Plants grown in the presence of CeCl(3) and LaCl(3) had higher biomass, longer roots and greater root number. Analysis of transformation efficiency showed that exposure of tobacco plants to 50 mM KCl resulted in ~6.0-fold increase in the number of regenerated calli and transgenic plants as compared to control plants. Exposure to various concentrations of CeCl(3) showed a maximum increase of ~3.0-fold in both the number of calli and transgenic plants. Segregation analysis showed that exposure to KCl and cerium (III) chloride leads to more frequent integrations of the transgene at a single locus. Analysis of transgene intactness showed better preservation of right T-DNA border during transgene integration. Our data suggest that KCl and CeCl(3) can be effectively used to improve quantity and quality of transgene integrations. PMID:21132499
Development of Agrobacterium-mediated transformation technology for mature seed-derived callus tissues of indica rice cultivar IR64.
Sahoo, Ranjan Kumar; Tuteja, Narendra
Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600=1.0) and co-cultured at 26±2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 ?m acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration. PMID:22538224
Agrobacterium tumefaciens Type IV Secretion Protein VirB3 Is an Inner Membrane Protein and Requires VirB4, VirB7, and VirB8 for Stabilization?
Mossey, Pamela; Hudacek, Andrew; Das, Anath
Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures. PMID:20348257
Genetic transformation of selected mature cork oak (Quercus suber L.) trees.
Alvarez, R; Alonso, P; Cortizo, M; Celestino, C; Hernández, I; Toribio, M; Ordás, R J
A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil. PMID:15185122
Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum.
Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein
Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand (TRAIL) as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV (Cauliflower Mosaic Virus) helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay (Methylthiazol Tetrazolium Assay). The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable. PMID:25561925
Barley Transformation Using Agrobacterium-Mediated Techniques
NASA Astrophysics Data System (ADS)
Harwood, Wendy A.; Bartlett, Joanne G.; Alves, Silvia C.; Perry, Matthew; Smedley, Mark A.; Leyland, Nicola; Snape, John W.
Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.
Agrobacterium tumefaciens -Mediated Transformation of Ectomycorrhizal Fungi
Minna J. Kemppainen; Maria C. Alvarez Crespo; Alejandro G. Pardo
\\u000a One of the most significant discoveries revealed by the genomic analysis of different organisms is the high number of putative\\u000a genes without a predicted function. Therefore, an essential tool for the functional analysis of full-sequenced genomes is\\u000a the capacity to generate loss of function-mutants of every gene of the genome. The fungal kingdom is now entering into the\\u000a genomic era
Genetic transformation of Begonia tuberhybrida by Ri rol genes.
Kiyokawa, S; Kikuchi, Y; Kamada, H; Harada, H
We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar medium supplemented with 0.1 mg/l NAA, 0.5 mg/l BA, 500 mg/l claforan and 100 mg/l kanamycin. These shoots exhibited GUS activity and Southern analysis showed a single copy insertion into the genome. When the transgenic plants were transferred to soil, they displayed the phenotype specific to the transgenic plants by A. rhizogenes such as dwarfness, delay of flowering, and wrinkled leaves and petals. PMID:24178527
Isolation of Agrobacterium Ti-plasmid regulatory mutants
Annik Petit; Jacques Tempé
Crown gall tumors incited by Agrobacterium tumefaciens synthesize basic amino acid derivatives called opines. Opine production in tumours and opine catabolism by A. tumefaciens are coded by Ti-plasmids which confer oncogenicity on this bacterium. Catabolism of opines is inducible, and a method for isolation of regulatory mutants is described. From octopine-type bacteria, by plating on non-inducing substrates (noroctopine, noroctopine acid,
Transgenic plant production mediated by Agrobacterium in Indica rice
Hamid Rashid; Shuuji Yokoi; Kinya Toriyama; Kokichi Hinata
A reproducible system has been developed for the production of transgenic plants in indica rice using Agrobacterium-mediated gene transfer. Three-week-old scutella calli served as an excellent starting material. These were infected with an Agrobacterium tumefaciens strain EHA101 carrying a plasmid pIG121Hm containing genes for ß-glucuronidase (GUS) and hygromycin resistnace (HygR). Hygromycin (50 mg\\/l) was used as a selectable agent. Inclusion
Agrobacterium -mediated transformation of bottle gourd ( Lagenaria siceraria Standl.)
J.-S. Han; C. K. Kim; S. H. Park; K. D. Hirschi; I.-G. Mok
We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the ß-d-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6–8 days on co-cultivation medium supplemented
Appropriate choice of antibiotic and Agrobacterium strain improves transformation of antibiotic-sensitive Fragaria vesca and F. v. semperflorens
M. Alsheikh; H.-P. Suso; M. Robson; N. Battey; A. Wetten
By identifying antibiotics that eliminated Agrobacterium tumefaciens with the least phytotoxic effects, we were able to select appropriate A. tumefaciens strains for a more efficient transformation of seasonal Fragaria vesca and everbearing F. v. semperflorens. An efficient and reproducible method of shoot regeneration from leaf discs was developed with optimal shoot regeneration obtained on medium supplemented with 0.25 mg l-1
Susceptibility of Paulownia elongata to Agrobacterium and production of transgenic calli and hairy roots by in vitro inoculation
Ben A. Bergmann; Xiaohong Lin; Rebecca Whetten
Susceptibility of Paulownia elongata S.Y. Hu (princess tree) to Agrobacterium tumefaciens and A. rhizogenes was demonstrated\\u000a by inoculating in vitro shoots. Shoots had a gall formation frequency of ?83% when inoculated with any of three A. tumefaciens\\u000a strains (542, A281, or C58). Timing of gall appearance and type of callus proliferation differed among A. tumefaciens strains.\\u000a Rapidly proliferating callus was
Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus.
Krastanova, S; Perrin, M; Barbier, P; Demangeat, G; Cornuet, P; Bardonnet, N; Otten, L; Pinck, L; Walter, B
Control of fanleaf disease induced by the Grapevine Fanleaf Nepovirus (GFLV) today is based on sanitary selection and soil disinfection with nematicides. This way of control is not always efficient and nematicides can be dangerous pollutants. Coat protein (CP) mediated protection could be an attractive alternative. We have transferred a chimeric CP gene of GFLV-F13 via Agrobacterium tumefaciens LBA4404 into two rootstock varieties: Vitis rupestris and 110 Richter (V. rupestris X V. Berlandieri). Transformation was performed on embryogenic callus obtained from anthers and on hypocotyl fragments from mature embryos. Success of the transformation was assessed by polymerase chain reaction and Southern analyses. Transformants with a number of copies of the CP gene varying from one to five were obtained. Enzyme-linked immunosorbent assay with virus-specific antibodies revealed various levels of expression of the coat protein in the different transformants. PMID:24185595
Relationship between transcript production and virus resistance in transgenic tobacco expressing the potato leafroll virus coat protein gene.
Barker, H; Reavy, B; Webster, K D; Jolly, C A; Kumar, A; Mayo, M A
The coat protein (CP) gene of potato leafroll luteovims (PLRV) was inserted into tobacco (Nicotiana tabacum) using disarmed Agrobacterium tumefaciens (LBA4404) containing a binary expression vector. PLRV CP gene transcript was detected in transgenic plants but its abundance differed between transformed lines. CP was not detected in virus-free transgenic plants. The segregation of kanamycin resistance in S1 seedling progenies (obtained by selfing transformed plants) indicated that multiple (up to five) integration events involving vector T-DNA had occurred in most transformants. However, the amount of detectable CP transcript was not related to the neomycin phosphotransferase II gene copy number. Multiplication of PLRV in mature transgenic plants was diminished by up to 6-fold; the greatest diminution was in those transformed lines in which most CP gene transcript was detected. However, S1 progeny seedlings of transgenic plants were no more resistant to infection, following inoculation with viruliferous aphids, than seedlings of non-transformed control plants. PMID:24196184
ORIGINAL PAPER Agrobacterium-mediated genetic transformation and plant
an integral framework for the genetic improvement of Fraxinus profunda (pumpkin ash) for future development, an Agrobacterium tumefaciens-mediated genetic transfor- mation system was successfully developed for pumpkin ash modifications of F. profunda to provide resistance to EAB. Keywords Fraxinus Á Genetic transformation Á Pumpkin
Unit 3D.2 Genetic Manipulation of Agrobacterium
Morton, Elise R.; Fuqua, Clay
Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for genetic manipulation of A. tumefaciens. PMID:22549163
Agrobacterium-mediated transformation of round leaved sundew ( Drosera rotundifolia L.)
Merja Hirsikorpi; Terttu Kämäräinen; Teemu Teeri; Anja Hohtola
Agrobacterium tumefaciens-mediated genetic transformation method of the carnivorous medicinal plant round leaved sundew (Drosera rotundifolia L.) was developed. The micropropagation conditions of sundew aseptically germinated seeds were defined and the internal kanamycin resistance of sundew was tested. Transformation was made by cocultivation of micropropagated sundew leaves with A. tumefaciens strain C58C1 containing a cointegrate plasmid vector with neomycin phosphotransferase and
Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.
Song, Geun Cheol; Lee, Soohyun; Hong, Jaehwa; Choi, Hye Kyung; Hong, Gun Hyong; Bae, Dong-Won; Mysore, Kirankumar S; Park, Yong-Soon; Ryu, Choong-Min
Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium. PMID:25676198
Multiple transformation of plant cells by Agrobacterium may be responsible for the complex organization of T-DNA in crown gall and hairy root
Annik Petit; André Berkaloff; Jacques Tempé
Inoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the
Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium -mediated plant transformation
G. Vancanneyt; R. Schmidt; A. O'Connor-Sanchez; L. Willmitzer; M. Rocha-Sosa
Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to
Agrobacterium -mediated transformation of quaking aspen ( Populus tremuloides ) and regeneration of transgenic plants
Chung-Jui Tsai; Gopi K. Podila; Vincent L. Chiang
Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification
Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells
Mary-Dell Chilton; David A. Tepfer; Annik Petit; Chantal David; Francine Casse-Delbart; Jacques Tempé
Agrobacterium rhizogenes, which induces hairy root disease of dicotyledonous plants1, is closely related to Agrobacterium tumefaciens, the causative agent of crown gall disease1-3. Virulence in both species is conferred by large plasmids4-7. Infected plant tissue synthesizes novel metabolites, opines8-11, that are not found in normal plant tissues. The pattern of opines synthesized is determined by the type of virulence plasmid
Agrobacterium -mediated transformation using embryogenic calli in Satsuma mandarin ( Citrus unshiu Marc.) cv. Miyagawa wase
Seong Beom Jin; Jeong Won Park; Hyeon Jin Sun; Su Hyun Yun; Hyo Yeon Lee; Dong Sun Lee; Quan Chun Hong; Yong Woo Kim; Key Zung Riu; Jae Hoon Kim
Agrobacterium-mediated transformation in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase was achieved with reasonable transformation efficiency of about 22%, which was the percentage of\\u000a transgenic plantlets confirmed by genomic PCR (37 plantlets\\/168 hygromycin-resistant calli). Embryogenic calli of Miyagawa\\u000a wase were infected with Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA1300 that contained miraculin gene (a taste-modifying protein) and hygromycin
The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean
Zhanyuan Zhang; Aiqiu Xing; Paul Staswick; Thomas E. Clemente
The soybean transformation procedure using the Agrobacterium-cotyledonary node transformation system and the bar gene as the selectable marker coupled with glufosinate as a selective agent is described. Soybean cotyledonary explants were derived from 5 day old seedlings and co-cultivated with Agrobacterium tumefaciens for 3 days. Explants were cultured on Gamborg's B5 medium supplemented with 1.67 mg l-1 BAP and glufosinate
Shoot regeneration and Agrobacterium -mediated transformation of Fragaria vesca L
Iman Mansouri; José A. Mercado; Victoriano Valpuesta; José M. López-Aranda; Fernando Pliego-Alfaro; Miguel A. Quesada
An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg\\/l) and indole-3-butyric acid (0.25 mg\\/l) induced the maximum percentage of shoot regeneration (98%) and the highest
Transformation of Medicago by Agrobacterium mediated gene transfer
Maria Deak; Gyorgy B. Kiss; Csaba Koncz; Denes Dudits
Shoot segments of Medicago varia genotype A2 were co-cultivated with Agrobacterium tumefaciens strain bo42 carrying pGA471, a plasmid coding for the kanamycin resistant determinant as transferable positive selection marker in plant cells (An et al., 1985). Resistant plants were regenerated at high frequency from green calli developed on inoculated stem cuttings under kanamycin selection. DNA-DNA hybridization analysis showed the presence
Agrobacterium -mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration
Ningxia Du; Paula M. Pijut
A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and ?-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were\\u000a transformed in the presence of 100 ?M acetosyringone using 90 s sonication plus 10 min
Agrobacterium -mediated transformation of strawberry calli and recovery of transgenic plants
Narender S. Nehra; Ravindra N. Chibbar; Kutty K. Kartha; Raju S. S. Datla; William L. Crosby; Cecil Stushnoff
Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 µg\\/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS
Agrobacterium -mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana
Andreas Leclerque; Hong Wan; Anette Abschütz; Siwei Chen; Galina V. Mitina; Gisbert Zimmermann; HansUlrich Schairer
Agrobacterium tumefaciens was used to stably transform the entomopathogenic deuteromycete Beauveria bassiana to hygromycin B resistance by integration of the hph gene of Escherichia coli into the fungal genome. The transformation protocol was optimized to generate a library of insertion mutants of Beauveria. Transformation frequencies around 10 -4 and suppression of background growth were achieved. Over 90% of the AIM mutants
Agrobacterium T-DNA-mediated integration and gene replacement in the brown rot pathogen Monilinia fructicola
Miin-Huey Lee; Richard M. Bostock
A transformation system utilizing Agrobacterium tumefaciens was developed for targeted gene disruption in Monilinia fructicola, a fungal pathogen that causes brown rot disease of stone fruits. Transformation with a vector containing the neomycin phosphotransferase II (nptII) cassette flanked with 4 kb cutinase gene (Mfcut1) flanking sequences resulted in an average of 13 transformants per 105 spores. When assayed by PCR and
Nodulation of Sesbania Species by Rhizobium (Agrobacterium) Strain IRBG74 and Other Rhizobia
Technology Transfer Automated Retrieval System (TEKTRAN)
Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridisation with R. ...
The effects of Agrobacterium rhizogenes rolAB genes in lettuce
Ian S. Curtis; Caiping He; J. Brian Power; Domenico Mariotti; Ad de Laat; Michael R. Davey
Agrobacterium rhizogenes rolAB genes were transferred into lettuce (Lactuca sativa L.) cultivar 'Lake Nyah' using a supervirulent strain of A. tumefaciens. Southern hybridisation confirmed the presence of the rol genes in kanamycinresistant plants. In culture, transgenic plants exhibited extensive root development and an increased response to auxin. Phenotypic characterisation of transgenic populations indicated significant alterations in plant development, especially in
Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus
Technology Transfer Automated Retrieval System (TEKTRAN)
Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in stored bulbs. In order to study the pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B...
Efficient Agrobacterium-mediated Transformation of Prunus serotina via Sonication and Vacuum-Infiltration
-mediated transformation of an elite mature genotype (#3) of black cherry using an RNAi construct for the AGAMOUS (AG) gene. Agrobacterium tumefaciens strain EHA105 harbouring an RNAi construct for the AGAMOUS (AG) gene (silencing, prunasin hydrolase (PH) and mandelonitrile lyase (MDL), and develop constructs for future transformation
A case of promiscuity: Agrobacterium's endless hunt for new partners.
Lacroix, Benoît; Tzfira, Tzvi; Vainstein, Alexander; Citovsky, Vitaly
Agrobacterium tumefaciens is a phytopathogenic bacterium that induces the 'crown gall' disease in plants by transfer and integration of a segment of its tumor-inducing (Ti) plasmid DNA into the genome of numerous plant species that represent most of the higher plant families. Recently, it has been shown that, under laboratory conditions, the host range of Agrobacterium can be extended to non-plant eukaryotic organisms. These include yeast, filamentous fungi, cultivated mushrooms and human cultured cells. In this article, we present Agrobacterium-mediated transformation of non-plant organisms as a source of new protocols for genetic transformation, as a unique tool for genomic studies (insertional mutagenesis or targeted DNA integration) and as a useful model system to study bacterium-host cell interactions. Moreover, better knowledge of the DNA-transfer mechanisms from bacteria to eukaryotic organisms can also help in understanding horizontal gene transfer--a driving force throughout biological evolution. PMID:16289425
An efficient Agrobacterium-mediated transient transformation of Arabidopsis.
Tsuda, Kenichi; Qi, Yiping; Nguyen, Le V; Bethke, Gerit; Tsuda, Yayoi; Glazebrook, Jane; Katagiri, Fumiaki
Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a ?-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana. PMID:22004025
Universal PCR primers for detection of phytopathogenic Agrobacterium strains.
Haas, J H; Moore, L W; Ream, W; Manulis, S
Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants. PMID:7487020
Substrate induction of conjugative activity of Agrobacterium tumefaciens Ti plasmids
A. Petit; J. Tempe; A. KERR; M. HOLSTERS; M. VAN MONTAGU; J. SCHELL
CONJUGATIVE plasmids of bacteria are extrachromosomal genetic elements able to bring about DNA transfer by conjugation. The best-studied system is that of F-like plasmids in Escherichia coli, where at least 12 genes are involved1. Expression of the transfer system in this case is subject to negative control2,3. We report here that the newly discovered conjugative activity of Ti plasmids is
Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens
Ming Cheng; Joyce E. Fry; Shengzhi Pang; Huaping Zhou; Catherine M. Hironaka; David R. Duncan; Timothy W. Conner; Yuechun Wan
A rapid Agrobacferium fumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. lhe explants were inoculated with a disarmed A. tumefa- ciens strain C58 (ABI) harboring the binary vector pMON18365 containing the p-glucuronidase gene with an intron, and a select- able marker, the neomycin phosphotransferase II gene. Various factors
High-frequency transformation of Arabidopsis thaliana by Agrobacterium tumefaciens
Michael C. Clarke; Wenbin Wei; Keith Lindsey
Incorporation of 5 mg\\/L silver thiosulphate into media for seed germination and callus induction, as used in the transformation\\u000a protocol originally described by Valvekens et al. (1988), was found to increase the frequency of regeneration of transformants\\u000a ofArabidopsis thaliana ecotypes C24 and Landsbergerecta by at least 10- to 100-fold. Other factors, such as density of the bacterial inoculation culture, density
Transfer of citrus tristeza virus (CTV)-derived resistance candidate sequences to four grapefruit cultivars through Agrobacterium -mediated genetic transformation
G. Ananthakrishnan; V. Orbovi?; G. Pasquali; M. ?alovi?; J. W. Grosser
Transgenic plants of grapefruit (Citrus paradisi Macf.) cvs. 'Duncan', 'Flame', 'Marsh', and 'Ruby Red' were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Two citrus tristeza virus (CTV)-derived candidate resistance genes:\\u000a '392' (3? region of the p23 ORF plus 3? untranslated region—UTR) and 'p23 hairpin' (sense-p23 ORF plus UTR plus antisense-p23\\u000a ORF) were introduced into grapefruit using Agrobacterium strains
Mechanism of Phenolic Activation of Agrobacterium Virulence Genes: Development of a Specific Inhibitor of Bacterial Sensor\\/Response Systems
Kathleen M. Hess; Matthew W. Dudley; David G. Lynn; Rolf D. Joerger; Andrew N. Binns
The aglycone of the dihydrodiconiferyl alcohol glycosides, a series of phenolic growth factors able to substitute for some of the hormone requirements of tobacco cell division, are also potent inducers of virulence gene expression in Agrobacterium tumefaciens. However, these factors do not conform to the previously established structural requirements necessary for vir expression. Systematic evaluation of the structural requirements of
Agrobacterium and PEG-mediated transformation of the phytopathogen Venturia inaequalis.
Fitzgerald, Anna M; Mudge, Agnieszka M; Gleave, Andrew P; Plummer, Kim M
We report the development of two new transformation systems, polyethylene glycol (PEG)-mediated transformation of protoplasts and Agrobacterium tumefaciens-mediated transformation of mycelium, for the filamentous ascomycete Venturia inaequalis. New binary vectors have been created for the latter. Although transformation was initially achieved using a PEG-mediated method, this was superseded by the A. tumefaciens-mediated approach. The advantages of the latter include: ease of the protocol, no requirement for protoplasts; higher transformation efficiency; and single-site integration. A comparison between the two transformation methods is presented. PMID:12967207
A Novel Phenolic Compound, Chloroxynil, Improves Agrobacterium-Mediated Transient Transformation in Lotus japonicus
Kimura, Mitsuhiro; Cutler, Sean; Isobe, Sachiko
Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 ?M chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 ?M chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops. PMID:26176780
Attachment of Agrobacterium to plant surfaces
Matthysse, Ann G.
Agrobacterium tumefaciens binds to the surfaces of inanimate objects, plants, and fungi. These bacteria are excellent colonizers of root surfaces. In addition, they also bind to soil particles and to the surface of artificial or man-made substances, such as polyesters and plastics. The mechanisms of attachment to these different surfaces have not been completely elucidated. At least two types of binding have been described unipolarpolysaccharide-dependent polar attachment and unipolar polysaccharide-independent attachment (both polar and lateral). The genes encoding the enzymes for the production of the former are located on the circular chromosome, while the genes involved in the latter have not been identified. The expression of both of these types of attachment is regulated in response to environmental signals. However, the signals to which they respond differ so that the two types of attachment are not necessarily expressed coordinately. PMID:24926300
Nucleotide sequence of the T-DNA region from the A grobacterium tumefaciens octopine Ti plasmid pTi15955
R. F. Barker; K. B. Idler; D. V. Thompson; J. D. Kemp
The complete nucleotide sequence of the transferred region (T-DNA) of an octopine tumor inducing (Ti) plasmid fromAgrobacterium tumefaciens (pTi15955) has been determined. A total of 24 595 nucleotides extending approximately 900 bases to either side of the outermost, T-DNA boundaries was sequenced. Computer analysis of the sequenced portion of the Ti plasmid revealed that recognition sites for 72 restriction endonucleases
Agrobacterium-Mediated Transformation of Fusarium oxysporum: An Efficient Tool for Insertional Mutagenesis and Gene Transfer.
Mullins, E D; Chen, X; Romaine, P; Raina, R; Geiser, D M; Kang, S
ABSTRACT Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an efficient tool for insertional mutagenesis. In this paper, we report the construction of four novel binary vectors for fungal transformation and the optimization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fusarium oxysporum and other phytopathogenic fungi to be achieved. Employing the binary vectors, carrying the bacterial hygromycin B phosphotrans-ferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 300 to 500 hygromycin B resistant transformants per 1 x 10(6) conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaciens cells and significantly with the number of Agrobacteruium tumefaciens cells present during the cocultivation period (r = 0.996; n = 3; P < 0.01). All transformants tested remained mitotically stable, maintaining their hygromycin B resistance. Growing Agrobacterium tumefaciens cells in the presence of acetosyringone (AS) prior to cocultivation shortened the time required for the formation of transformants but decreased to 53% the percentage of transformants containing a single T-DNA insert per genome. This increased to over 80% when Agrobacterium tumefaciens cells grown in the absence of AS were used. There was no correlation between the average copy number of T-DNA per genome and the colony diameter of the transformants, the period of cocultivation or the quantity of Agrobacterium tumefaciens cells present during cocultivation. To isolate the host sequences flanking the inserted T-DNA, we employed a modified thermal asymmetric interlaced PCR (TAIL-PCR) technique. Utilizing just one arbitrary primer resulted in the successful amplification of desired products in 90% of those transformants analyzed. The insertion event appeared to be a random process with truncation of the inserted T-DNA, ranging from 1 to 14 bp in size, occurring on both the right and left border sequences. Considering the size and design of the vectors described here, coupled with the efficiency and flexibility of this ATMT protocol, it is suggested that ATMT should be regarded as a highly efficient alternative to other DNA transfer procedures in characterizing those genes important for the pathogenicity of F. oxysporum and potentially those of other fungal pathogens. PMID:18944391
Constitutive Expression of the Virulence Genes Improves the Efficiency of Plant Transformation by Agrobacterium
Genevieve Hansen; Anath Das; Mary-Dell Chilton
Inducible virulence (vir) genes of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid are under control of a two-component regulatory system. In response to environmental factors (phenolic compounds, sugars, pH) VirA protein phosphorylates VirG, which in turn interacts with the promoters of other vir genes, causing induction. A mutation of virG, virGN54D (which codes for a Asn-54 --> Asp amino acid change
Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium
Bart-Jan Janssen; Richard C. Gardner
A chimaeric gene has been constructed that expresses ß-D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3–4 days and then declined; if selection was imposed expression increased again
Factors influencing Agrobacterium-mediated transient expression of gusA in rice
Xiu-Qing Li; Chang-Nong Liu; Steven W. Ritchie; Jian-ying Peng; Stanton B. Gelvin; Thomas K. Hodges
Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains
A one-step method to convert vectors into binary vectors suited for Agrobacterium -mediated transformation
Frank L. W. Takken; Ringo van Wijk; Caroline B. Michielse; Petra M. Houterman; Arthur F. J. Ram; Ben J. C. Cornelissen
Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps, positional-cloning and whole-genome sequencing strategies. Unfortunately, their use for functional genomics is limited, as currently there is no efficient method to use BACs directly for complementation. We describe a novel strategy for one-step conversion of any BAC into a binary BAC (BIBAC). Using Agrobacterium tumefaciens, these BIBACs
Temperature, acetosyringone, virulence genes and wounding effects on Agrobacterium-mediated transformation efficiency
Salas Fernandez, Maria Guadalupe
compounds. Bolton et al. (1986) broadened the spectrum of phenolic compounds that can induce A. tumefaciens when they found that at least seven different compounds can produce the same effect as acetosyringone (catechol, gallic acid, pyrogallic acid, p...-hydroxybenzoic acid, photocatechnic acid, P-resorcylic acid and vanillin). This finding was not unexpected since Agrobacterium has a wide host range and is able to infect many dicotyledonous plants, Ashby et al. (1988) reported that the same group of phenolic...
Role of Agrobacterium VirB11 ATPase in T-Pilus Assembly and Substrate Selection
The VirB11 ATPase is a subunit of the Agrobacterium tumefaciens transfer DNA (T-DNA) transfer system, a type IV secretion pathway required for delivery of T-DNA and effector proteins to plant cells during infection. In this study, we examined the effects of virB11 mutations on VirB protein accumulation, T-pilus production, and substrate translocation. Strains synthesizing VirB11 derivatives with mutations in the
Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.)
Z. N. Yang; I. L. Ingelbrecht; E. Louzada; M. Skaria; T. E. Mirkov
Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation\\u000a procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic\\u000a shoots. Transformed shoots are invigorated and multiplied on a non-selective medium
The promoter of T L DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector
Csaba Koncz; Jeff Schell
A "plant gene vector cassette" to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also
Agrobacterium-mediated transformation of meadow fescue (Festuca pratensis Huds.).
Gao, Caixia; Liu, Jinxing; Nielsen, Klaus Kristian
Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and beta-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene. PMID:19603171
In planta transformation method for T-DNA transfer in orchids
NASA Astrophysics Data System (ADS)
Semiarti, Endang; Purwantoro, Aziz; Mercuriani, Ixora S.; Anggriasari, Anida M.; Jang, Seonghoe; Suhandono, Sony; Machida, Yasunori; Machida, Chiyoko
Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro?PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1?2 pro?GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.
Wally, Owen S D; Punja, Zamir K
Plants are susceptible to infection by a broad range of fungal pathogens. A range of proteins have been evaluated that can enhance tolerance to these pathogens by heterologous expression in transgenic carrot tissues. The protocols for carrot transformation with Arabidopsis NPR1 (Non-Expressor of Pathogenesis-Related Proteins 1) are described in this chapter, using the herbicide resistance gene bar, which encodes phosphinothricin acetyltransferase, as a selectable marker. In this protocol, petiole segments (0.5-1.0 cm long) from aseptically grown carrot seedlings are exposed to Agrobacterium tumefaciens strain LBA4404 for 10-30 min and cocultivated for 2-3 days. Herbicide selection is then imposed for 8-12 weeks on a series of different tissue culture media until embryogenic calli are produced. The transfer of the embryogenic calli to hormone-free medium results in embryo development which eventually gives rise to transgenic plantlets. Embryogenic calli can also be propagated in suspension cultures. This protocol has yielded transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern-positive independent events out of 100. PMID:25416249
Transcription factor AtbZIP60 regulates expression of Ca2+ -dependent protein kinase genes in transgenic cells.
Tang, Wei; Page, Michael
The Arabidopsis thaliana bZIP60 (AtbZIP60) transcription factor regulates stress signaling. However, its molecular mechanism remains to be elucidated. In this investigation, cell suspension cultures of two different plant species rice (Oryza sativa L.) and white pine (Pinus strobes L.) were transformed using Agrobacterium tumefaciens strain LBA4404 harboring pBI-AtZIP60. Integration of the AtbZIP60 gene into the genome of rice and white pine has been confirmed by polymerase chain reaction (PCR), southern blotting, and northern blotting analyses. Six transgenic cell lines from O. sativa and three transgenic cell lines from P. strobus were used to analyze the salt, drought, and cold tolerance conferred by the overexpression of the AtbZIP60 gene. Our results demonstrated that expression of the AtbZIP60 gene enhanced salt, drought, and cold tolerance in rice and white pine transgenic cell lines. In rice, transcription factor AtbZIP60 increased expression of Ca(2+)-dependent protein kinase genes OsCPK6, OsCPK9, OsCPK10, OsCPK19, OsCPK25, and OsCPK26 under treatment of salt, drought, and cold. These results demonstrated that overexpression of the AtbZIP60 gene in transgenic cell lines improved salt, drought, and cold stress tolerances by regulating expression of Ca(2+)-dependent protein kinase genes. Overexpression of the AtbZIP60 gene could be an alternative choice for engineering plant abiotic stress tolerance. PMID:23275191
Genetic transformation of mature embryos of bread (T. aestivum) and pasta (T. durum) wheat genotypes.
Moghaieb, Reda E A; El-Arabi, Nagwa I; Momtaz, Osama A; Youssef, Sawsan S; Soliman, Mohamed H
The objective of the present study is to develop an efficient protocol for regeneration of transgenic wheat plants using Agrobacterium- mediated transformation of mature embryos of hexaploid bread wheat (Triticum aestivum) and tetraploid pasta wheat (Triticum durum). The data indicated that embryogenic calli were formed within 7 days in the presence of 2 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic calli in the presence of 2 mgl-1 BA. Shoot regeneration frequency varied between wheat cultivars according to their genetic background differences. Regeneration frequency was higher in the cultivar Gemmiza 10 (95 %) compared with the other cultivars tested. Mature embryos derived callus of the cultivars Gemmiza 10 and Gemmiza 9 were co-cultivated with A. tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (npt-II). The resulted putative transgenic plantlets were able to grow on kanamycin containing medium. A successful integration of the transgene was confirmed by analyzing the T0 plantlets using Southern hybridization and PCR amplification. The gus gene expression can be detected only in the transgenic plants. The reported protocol is reproducible and can be used to regenerate transgenic wheat plants expressing the genes present in A. tumifaciens binary vectors. PMID:21865876
Activity of T-DNA borders in plant cell transformation by mini-T plasmids.
Jen, G C; Chilton, M D
By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoë leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T-DNA transfer and integration are discussed. Images PMID:3009403
Curtis, Ian S
Lettuce is a globally important leafy vegetable with the United States being the largest world producers. The crop is susceptible to a number of viruses that are aphid transmitted and also highly vulnerable to post harvest diseases. Although wild species of lettuce are an important source of disease resistance genes, their introgression into commercial lettuce has been limited owing to sexual incompatibilities. Hence, the development of a gene transfer system for lettuce would be extremely valuable both in improving the genetic diversity of the crop and also for the transfer of useful agronomic traits. This chapter describes an Agrobacterium-mediated gene delivery system that is highly adaptable for the production of transgenic plants using a wide range of lettuce germplasms. The system described, commonly referred to as the genotype-independent transformation system, has been used for the transfer of several agriculturally useful traits into commercial varieties of lettuce. In this case, A. tumefaciens strain LBA4404 carrying a binary vector with supervirulent pToK47 was used for infecting excised cotyledonary explants. The plant selectable marker gene neomycin phosphotransferase II (nptII) was used, and transformed plants were selected using kanamycin in the culture medium. The beta-glucuronidase gene with intron (gus-intron) was also used in the gene transfer study to confirm the transgenicity of regenerated plants further. PMID:16988367
Agrobacterium -mediated transformation as a useful tool for the molecular genetic study of the phytopathogen Curvularia lunata
Tong Liu; Lixing Liu; Xue Jiang; Jumei Hou; Kehe Fu; Feihong Zhou; Jie Chen
In order to explore the molecular mechanisms of virulence and genetic variance of Curvularia lunata in maize, an ATMT (Agrobacterium tumefaciens-mediated transformation) system was established in order to create a wide range of insertional transformants of C. lunata. Our results showed that the germinating conidia were the ideal starting material for transformation. Based on our optimised\\u000a transformation condition, the transformation
Agrobacterium -mediated transient expression in citrus leaves: a rapid tool for gene expression and functional gene assay
Jose F. L. Figueiredo; Patrick Römer; Thomas Lahaye; James H. Graham; Frank F. White; Jeffrey B. Jones
In this study, we present a method for transient expression of the type III effector AvrGf1 from Xanthomonas citri subsp. citri strain Aw in grapefruit leaves (Citrus paradisi) via Agrobacterium tumefaciens. The coding sequence of avrGf1 was placed under the control of the constitutive CaMV 35S promoter in the binary vectors pGWB2 and pGWB5. Infiltration of\\u000a grapefruit leaves with A.
T-DNA from Agrobacterium Ti Plasmid is in the Nuclear DNA Fraction of Crown Gall Tumor Cells
Mary-Dell Chilton; Randall K. Saiki; Narendra Yadav; Milton P. Gordon; Francis Quetier
The crown gall teratoma tumor line BT37, incited by Agrobacterium tumefaciens strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether
Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium -mediated soybean transformation
Margie M. Paz; Juan Carlos Martinez; Andrea B. Kalvig; Tina M. Fonger; Kan Wang
The utility of transformation for soybean improvement requires an efficient system for production of stable transgenic lines.\\u000a We describe here an improved cotyledonary node method using an alternative explant for Agrobacterium tumefaciens-mediated soybean transformation. We use the term "half-seed" to refer to this alternative cotyledonary explant that is derived\\u000a from mature seed of soybean following an overnight imbibition and to
Agrobacterium -mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants
Bruno Delbreil; Philippe Guerche; M. Jullien
Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them
Temperature Effects on Agrobacterium Phytochrome Agp1
Njimona, Ibrahim; Lamparter, Tilman
Phytochromes are widely distributed biliprotein photoreceptors with a conserved N-terminal chromophore-binding domain. Most phytochromes bear a light-regulated C-terminal His kinase or His kinase-like region. We investigated the effects of light and temperature on the His kinase activity of the phytochrome Agp1 from Agrobacterium tumefaciens. As in earlier studies, the phosphorylation activity of the holoprotein after far-red irradiation (where the red-light absorbing Pr form dominates) was stronger than that of the holoprotein after red irradiation (where the far red-absorbing Pfr form dominates). Phosphorylation activities of the apoprotein, far red-irradiated holoprotein, and red-irradiated holoprotein decreased when the temperature increased from 25°C to 35°C; at 40°C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40°C was nearly completely restored when the temperature returned to 25°C. UV/visible spectroscopy indicated that the protein was not denatured up to 45°C. At 50°C, however, Pfr denatured faster than the dark-adapted sample containing the Pr form of Agp1. The Pr visible spectrum was unaffected by temperatures of 20–45°C, whereas irradiated samples exhibited a clear temperature effect in the 30–40°C range in which prolonged irradiation resulted in the photoconversion of Pfr into a new spectral species termed Prx. Pfr to Prx photoconversion was dependent on the His-kinase module of Agp1; normal photoconversion occurred at 40°C in the mutant Agp1-M15, which lacks the C-terminal His-kinase module, and in a domain-swap mutant in which the His-kinase module of Agp1 is replaced by the His-kinase/response regulator module of the other A. tumefaciens phytochrome, Agp2. The temperature-dependent kinase activity and spectral properties in the physiological temperature range suggest that Agp1 serves as an integrated light and temperature sensor in A. tumefaciens. PMID:22043299
Strawberry FaEtr2 gene RNAi expression vector construction and genetic transformation
Chunli Song; Junlian Ma; Xia Tang; Zide Zhang; Zhixia Hou
The short hairpin RNA (shRNA) expression vector of the FaEtr2 gene was constructed by inserting the sense fragment into the constructed antisense vector of FaEtr2 (pBI121-Anti-Etr2) in sense orientation. The constructed RNA interference (RNAi) expression vector was transformed into Agrobacterium fumefeciens LBA4404 and used to infect strawberry leaves. Using in vitro plantlet leaves as explants, the transformation conditions of All-Star
Agrobacterium rhizogenes GALLS Protein Contains Domains for ATP Binding, Nuclear Localization, and Type IV Secretion?
Hodges, Larry D.; Vergunst, Annette C.; Neal-McKinney, Jason; den Dulk-Ras, Amke; Moyer, Deborah M.; Hooykaas, Paul J. J.; Ream, Walt
Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2. PMID:17012398
Tissue culture independent transformation of the forage crop sunnhemp (Crotalaria juncea L.): an easy method towards generation of transgenics.
Rao, Jyothsna P; Agrawal, Pushpa; Mahmood, Riaz; Sreevathsa, Rohini; Rao, K Sankara; Reddy, G R; Suryanarayana, V V S
A transformation system which is free of in vitro plant regeneration following Agrobacterium infection is established for the forage legume, Sunnhemp (Crotalaria juncea L.) where in the entire embryo axis of the germinating seed was used as the target tissue for transformation. After standardization of transformation conditions, the cotyledonary node of the embryo axis was infected with Agrobacterium host LBA 4404 harboring the recombinant vector pCAMBIA 2301. The bivalent 1D gene of the two major foot and mouth disease virus (FMDV) serotypes 'O' and 'A22' and the neomycin phosphotransferase (nptII) gene were used as the markers for optimization of the protocol. The embryo axes were pricked randomly on the cotyledonary node and co-cultivated with Agrobacterium. The germlings were then allowed to grow under standard growth room conditions in to mature fertile plants. 60 T0 plants were established from 3 separate experiments. Three hundred seeds from the 60 T0 plants were sown to raise the T1 generation of which 180 were analyzed for integration of bivalent FMDV gene 1D "O" and "A22" and the nptII gene. Eighteen out of these 180 plants amplified both the marker genes. Two independent transgenic lines 24 and 37, showed elevated levels of expression of 12 ?g and 8 ?g (per gm of fresh leaf) of the bivalent ID antigen "O" and "A22" . The results showed that the transformation efficiency was 3 %. To the best of our knowledge, this is the first successful attempt of Agrobacterium tumefaciens mediated transformation of Sunnhemp. The protocol can generate whole plant transformants with relative ease and should be compatible to all genotypes of Sunnhemp. PMID:23573040
Mechanisms and regulation of surface interactions and biofilm formation in Agrobacterium
Heindl, Jason E.; Wang, Yi; Heckel, Brynn C.; Mohari, Bitan; Feirer, Nathan; Fuqua, Clay
For many pathogenic bacteria surface attachment is a required first step during host interactions. Attachment can proceed to invasion of host tissue or cells or to establishment of a multicellular bacterial community known as a biofilm. The transition from a unicellular, often motile, state to a sessile, multicellular, biofilm-associated state is one of the most important developmental decisions for bacteria. Agrobacterium tumefaciens genetically transforms plant cells by transfer and integration of a segment of plasmid-encoded transferred DNA (T-DNA) into the host genome, and has also been a valuable tool for plant geneticists. A. tumefaciens attaches to and forms a complex biofilm on a variety of biotic and abiotic substrates in vitro. Although rarely studied in situ, it is hypothesized that the biofilm state plays an important functional role in the ecology of this organism. Surface attachment, motility, and cell division are coordinated through a complex regulatory network that imparts an unexpected asymmetry to the A. tumefaciens life cycle. In this review, we describe the mechanisms by which A. tumefaciens associates with surfaces, and regulation of this process. We focus on the transition between flagellar-based motility and surface attachment, and on the composition, production, and secretion of multiple extracellular components that contribute to the biofilm matrix. Biofilm formation by A. tumefaciens is linked with virulence both mechanistically and through shared regulatory molecules. We detail our current understanding of these and other regulatory schemes, as well as the internal and external (environmental) cues mediating development of the biofilm state, including the second messenger cyclic-di-GMP, nutrient levels, and the role of the plant host in influencing attachment and biofilm formation. A. tumefaciens is an important model system contributing to our understanding of developmental transitions, bacterial cell biology, and biofilm formation. PMID:24834068
Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation
NASA Astrophysics Data System (ADS)
Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina
This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.
Component identification of electron transport chains in curdlan-producing Agrobacterium sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis.
Zhang, Hongtao; Setubal, Joao Carlos; Zhan, Xiaobei; Zheng, Zhiyong; Yu, Lijun; Wu, Jianrong; Chen, Dingqiang
Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed. PMID:20730594
Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor
Yong-Sun Moon; Bruno G. G. Donzelli; Stuart B. Krasnoff; Heather McLane; Mike H. Griggs; Peter Cooke; John D. Vandenberg; Donna M. Gibson; Alice C. L. Churchill
Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium aniso- pliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence
Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)
Ahangarzadeh, Shahrzad; Daneshvar, Mohammad Hosein; Rajabi-Memari, Hamid; Galehdari, Hamid; Alamisaied, Khalil
Background Molecular farming is the production of important recombinant proteins in transgenic organisms on an agricultural scale. Interferons are proteins with antiviral and antitumor activities and can be used for viral infections and cancers treatments. Objectives This study reports the transformation of INF ?2b gene in tobacco plant for the first time in Iran. Materials and Methods Interferon ?2b gene was amplified by PCR using specific primers containing appropriate restriction enzymes, plant highly expression sequence and Histidine tag sequence. Target sequence was cloned in plant expression vector pCAMBIA1304 and the construct named pCAMINF?. pCAMINF? was transferred to E. coli strain DH5? and plated on LB agar medium containing kanamycin 50 mgl-1. The colonies were confirmed by colony PCR and sequencing. The construct was transferred into Agrobacterium tumefaciens by freeze-thaw method and transformed colonies were confirmed by colony PCR. Tobacco plants (cultivar xanthi) were inoculated with A. tumefaciens strain LBA4404 by leaf disc method. Inoculated explants were cultured on MSII (MS + BAP 1mgl-1 + NAA 0.1 mgl-1) at 28°C and darkness for 48 hours. Then explants were transferred to selection medium containing cephotaxime (250 mgl-1) and hygromycin (15 mgl-1) in a 16/8 (day/night) h photoperiod in growth room with an irradiance of 5000 lux. Transgenic plants were regenerated and transferred to perlite. Genomic DNA was extracted from regenerated plants by Dellaporta method at 5-leaf step and transgenic lines were confirmed by PCR with specific primers. Expression of Interferon ?2b gene was confirmed by dot blotting. Conclusions Since no report of interferon alpha production in plants in Iran has been expressed yet, this research could create a field of producing this drug in tobacco, in Iran. PMID:24624166
Agrobacterium infection and plant defense—transformation success hangs by a thread
Pitzschke, Andrea
The value of Agrobacterium tumefaciens for plant molecular biologists cannot be appreciated enough. This soil-borne pathogen has the unique capability to transfer DNA (T-DNA) into plant systems. Gene transfer involves both bacterial and host factors, and it is the orchestration of these factors that determines the success of transformation. Some plant species readily accept integration of foreign DNA, while others are recalcitrant. The timing and intensity of the microbially activated host defense repertoire sets the switch to "yes" or "no." This repertoire is comprised of the specific induction of mitogen-activated protein kinases (MAPKs), defense gene expression, production of reactive oxygen species (ROS) and hormonal adjustments. Agrobacterium tumefaciens abuses components of the host immunity system it mimics plant protein functions and manipulates hormone levels to bypass or override plant defenses. A better understanding of the ongoing molecular battle between agrobacteria and attacked hosts paves the way toward developing transformation protocols for recalcitrant plant species. This review highlights recent findings in agrobacterial transformation research conducted in diverse plant species. Efficiency-limiting factors, both of plant and bacterial origin, are summarized and discussed in a thought-provoking manner. PMID:24391655
Female Reproductive Tissues Are the Primary Target of Agrobacterium-Mediated Transformation by the Arabidopsis Floral-Dip Method1
Desfeux, Christine; Clough, Steven J.; Bent, Andrew F.
The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding ?-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved. PMID:10889238
Root and shoot parts of strawberry: factories for production of functional human pro-insulin.
Tavizi, Ashkan; Javaran, Mokhtar Jalali; Moieni, Ahmad; Mohammadi-Dehcheshmeh, Manijeh; Mohebodini, Mehdi; Ebrahimie, Esmaeil
Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry. PMID:25403333
The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].
Ellul, P; Ríos, G; Atarés, A; Roig, L A; Serrano, R; Moreno, V
An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species. PMID:12783167
Cloning, Transformation and Expression of Proinsulin Gene in Tomato (Lycopersicum esculentum Mill.)
Soltanmohammadi, Behnoush; Jalali-Javaran, Mokhtar; Rajabi-Memari, Hamid; Mohebodini, Mehdi
Background: Plants are among promising and suitable platform systems for production of recombinant biopharmaceutical proteins due to several features such as safety, no need for fermentation, inexpensive investment, and fast and easy scale-up. Human insulin is one of the most widely used medicines in the world. Up to now different expression systems including Escherichia coli, yeast and CHO have been exploited for producing recombinant human insulin and a variety of different recombinant insulin are extensively used. Objectives: This study reports on the transformation and expression of proinsulin gene in tomato plants for the first time in Iran. Materials and Methods: This study reports the cloning, transformation and expression of proinsulin gene in tomato plants. Specific primers were designed and used for PCR amplification and cloning of the proinsulin gene in the plant expression vector pCAMBIA1304. The recombinant construct was transferred into Agrobacterium tumefaciens strain LBA4404, and used for Agrobacterium mediated stable transformation of tomato plants. Presence of the desired gene in transgenic lines was confirmed through colony PCR and sequencing. The expression of the protein in transgenic lines was confirmed by immunodot blot assay. Results: The presence of the proinsulin gene in the genomic DNA of transgenic tomato was confirmed by PCR. Also total protein of transgenic tomato was extracted and the expression of proinsulin was detected using dotblot assay. Conclusions: This survey addresses the possibility of proinsulin gene transfer and expression in tomato transgenic lines. This study can be used as a basis for future researches to produce human proinsulin in tomato and other candidate plants. PMID:24644433
RP4 promotion of transfer of a large Agrobacterium plasmid which confers virulence.
Chilton, M D; Farrand, S K; Levin, R; Nester, E W
Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes. PMID:971805
Ecological dynamics and complex interactions of Agrobacterium megaplasmids
Platt, Thomas G.; Morton, Elise R.; Barton, Ian S.; Bever, James D.; Fuqua, Clay
As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it's Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together. PMID:25452760
Identification of Genomic Species in Agrobacterium Biovar 1 by AFLP Genomic Markers?
Portier, Perrine; Fischer-Le Saux, Marion; Mougel, Christophe; Lerondelle, Catherine; Chapulliot, David; Thioulouse, Jean; Nesme, Xavier
Biovar 1 of the genus Agrobacterium consists of at least nine genomic species that have not yet received accepted species names. However, rapid identification of these organisms in various biotopes is needed to elucidate crown gall epidemiology, as well as Agrobacterium ecology. For this purpose, the AFLP methodology provides rapid and unambiguous determination of the genomic species status of agrobacteria, as confirmed by additional DNA-DNA hybridizations. The AFLP method has been proven to be reliable and to eliminate the need for DNA-DNA hybridization. In addition, AFLP fragments common to all members of the three major genomic species of agrobacteria, genomic species G1 (reference strain, strain TT111), G4 (reference strain, strain B6, the type strain of Agrobacterium tumefaciens), and G8 (reference strain, strain C58), have been identified, and these fragments facilitate analysis and show the applicability of the method. The maximal infraspecies current genome mispairing (CGM) value found for the biovar 1 taxon is 10.8%, while the smallest CGM value found for pairs of genomic species is 15.2%. This emphasizes the gap in the distribution of genome divergence values upon which the genomic species definition is based. The three main genomic species of agrobacteria in biovar 1 displayed high infraspecies current genome mispairing values (9 to 9.7%). The common fragments of a genomic species are thus likely "species-specific" markers tagging the core genomes of the species. PMID:16936063
Technology Transfer Automated Retrieval System (TEKTRAN)
Stem rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of many economically important crops under conducive conditions. Despite extensive investigations on the disease, the genetic factors that control the pathogenesis of S. sclerotiorum are incompletely understood, except ...
Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens.
Zale, Janice M; Agarwal, S; Loar, S; Steber, C M
Hexaploid wheat, one of the world's most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation. PMID:19308413
Optimization of the uidA gene transfer into somatic embryos of rose via Agrobacterium tumefaciens
Korban, Schuyler S.
, transgenic plants were only induced from transformed primary embryogenic callus. Stable integration] produced transgenic plants by trans- forming friable embryogenic tissues of rose, induced from filament transgenic plants from roots derived from stem sections of the rootstock R. hybrida cv. Moneyway following co
Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for efficient transient expression in plants
Sylvestre Marillonnet; Carola Thoeringer; Romy Kandzia; Victor Klimyuk; Yuri Gleba
Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants: stable genetic transformation and transient expression using viral vectors. Although much faster, the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes. We have developed constructs for the efficient delivery of RNA viral vectors as DNA precursors and show
Organogenesis and Agrobacterium tumefaciens -mediated transformation of Eucalyptus saligna with P5CS gene
R. Dibax; C. Deschamps; J. C. Bespalhok Filho; L. G. E. Vieira; H. B. C. Molinari; M. K. F. De Campos; M. Quoirin
The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes ?1-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced\\u000a on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots\\u000a were subsequently
First report of crown gall caused by Agrobacterium tumefaciens on Euphorbia esula/virgata in Europe
Technology Transfer Automated Retrieval System (TEKTRAN)
Hypertrophy and hyperplasia resembling crown galls were found on roots of Euphorbia esula virgata occurring at a single site (47°34'32.52"N, 21° 27' 38.31"E) in east-central Hungary in 2005. Leafy spurge (E. esula/virgata) is an invasive species causing substantial economic losses to the value of gr...
Evidence for stable transformation of wheat by floraldip in Agrobacterium tumefaciens
Technology Transfer Automated Retrieval System (TEKTRAN)
Hexaploid wheat is one of the world's most important staple crops but genetic transformation is still challenging. We have developed a floral transformation protocol that does not utilize tissue culture. Three T-DNA wheat transformants have been produced in the germplasm line, Crocus, using this p...
Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)
Hammock, Bruce D.
Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter , Bruce D. Hammockb a Department of Land, Air, and Water Resources, University of California, One Shields) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has
Agrobacterium tumefaciens is a plant-patho-genic bacterium notorious for its ability to
Ulm, Roman
by thestablegeneticmodificationofthehostplant after integration of the bacterial T-DNA into the plant genome, a unique example of inter- kingdom that the pores would be closed in planta, therefore not necessarily detrimental to the plant cell. Although
Agrobacterium tumefaciens T-DNA integrates into multiple sites of the sunflower crown gall genome
Doris Ursic; Jerry L. Slightom; John D. Kemp
We analyzed the integration of a tumor inducing (Ti) plasmid into an octopine producing crown gall tumor of sunflower, line PSCG 15955. A continuous Ti plasmid segment (T-DNA) of about 19.5 kilo base pairs (kbp) is transferred and integrated into a small number of sites of the plant DNA.
Leaf disc transformation of cultivated tomato ( L. esculentum ) using Agrobacterium tumefaciens
Sheila McCormick; Jeanne Niedermeyer; Joyce Fry; Arlene Barnason; Robert Horsch; Robert Fraley
The leaf disc transformation\\/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon\\/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios.
Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid.
Ramírez-Bahena, Martha H; Vial, Ludovic; Lassalle, Florent; Diel, Benjamin; Chapulliot, David; Daubin, Vincent; Nesme, Xavier; Muller, Daniel
Linear chromosomes are atypical in bacteria and likely a secondary trait derived from ancestral circular molecules. Within the Rhizobiaceae family, whose genome contains at least two chromosomes, a particularity of Agrobacterium fabrum (formerly A. tumefaciens) secondary chromosome (chromid) is to be linear and hairpin-ended thanks to the TelA protelomerase. Linear topology and telA distributions within this bacterial family was screened by pulse field gel electrophoresis and PCR. In A. rubi, A. larrymoorei, Rhizobium skierniewicense, A. viscosum, Agrobacterium sp. NCPPB 1650, and every genomospecies of the biovar 1/A. tumefaciens species complex (including R. pusense, A. radiobacter, A. fabrum, R. nepotum plus seven other unnamed genomospecies), linear chromid topologies were retrieved concomitantly with telA presence, whereas the remote species A. vitis, Allorhizobium undicola, Rhizobium rhizogenes and Ensifer meliloti harbored a circular chromid as well as no telA gene. Moreover, the telA phylogeny is congruent with that of recA used as a marker gene of the Agrobacterium phylogeny. Collectively, these findings strongly suggest that single acquisition of telA by an ancestor was the founding event of a large and diverse clade characterized by the presence of a linear chromid. This clade, characterized by unusual genome architecture, appears to be a relevant candidate to serve as a basis for a possible redefinition of the controversial Agrobacterium genus. In this respect, investigating telA in sequenced genomes allows to both ascertain the place of concerned strains into Agrobacterium spp. and their actual assignation to species/genomospecies in this genus. PMID:24440816
Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation.
Chattopadhyay, Tirthartha; Roy, Sheuli; Mitra, Adinpunya; Maiti, Mrinal K
Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants. PMID:21153028
Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica
Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu
The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121
Plant expression signals of the Agrobacterium T-cyt gene.
de Pater, B S; Klinkhamer, M P; Amesz, P A; de Kam, R J; Memelink, J; Hoge, J H; Schilperoort, R A
Within the 5' and 3' non-coding regions of the T-cyt gene from the octopine T-DNA of Agrobacterium tumefaciens sequences required for expression of this gene in plant cells were identified by deletion mutagenesis. The results show that 184 bp of the 5' non-coding region and 270 bp of the 3' non-coding region are sufficient for wild-type expression. Within the 5' non-coding region two essential expression signals were identified: (1.) an activator element located between -185 and -129 with respect to the ATG start codon and (2.) one out of two TATA boxes. Deletions of the activator element or the two TATA boxes resulted in nonfunctional genes. Deletion of the upstream TATA box and both putative CAAT boxes did not significantly affect expression. Within the 3' non-coding region, the polyadenylation box most distal to the stop codon was not essential for expression, but sequences more upstream, including a second polyadenylation box were found to be required for wild-type expression. Images PMID:3671083
Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer.
Michielse, C B; Salim, K; Ragas, P; Ram, A F J; Kudla, B; Jarry, B; Punt, P J; van den Hondel, C A M J J
Two transformation systems, based on the use of CaCl(2)/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS(+) marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl(2)/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl(2)/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome. PMID:15067540
Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility
Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 22.214.171.124) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274
Direct visualization of Agrobacterium-delivered VirE2 in recipient cells
Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q
Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 ?m sec?1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048
Efficient Agrobacterium-based transient expression system for the production of biopharmaceuticals in plants.
Circelli, Patrizia; Donini, Marcello; Villani, Maria Elena; Benvenuto, Eugenio; Marusic, Carla
We have recently described an efficient transient expression system mediated by Agrobacterium tumefaciens for the production of HIV-1 Nef protein in Nicotiana benthamiana plants. In order to enhance the yield of recombinant protein we assayed the effect of three gene-silencing viral suppressor proteins (P25 of Potato Virus X, P19 of Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef expression levels. Results demonstrated that AMCV-P19 gave the highest Nef yield (1.3% of total soluble protein) and that this effect was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms. Here we report additional data on the production of different heterologous proteins including human immunoglobulin heavy and light chains and a virus coat protein that demonstrate the robustness of this co-agroinfiltration expression system boosted by the AMCV-P19 gene-silencing suppressor. PMID:21326930
Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.
Kobayashi, M; Suzuki, T; Fujita, T; Masuda, M; Shimizu, S
The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM. Images Fig. 1 PMID:11607511
Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement
Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena
Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop. PMID:25309562
Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.
Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J
Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli. PMID:24376037
Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration.
Du, Ningxia; Pijut, Paula M
A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were transformed in the presence of 100 microM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l(-1) was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with 13.3 microM 6-benzylaminopurine, 4.5 microM thidiazuron, 50 mg l(-1) adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse. This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash. PMID:19343350
Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment
Ibrahim, Evra Raunie
Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30?mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280?psi of helium pressure at 6 to 8?cm distance. PMID:25295258
An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.
Zhang, Jin jing; Shi, Liang; Chen, Hui; Sun, Yun qi; Zhao, Ming wen; Ren, Ang; Chen, Ming jie; Wang, Hong; Feng, Zhi yong
Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species. PMID:24612605
Finger millet [Eleusine coracana (L.) Gaertn].
Ceasar, Stanislaus Antony; Ignacimuthu, Savarimuthu
Millets are the primary food source for millions of people in tropical regions of the world supplying mineral nutrition and protein. In this chapter, we describe an optimized protocol for the Agrobacterium-mediated transformation of finger millet variety GPU 45. Agrobacterium strain LBA4404 harboring plasmid pCAMBIA1301 which contains hygromycin phosphotransferase (hph) as selectable marker gene and ?-glucuronidase (GUS) as reporter gene has been used. This protocol utilizes the shoot apex explants for the somatic embryogenesis and regeneration of finger millet after the transformation by Agrobacterium. Desiccation of explants during cocultivation helps for the better recovery of transgenic plants. This protocol is very useful for the efficient production of transgenic plants in finger millet through Agrobacterium-mediated transformation. PMID:25300836
Agrobacterium-Mediated Gene Transfer Results Mainly in Transgenic Plants Transmitting T-DNA as a Single Mendelian Factor
Budar, F.; Thia-Toong, L.; Van Montagu, M.; Hernalsteens, J.-P.
Forty-four independent transformed tobacco plants were obtained from a cocultivation experiment with Agrobacterium tumefaciens strains carrying modified Ti-plasmids. The transformed plants were either self-fertilized or crossed with nontransformed plants or with other transformed plants. The segregation of a phenotypic marker (kanamycin resistance) in the progenies of these plants was determined. In 40 cases out of 44, the segregation of the kanamycin resistance marker is consistent with Mendelian genetics. Among these 40 clones, 35 contain a single kanamycin resistance locus. The five others segregate two independent resistance loci. In two of the single insert clones, the segregation ratio after selfing indicates that the T-DNA insertion may have caused a recessive lethal mutation. PMID:17246346
Development of an Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos
Jube, Sandro
The tree-legume Leucaena leucocephala (leucaena) is used as a perennial fodder because of its fast-growing foliage, which is high in protein content. The use of leucaena as a fodder is however restricted due to the presence of the toxin mimosine. Improvements in the nutritional contents as well as other agronomic traits of leucaena can be accomplished through genetic transformation. The objective of this research was to develop a transformation protocol for leucaena using phosphinothricin resistance as the plant selectable marker. Explants obtained from immature zygotic embryos infected with the Agrobacterium tumefaciens strain C58C1 containing the binary plasmid pCAMBIA3201 produced four putative transformed leucaena plants. Transformation was con- firmed by PCR, RT-PCR, Southern blot, Western analyses, GUS-specific enzyme activity and herbicide leaf spraying assay. A transformation efficiency of 2% was established using this protocol. PMID:20041041
Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays
Background Agrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain 'normal' sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or 'stroma-filled-tubules' emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes. Results Using a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation. Conclusion Although we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as 'normal' as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy. PMID:24886417
Biological systems of the host cell involved in Agrobacterium infection
Vitaly Citovsky; Stanislav V. Kozlovsky; Benoît Lacroix; Adi Zaltsman; Mery Dafny-Yelin; Shachi Vyas; Andriy Tovkach; Tzvi Tzfira
Summary Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, repre- sents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant
Draft Genome Sequences of Agrobacterium nepotum Strain 39/7T and Agrobacterium sp. Strain KFB 330
Pu?awska, Joanna; Proki?, An?elka; Ivanovi?, Milan; Zlatkovi?, Nevena; Gaši?, Katarina; Obradovi?, Aleksa
Tumorigenic strains of Agrobacterium spp. are responsible for crown gall disease of numerous plant species. We present here draft genome sequences of nonpathogenic Agrobacterium nepotum strain 39/7T (CFBP 7436T, LMG 26435T), isolated from crown gall tumor on Prunus cerasifera, and tumorigenic Agrobacterium sp. strain KFB 330 (CFBP 8308, LMG 28674), isolated from galls on raspberry. PMID:25908139
Draft Genome Sequences of Agrobacterium nepotum Strain 39/7T and Agrobacterium sp. Strain KFB 330.
Kuzmanovi?, Nemanja; Pu?awska, Joanna; Proki?, An?elka; Ivanovi?, Milan; Zlatkovi?, Nevena; Gaši?, Katarina; Obradovi?, Aleksa
Tumorigenic strains of Agrobacterium spp. are responsible for crown gall disease of numerous plant species. We present here draft genome sequences of nonpathogenic Agrobacterium nepotum strain 39/7(T) (CFBP 7436(T), LMG 26435(T)), isolated from crown gall tumor on Prunus cerasifera, and tumorigenic Agrobacterium sp. strain KFB 330 (CFBP 8308, LMG 28674), isolated from galls on raspberry. PMID:25908139
Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.
Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Nesme, Xavier; Lavire, Céline; Hommais, Florence
The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-?-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-?-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-?-ketopropionic acid (HMPKP)-CoA ?-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent ?-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials. PMID:24657856
Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants
NASA Technical Reports Server (NTRS)
Cardoza, V.; Stewart, C. N.
An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.
Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent, Beta-Oxidative Deacetylation Pathway
Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Lavire, Céline; Hommais, Florence
The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-?-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-?-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-?-ketopropionic acid (HMPKP)–CoA ?-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent ?-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials. PMID:24657856
The Genome of the Natural Genetic Engineer Agrobacterium
Liao, Li
,12 Maynard V. Olson,5 Eugene W. Nester1,13 § The 5.67-megabase genome of the plant pathogen. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont in their genome structure and virulence gene com- plement. Availability of the A. tumefaciens sequence
Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens -mediated plant transformation
Alex Boyko; Aki Matsuoka; Igor Kovalchuk
Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors.\\u000a Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency\\u000a of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases\\u000a the frequency of both homologous
Agrobacterium tumefaciens -mediated transformation of Oncidium and Odontoglossum orchid species with the ethylene receptor mutant gene etr1-1
Barbara Raffeiner; Margrethe Serek; Traud Winkelmann
Oncidium and Odontoglosum orchid species have reduced display lives and are thus commercially less important than Phalaenopsis. One approach to prolonging display life permanently is to transform Oncidium and Odontoglossum with the ethylene receptor mutant gene etr1-1 from Arabidopsis under control of a flower specific promoter; this should reduce their sensitivity to exogenous ethylene. To achieve this\\u000a it will be
Isolation of pathogenicity- and gregatin-deficient mutants of Phialophora gregata f. sp. adzukicola through Agrobacterium tumefaciens -mediated transformation
Soichi Tanaka; Norio Kondo; Shigeo Naito
Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins\\u000a A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are\\u000a considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and
Development of Efficient Plant Regeneration and Transformation System for Impatiens Using Agrobacterium tumefaciens and Multiple Bud Cultures as Explants
Yinghui Dan; Aaron Baxter; Song Zhang; Christopher J Pantazis; Richard E Veilleux
BACKGROUND: Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. RESULTS: In
Iron-Binding Compounds from Agrobacterium spp.: Biological Control Strain Agrobacterium rhizogenes K84 Produces a Hydroxamate Siderophore
Penyalver, Ramón; Oger, Philippe; López, María M.; Farrand, Stephen K.
Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound. PMID:11157228
Agrobacterium: nature's genetic engineer
Nester, Eugene W.
Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun's old observations and also explain why Agrobacterium is nature's genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering. PMID:25610442
Horticultural characteristics of transgenic tobacco expressing the rolC gene from Agrobacterium rhizogenes
Scorza, R.; Zimmerman, T.W.; Cordts, J.M.; Footen, K.J. (Dept. of Agriculture, Kearneysville, WV (United States)); Ravelonandro, M. (Inst. National Recherche Agronomique, Villenave d'Ornon (France). Station de Pathologie Vegetale)
Wisconsin 38 tobacco (Nicotiana tabacum L.) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying the rolC gene from A. rhizogenes and NPT II and GUS genes. Shoots that regenerated on kanamycin-containing medium were confirmed as transgenic through GUS assays, polymerase chain reaction (PCR), Southern blot analyses, and transmission of the foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants; were earlier flowering by up to 35 days; and had smaller leaves, shorter internodes, smaller seed capsules, fewer seeds, smaller flowers, and reduced pollen viability. The number of seed capsules, leaf number, and specific root length were similar between transgenic and control plants. Transgenic clones varied in the expression of the rolC-induced growth alterations as did the first generation of seedlings from these clones. Such differences suggested the potential for selecting for different levels of expression. Transformation with the rolC gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size. Chemical names used: neomycin phosphotransferase (NPTII), [beta]-glucuronidase (GUS).
Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles.
Talhinhas, Pedro; Muthumeenakshi, S; Neves-Martins, Joăo; Oliveira, Helena; Sreenivasaprasad, S
Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 10(4) conidia, with >70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles. PMID:18183501
Root induction by Agrobacterium rhizogenes in walnut
Emilia Caboni; Paola Lauri; Mariagrazia Tonelli; Giuseppina Falasca; Carmine Damiano
Agrobacterium rhizogenes (wild-type, strain 1855), when applied to the basal part of microcuttings of walnut (J. regia L.), produced numerous adventitious roots in vitro: 58.6% of rooting was induced in microcuttings in hormone free medium and 62.9% and abundant callus formation in the presence of IBA. A. rhizogenes did not induce rooting when IAA was present in the rooting medium.
A highly efficient Agrobacterium mediated transformation system for chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri using DsRed-Express to follow root colonisation.
Islam, Md Nazrul; Nizam, Shadab; Verma, Praveen K
The soil-borne fungus Fusarium oxysporum f. sp. ciceri (Foc) causes vascular wilt of chickpea (Cicer arietinum L.), resulting in substantial yield losses worldwide. Agrobacterium tumefaciens mediated transformation (ATMT) has served as a resourceful tool for plant-pathogen interaction studies and offers a number of advantages over conventional transformation systems. Here, we developed a highly efficient A. tumefaciens mediated transformation system for Foc. In addition, a binary vector for constitutive expression of red fluorescent protein (DsRed-Express) was used to study developmental stages and host-pathogen interactions. Southern hybridisation was performed to confirm the transformation event and the presence of T-DNA in selected hygromycin resistant transformants. Most of the transformants showed single copy integrations at random positions. Microscopic studies revealed significant levels of fluorescent protein, both in conidia and mycelia. Confocal microscopy of chickpea roots infected with the transformed Foc showed rapid colonisation. These studies will allow us to develop strategies to determine the mechanisms of Foc-chickpea interaction in greater detail and to apply functional genomics for the characterisation of involved genes at the molecular level either by insertional mutagenesis or gene knock-out. PMID:22397973
Isolation of a New Broad-Host-Range IncQ-Like Plasmid, pTC-F14, from the Acidophilic Bacterium Acidithiobacillus caldus and Analysis of the Plasmid Replicon
Gardner, Murray N.; Deane, Shelly M.; Rawlings, Douglas E.
A moderately thermophilic (45 to 50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid. PMID:11344137
Biological systems of the host cell involved in Agrobacterium infection.
Citovsky, Vitaly; Kozlovsky, Stanislav V; Lacroix, Benoît; Zaltsman, Adi; Dafny-Yelin, Mery; Vyas, Shachi; Tovkach, Andriy; Tzfira, Tzvi
Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, represents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant and diverse species? The answer to this question lies in the ability of Agrobacterium to hijack fundamental cellular processes which are shared by most eukaryotic organisms. Our knowledge of these host cellular functions is critical for understanding the molecular mechanisms that underlie genetic transformation of eukaryotic cells. This review outlines the bacterial virulence machinery and provides a detailed discussion of seven major biological systems of the host cell-cell surface receptor arrays, cellular motors, nuclear import, chromatin targeting, targeted proteolysis, DNA repair, and plant immunity--thought to participate in the Agrobacterium-mediated genetic transformation. PMID:17222189
Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants
Padavannil, Abhilash; Jobichen, Chacko; Qinghua, Yang; Seetharaman, Jayaraman; Velazquez-Campoy, Adrian; Yang, Liu; Pan, Shen Q.; Sivaraman, J.
The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of ?-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems. PMID:24626239
Dimerization of VirD2 binding protein is essential for Agrobacterium induced tumor formation in plants.
Padavannil, Abhilash; Jobichen, Chacko; Qinghua, Yang; Seetharaman, Jayaraman; Velazquez-Campoy, Adrian; Yang, Liu; Pan, Shen Q; Sivaraman, J
The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2-T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of ?-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems. PMID:24626239
Agrobacterium-mediated gene transfer in plants and biosafety considerations.
Mehrotra, Shweta; Goyal, Vinod
Agrobacterium, the natures' genetic engineer, has been used as a vector to create transgenic plants. Agrobacterium-mediated gene transfer in plants is a highly efficient transformation process which is governed by various factors including genotype of the host plant, explant, vector, plasmid, bacterial strain, composition of culture medium, tissue damage, and temperature of co-cultivation. Agrobacterium has been successfully used to transform various economically and horticulturally important monocot and dicot species by standard tissue culture and in planta transformation techniques like floral or seedling infilteration, apical meristem transformation, and the pistil drip methods. Monocots have been comparatively difficult to transform by Agrobacterium. However, successful transformations have been reported in the last few years based on the adjustment of the parameters that govern the responses of monocots to Agrobacterium. A novel Agrobacterium transferred DNA-derived nanocomplex method has been developed which will be highly valuable for plant biology and biotechnology. Agrobacterium-mediated genetic transformation is known to be the preferred method of creating transgenic plants from a commercial and biosafety perspective. Agrobacterium-mediated gene transfer predominantly results in the integration of foreign genes at a single locus in the host plant, without associated vector backbone and is also known to produce marker free plants, which are the prerequisites for commercialization of transgenic crops. Research in Agrobacterium-mediated transformation can provide new and novel insights into the understanding of the regulatory process controlling molecular, cellular, biochemical, physiological, and developmental processes occurring during Agrobacterium-mediated transformation and also into a wide range of aspects on biological safety of transgenic crops to improve crop production to meet the demands of ever-growing world's population. PMID:23090683
Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.
Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S; Mirza, Bushra
Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens. PMID:24817272
Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens
Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S.; Mirza, Bushra
Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens. PMID:24817272
Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model1[W][OPEN
Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A.; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B.; Bailey-Serres, Julia; Brady, Siobhan M.
Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032
Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype.
Kumar, Vinod; Satyanarayana, K V; Sarala Itty, S; Indu, E P; Giridhar, P; Chandrashekar, A; Ravishankar, G A
A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders. PMID:16331458
Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures
Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes. PMID:21595964
Unmasking host and microbial strategies in the Agrobacterium-plant defense tango
Hwang, Elizabeth E.; Wang, Melinda B.; Bravo, Janis E.; Banta, Lois M.
Coevolutionary forces drive adaptation of both plant-associated microbes and their hosts. Eloquently captured in the Red Queen Hypothesis, the complexity of each plant–pathogen relationship reflects escalating adversarial strategies, but also external biotic and abiotic pressures on both partners. Innate immune responses are triggered by highly conserved pathogen-associated molecular patterns, or PAMPs, that are harbingers of microbial presence. Upon cell surface receptor-mediated recognition of these pathogen-derived molecules, host plants mount a variety of physiological responses to limit pathogen survival and/or invasion. Successful pathogens often rely on secretion systems to translocate host-modulating effectors that subvert plant defenses, thereby increasing virulence. Host plants, in turn, have evolved to recognize these effectors, activating what has typically been characterized as a pathogen-specific form of immunity. Recent data support the notion that PAMP-triggered and effector-triggered defenses are complementary facets of a convergent, albeit differentially regulated, set of immune responses. This review highlights the key players in the plant's recognition and signal transduction pathways, with a focus on the aspects that may limit Agrobacterium tumefaciens infection and the ways it might overcome those defenses. Recent advances in the field include a growing appreciation for the contributions of cytoskeletal dynamics and membrane trafficking to the regulation of these exquisitely tuned defenses. Pathogen counter-defenses frequently manipulate the interwoven hormonal pathways that mediate host responses. Emerging systems-level analyses include host physiological factors such as circadian cycling. The existing literature indicates that varying or even conflicting results from different labs may well be attributable to environmental factors including time of day of infection, temperature, and/or developmental stage of the host plant. PMID:25873923
Quorum-Dependent Mannopine-Inducible Conjugative Transfer of an Agrobacterium Opine-Catabolic Plasmid
Wetzel, Margaret E.; Kim, Kun-Soo; Miller, Marilyn; Olsen, Gary J.
The Ti plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related plasmids. Sequence analysis of one such plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the plasmid and produced little to no quormone even when grown with MOP, indicating that TraRpAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved. PMID:24363349
T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells
Chilton, Mary-Dell; Saiki, Randall K.; Yadav, Narendra; Gordon, Milton P.; Quetier, Francis
The crown gall teratoma tumor line BT37, incited by Agrobacterium tumefaciens strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether T-DNA is in chloroplasts, mitochondria, or nuclei. Tumor cell chloroplast DNA exhibited EcoRI and Bst I endonuclease cleavage patterns identical to those of normal tobacco chloroplast DNA. Tumor cell mitochondrial DNA exhibited a complex Bst I cleavage pattern that did not differ detectably from that of normal tobacco mitochondrial DNA. Southern blots of tumor chloroplast and mitochondrial cleavage products did not hybridize with labeled pTi T37 DNA, whereas blots of tumor cell nuclear DNA cleavage products hybridized strongly. We conclude that T-DNA is located not in chloroplasts or mitochondria but rather in the nuclear fraction of this tumor line. Images PMID:16592850
A one-step method to convert vectors into binary vectors suited for Agrobacterium-mediated transformation.
Takken, Frank L W; Van Wijk, Ringo; Michielse, Caroline B; Houterman, Petra M; Ram, Arthur F J; Cornelissen, Ben J C
Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps, positional-cloning and whole-genome sequencing strategies. Unfortunately, their use for functional genomics is limited, as currently there is no efficient method to use BACs directly for complementation. We describe a novel strategy for one-step conversion of any BAC into a binary BAC (BIBAC). Using Agrobacterium tumefaciens, these BIBACs can be efficiently transformed to virtually all organisms, including plants, fungi, yeasts and human cells. As the strategy is based on in vivo recombineering and does not depend on restriction sites, it is applicable to any vector. To show the feasibility of the method five BACs, containing 0-75 kb of fungal DNA, were converted into BIBACs. These were subsequently transformed to the plant pathogenic fungus Fusarium oxysporum f.sp. lycopersici and to Aspergillus awamori, a filamentous fungus often used for large-scale protein production. Molecular characterisation of the transformants showed that the BIBACs were efficiently transferred to the fungi and stably integrated into their genomes. PMID:14745506
Horizontal gene transfer from Agrobacterium to plants.
Matveeva, Tatiana V; Lutova, Ludmila A
Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role. PMID:25157257
Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium
Tzvi Tzfira; Manjusha Vaidya; Vitaly Citovsky
Genetic transformation of plant cells by Agrobacterium represents a unique case of trans-kingdom DNA transfer. During this process, Agrobacterium exports its transferred (T) DNA and several virulence (Vir) proteins into the host cell, within which T-DNA nuclear import is mediated by VirD2 (ref. 3) and VirE2 (ref. 4) and their host cell interactors AtKAP-alpha and VIP1 (ref. 6), whereas its
Arginine catabolism: A new function of both octopine and nopaline Ti-plasmids of Agrobacterium
Jeffrey G. Ellis; Allen Kerr; Jacques Tempé; Annik Petit
The oncogenic plasmids of Agrobacterium, the Ti-plasmids, carry genes that enable their bacterial host to catabolize opines. Opines are unusual amino acid derivatives that are only produced in crown gall tumours incited by oncogenic strains of Agrobacterium. The 2 opines, octopine and nopaline, are degraded by Agrobacterium strains carrying the octopine or the nopoline Ti-plasmid, respectively, to arginine and pyruvic
Rhizobium pusense is the main human pathogen in the genus Agrobacterium/Rhizobium.
Aujoulat, F; Marchandin, H; Zorgniotti, I; Masnou, A; Jumas-Bilak, E
Rhizobium pusense was recently described after isolation from the rhizosphere of chickpea. Multilocus sequence-based analysis of clinical isolates identified as Agrobacterium (Rhizobium) radiobacter demonstrated that R. pusense is the main human pathogen within Agrobacterium (Rhizobium) spp. Clinical microbiology of Agrobacterium (Rhizobium) should be considered in the light of recent taxonomic changes. PMID:25669878
Vitreoscilla hemoglobin promotes Salecan production by Agrobacterium sp. ZX09*
Chen, Yun-mei; Xu, Hai-yang; Wang, Yang; Zhang, Jian-fa; Wang, Shi-ming
Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. ZX09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan a promising material for applications in coagulation, lubrication, protection against acute liver injury, and alleviating constipation. In this study, we cloned the Vitreoscilla hemoglobin gene into a broad-host-range plasmid pCM158. Without antibiotic selection, there was negligible loss of the plasmid in the host Agrobacterium sp. ZX09 after one passage of cultivation. The expression of Vitreoscilla hemoglobin was demonstrated by carbon monoxide (CO) difference spectrum. The engineered strain Agrobacterium sp. ZX09 increased Salecan yield by 30%. The other physiological changes included its elevated respiration rate and cellular invertase activity. PMID:25367790
Agrobacterium T-DNA integration: molecules and models.
Tzfira, Tzvi; Li, Jianxiong; Lacroix, Benoît; Citovsky, Vitaly
Genetic transformation mediated by Agrobacterium involves the transfer of a DNA molecule (T-DNA) from the bacterium to the eukaryotic host cell, and its integration into the host genome. Whereas extensive work has revealed the biological mechanisms governing the production, Agrobacterium-to-plant cell transport and nuclear import of the Agrobacterium T-DNA, the integration step remains largely unexplored, although several different T-DNA integration mechanisms have been suggested. Recent genetic and functional studies have revealed the importance of host proteins involved in DNA repair and maintenance for T-DNA integration. In this article, we review our understanding of the specific function of these proteins and propose a detailed model for integration. PMID:15262410
Two-way chemical signaling in Agrobacterium-plant interactions.
Winans, S C
The discovery in 1977 that Agrobacterium species can transfer a discrete segment of oncogenic DNA (T-DNA) to the genome of host plant cells has stimulated an intense interest in the molecular biology underlying these plant-microbe associations. This attention in turn has resulted in a series of insights about the biology of these organisms that continue to accumulate at an ever-increasing rate. This excitement was due in part to the notion that this unprecedented interkingdom DNA transfer could be exploited to create transgenic plants containing foreign genes of scientific or commercial importance. In the course of these discoveries, Agrobacterium became one of the best available models for studying the molecular interactions between bacteria and higher organisms. One extensively studied aspect of this association concerns the exchange of chemical signals between Agrobacterium spp. and host plants. Agrobacterium spp. can recognize no fewer than five classes of low-molecular-weight compounds released from plants, and other classes probably await discovery. The most widely studied of these are phenolic compounds, which stimulate the transcription of the genes needed for infection. Other compounds include specific monosaccharides and acidic environments which potentiate vir gene induction, acidic polysaccharides which induce one or more chromosomal genes, and a family of compounds called opines which are released from tumorous plant cells to the bacteria as nutrient sources. Agrobacterium spp. in return release a variety of chemical compounds to plants. The best understood is the transferred DNA itself, which contains genes that in various ways upset the balance of phytohormones, ultimately causing neoplastic cell proliferation. In addition to transferring DNA, some Agrobacterium strains directly secrete phytohormones. Finally, at least some strains release a pectinase, which degrades a component of plant cell walls. PMID:1579105
Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea
Mohan B Singh; Prem L Bhalla
Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe
ORIGINAL PAPER Agrobacterium-mediated transformation of black cherry
ORIGINAL PAPER Agrobacterium-mediated transformation of black cherry for flowering control cherry is one of the most valuable hardwood species for cabinetry, furniture, and veneer. The goal of this study was to develop transgenic black cherry plants with reproductive sterility and enhanced insect
The Nucleotide Sequence of a Soybean Mosaic Virus Coat Protein-coding Region and Its Expression in Escherichia coli, Agrobacterium tumefaciens and Tobacco Callus
SUMMARY A DNA complementary to the T-terminal 1168 nucleotides of the genome of the N strain of soybean mosaic virus (SMV) has been cloned and sequenced, cDNA sequence and coat protein analyses indicate that the SMV coat protein-coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein. The coat
Cloning and targeted disruption, via Agrobacterium tumefaciens -mediated transformation, of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae
Katherine F. Dobinson; Sandra J. Grant; Seogchan Kang
A gene encoding a trypsin protease was isolated from a tomato isolate of Verticillium dahliae. The gene, designated VTP1, contains two introns and is predicted to encode a protein of 256 amino acids. The gene is present in V. dahliae isolates from different host plants and in V. albo-atrum; weakly hybridizing sequences are present in V. tricorpus. VTP1 cDNA sequences were
Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor? †
Moon, Yong-Sun; Donzelli, Bruno G. G.; Krasnoff, Stuart B.; McLane, Heather; Griggs, Mike H.; Cooke, Peter; Vandenberg, John D.; Gibson, Donna M.; Churchill, Alice C. L.
Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in ?Manps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in ?Manps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. ?Manps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the ?Manps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor. PMID:18502925
One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE
Untergasser, Andreas; Bijl, Gerben J. M.; Liu, Wei; Bisseling, Ton; Schaart, Jan G.; Geurts, René
Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes. PMID:23112864
Molecular analysis of Agrobacterium T-DNA integration in tomato reveals a role for left border sequence homology in most integration events.
Thomas, Colwyn M; Jones, Jonathan D G
Studies in several plants have shown that Agrobacterium tumefaciens T-DNA can integrate into plant chromosomal DNA by different mechanisms involving single-stranded (ss) or double-stranded (ds) forms. One mechanism requires sequence homology between plant target and ssT-DNA border sequences and another double-strand-break repair in which preexisting chromosomal DSBs "capture" dsT-DNAs. To learn more about T-DNA integration in Solanum lycopersicum we characterised 98 T-DNA/plant DNA junction sequences and show that T-DNA left border (LB) and right border transfer is much more variable than previously reported in Arabidopsis thaliana and Populus tremula. The analysis of seven plant target sequences showed that regions of homology between the T-DNA LB and plant chromosomal DNA plays an important role in T-DNA integration. One T-DNA insertion generated a target sequence duplication that resulted from nucleolytic processing of a LB/plant DNA heteroduplex that generated a DSB in plant chromosomal DNA. One broken end contained a captured T-DNA that served as a template for DNA repair synthesis. We propose that most T-DNA integrations in tomato require sequence homology between the ssT-DNA LB and plant target DNA which results in the generation of DSBs in plant chromosomal DNA. PMID:17574477
Start | View At a Glance | Author Index 219-5 Coupled Biotic and Abiotic Arsenite Oxidation Kinetics with Heterotrophic Soil Bacteria and a Poorly Crystalline
Sparks, Donald L.
strains of previously isolated bacteria, Alcaligenes faecalis, Agrobacterium tumefaciens, Pseudomonas of bacteria (A. faecalis, A. tumefaciens, P. fluorescens, and V. paradoxus) are characterized. The kinetic
Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression
E. R. Santarém; H. N. Trick; J. S. Essig; J. J. Finer
Sonication-assisted Agrobacterium-mediated transformation (SAAT) tremendously improves the efficiency of Agrobacterium infection by introducing large numbers of microwounds into the target plant tissue. Using immature cotyledons of soybean\\u000a as explants, we evaluated the effects of the following parameters on transient ?-glucuronidase (GUS) activity: cultivars, binary vectors, optical density of Agrobacterium during infection, duration of sonication treatment, co-culture conditions, length of explant
Transfer of virulence in vivo and in vitro in Agrobacterium
Allen Kerr; PIERRE MANIGAULT; JACQUES TEMPÉ
TRANSFER of virulence from a pathogenic to a non-pathogenic strain of Agrobacterium has been reported to occur in vivo1,2 and is due to the transfer of a plasmid which codes for virulence3,4. In the experimental conditions described, virulence transfer can be detected only after several weeks. The reason for this delay is not clear. The most likely explanations are (1)
Gene disruption in Trichoderma atroviride via Agrobacterium -mediated transformation
Susanne Zeilinger
A modified Agrobacterium-mediated transformation method for the efficient disruption of two genes encoding signaling compounds of the mycoparasite Trichoderma atroviride is described, using the hph gene of Escherichia coli as selection marker. The transformation vectors contained about 1 kb of 5' and 3' non-coding regions from the tmk1 (encoding a MAP kinase) or tga3 (encoding an a-subunit of a heterotrimeric G protein) target
Trojan Horse Strategy in Agrobacterium Transformation: Abusing MAPK Defense Signaling
Djamei Armin; Pitzschke Andrea; Nakagami Hirofumi; Rajh Iva; Heribert Hirt
Nuclear import of transfer DNA (T-DNA) is a central event in Agrobacterium transformation of plant cells and is thought to occur by the hijacking of certain host cell proteins. The T-DNA-associated virulence protein VirE2 mediates this process by binding to the nuclear import machinery via the host cell factor VIP1, whose role in plants has been so far unknown. Here
Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes
Masako Akutsu; Takuma Ishizaki; Hiroji Sato
An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase
Transcription of Agrobacterium rhizogenes A4 T-DNA
Brian H. Taylor; Frank F. White; Eugene W. Nester; Milton P. Gordon
Two distinct segments of transferred DNA (T-DNA) were detected in tumor tissue incited by Agrobacterium rhizogenes strain A4 on Nicotiana glauca. RNA from these tumors was examined for the presence of T-DNA encoded transcripts. Six genetic loci involved in virulence and tumor morphology have been identified by transposon mutagenesis: rol A-D on the leftward T-DNA (TL-DNA) and tms1 and 2
Agrobacterium mediated transformation of gypsophila ( Gypsophila paniculata L.)
Michal Moyal Ben Zvi; Amir Zuker; Marianna Ovadis; Elena Shklarman; Hagit Ben-Meir; Shamir Zenvirt; Alexander Vainstein
As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility\\u000a of this species' genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment\\u000a of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium
Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake
Vibha Gupta; G. Lakshmi Sita; M. S. Shaila; V. Jagannathan
Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection
77 FR 60431 - Agrobacterium radiobacter strains K84/Kerr-84 and K1026; Notice of Availability
Federal Register 2010, 2011, 2012, 2013, 2014
...pathogenic strains of Agrobacterium spp. Bacteriocins are peptides that are produced by certain bacteria, and are known to inhibit the growth of other bacteria. In accordance with 40 CFR 155.57, a registration review decision is the Agency's...
The VirE3 protein of Agrobacterium mimics a host cell function required for plant
Citovsky, Vitaly
-DNA nuclear import and subsequent expres- sion within the infected cell. The EMBO Journal (2005) 24, 428; Tzfira and Citovsky, 2000; Tzfira et al, 2000; Zupan et al, 2000). Agrobacterium infection has is exported into the host yeast
Efektivitas Agrobacterium mentransfer gen P5CS ke dalam kalus tebu klon PS 851
F. NURILMALA; Hayati MINARSIH
Summary Transformation of a P5CS gene construct into plant cells coupled with regeneration for transgenic plantlets should develop sugarcane tolerant to drought stress. The purpose of the research is to increase the efectivity and efficiency of Agrobacterium to transfer into sugarcane callus. Gene transfer into the plant cells was performed biologically using Agrobacterium. In this method, recombinant plasmid of pBI-P5CS
rol genes of Agrobacterium rhizogenes cucumopine strain: sequence, effects and pattern of expression
G. Serino; D. Clerot; J. Brevet; P. Costantino; M. Cardarelli
By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA-which we refer to as rola, ß and ?-were utilized to infect carrot
High efficiency Agrobacterium rhizogenes -mediated transformation of Saponaria vaccaria L. (Caryophyllaceae) using fluorescence selection
Janice F. Schmidt; Maria D. Moore; Lawrence E. Pelcher; Patrick S. Covello
A highly efficient and convenient method for the Agrobacterium rhizogenes-dependent production of transformed roots of Saponaria vaccaria L. (Caryophyllaceae) is described. The parameters tested and optimized include S. vaccaria cultivar, explant type, Agrobacterium rhizogenes strain and culture conditions. For cotransformation using additional recombinant T-DNA-containing A. rhizogenes strains, use of neomycin phosphotransferase and enhanced green fluorescent protein genes as selectable markers
Phylogeny of the Rhizobium-Allorhizobium-Agrobacterium clade supports the delineation of Neorhizobium gen. nov.
Mousavi, Seyed Abdollah; Österman, Janina; Wahlberg, Niklas; Nesme, Xavier; Lavire, Céline; Vial, Ludovic; Paulin, Lars; de Lajudie, Philippe; Lindström, Kristina
The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the "R. galegae complex" (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera. PMID:24581678
Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.
Zhang, Wan-Jun; Dewey, Ralph E; Boss, Wendy; Phillippy, Brian Q; Qu, Rongda
Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species. PMID:23242917
Transgenic indica rice lines, expressing Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1), exhibit enhanced resistance to major pathogens.
Sadumpati, Vijayakumar; Kalambur, Muralidharan; Vudem, Dashavantha Reddy; Kirti, Pulugurtha Bharadwaja; Khareedu, Venkateswara Rao
Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into commercial indica rice varieties by Agrobacterium-mediated genetic transformation. Transgenic rice plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in various transgenic rice lines. Transgenes NPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Homozygous transgenic rice lines expressing BjNPR1 protein displayed enhanced resistance to rice blast, sheath blight and bacterial leaf blight diseases. Rice transformants with higher levels of NPR1 revealed notable increases in plant height, panicle length, flag-leaf length, number of seeds/panicle and seed yield/plant as compared to the untransformed plants. The overall results amply demonstrate the profound impact of BjNPR1 in imparting resistance against major pathogens of rice. The multipotent BjNPR1, as such, seems promising as a prime candidate gene to fortify crop plants with durable resistance against various pathogens. PMID:23664883
Development of transgenic finger millet (Eleusine coracana (L.) Gaertn.) resistant to leaf blast disease.
Ignacimuthu, S; Ceasar, S Antony
Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R0 and R1 progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R1 lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance. PMID:22357211
High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)
Allan Richard Wenck; Michelle Quinn; Ross W. Whetten; Gerald Pullman; Ronald Sederoff
Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large- scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to
Expression of the GUS-gene in the monocot tulip after introduction by particle bombardment and Agrobacterium
A. Wilmink; B. C. E. van de Ven; J. J. M. Dons
Gene transfer to the monocotyledon tulip (Tulipa sp. L.) was obtained both by particle bombardment and Agrobacterium transformation. Using a Particle Delivery System, transient expression of the reporter gene for ßglucuronidase was demonstrated. It was shown that the CAMV 35S as well as the TR2' promoter were active in flower stem expiants. Various wildtype and disarmed Agrobacterium strains, harbouring the
Single genes from Agrobacterium rhizogenes influence plant development
Schmülling, T.; Schell, J.; Spena, A.
The combined expression of the rol A, B and C loci of Agrobacterium rhizogens Ri-plasmids establishes, in transgenic tobacco plants, a pathological state called hairy-root syndrome. However, when expressed separately they provoke distinct developmental abnormalities characteristic for each rol gene. Moreover, changes in their mode of expression obtained by replacing the promoters of the rol B and C genes with the cauliflower mosaic virus 35S promoter elicit new and distinct developmental patterns. These results indicate that the different rol gene products have either different targets, or have a qualitatively different effect on the same target. The target(s) must be involved in the control of plant development. Although each of the three rol genes are independently able to promote root formation in tobacco, efficient root initiation and growth is best achieved through the combined activities of more than a single rol gene. Models explaining the biological effects of A. rhizogenes-derived TL-DNA genes are discussed. Images PMID:15977331
Single genes from Agrobacterium rhizogenes influence plant development.
Schmülling, T; Schell, J; Spena, A
The combined expression of the rol A, B, and C loci of Agrobacterium rhizogenes Ri-plasmids establishes, in transgenic tobacco plants, a pathological state called hairy-root syndrome. However, when expressed separately they provoke distinct developmental abnormalities characteristic for each rol gene. Moreover, changes in their mode of expression obtained by replacing the promoters of the rol B and C genes with the cauliflower mosaic virus 35S promoter elicit new and distinct developmental patterns. These results indicate that the different rol gene products have either different targets, or have a qualitatively different effect on the same target. The target(s) must be involved in the control of plant development. Although each of the three rol genes are independently able to promote root formation in tobacco, efficient root initiation and growth is best achieved through the combined activities of more than a single rol gene. Models explaining the biological effects of A. rhizogenes-derived TL-DNA genes are discussed. PMID:15977331
Genetic transformation of Ascochyta rabiei using Agrobacterium-mediated transformation.
White, David; Chen, Weidong
In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 h was optimal for generating transformants, but increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried transfer-DNA (T-DNA) as determined by polymerase chain reaction (PCR) and the T-DNA integrations appeared to be random and in single copy as detected by Southern hybridization. Transformants remained resistant to hygromycin B in the absence of selection. Variations in colony morphology were observed in the presence of hygromycin B under different culture conditions, and a variety of altered phenotypes including reduced virulence were observed among 550 transformants. Inverse PCR was more efficient than TAIL-PCR in identifying flanking genomic sequences from T-DNA borders, and the possible causes are discussed. This transformation technique and recovery of flanking DNA using inverse PCR will provide a useful tool for genetic studies of A. rabiei. PMID:16369840
Agrobacterium-Mediated Transformation of the Recalcitrant Vanda Kasem's Delight Orchid with Higher Efficiency
Gnasekaran, Pavallekoodi; James Antony, Jessica Jeyanthi; Uddain, Jasim; Subramaniam, Sreeramanan
The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4?mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A600nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200?𝜇M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250?mg/L cefotaxime and 30?mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes. PMID:24977213
Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha using regenerating thalli.
Kubota, Akane; Ishizaki, Kimitsune; Hosaka, Masashi; Kohchi, Takayuki
The thallus, the gametophyte body of the liverwort Marchantia polymorpha, develops clonal progenies called gemmae that are useful in the isolation and propagation of isogenic plants. Developmental timing is critical to Agrobacterium-mediated transformation, and high transformation efficiency has been achieved only with sporelings. Here we report an Agrobacterium-mediated transformation system for M. polymorpha using regenerating thalli. Thallus regeneration was induced by cutting the mature thallus across the apical-basal axis and incubating the basal portion of the thallus for 3 d. Regenerating thalli were infected with Agrobacterium carrying binary vector that contained a selection marker, the hygromycin phosphotransferase gene, and hygromycin-resistant transformants were obtained with an efficiency of over 60%. Southern blot analysis verified random integration of 1 to 4 copies of the T-DNA into the M. polymorpha genome. This Agrobacterium-mediated transformation system for M. polymorpha should provide opportunities to perform genetic transformation without preparing spores and to generate a sufficient number of transformants with isogenic background. PMID:23291762
Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes
Takafumi Yoshikawa; Tsutomu Furuya
Hairy root culture of Ginseng (Panax ginseng) was established after roots were induced on callus following infection with Agrobacterium rhizogenes. The transformed cultures of ginseng could be subcultured as an axenic root culture in the absence of phytohormones, and grew with extensive lateral branches more rapidly than the ordinary cultured roots induced by hormonal control from ginseng callus. The hairy
Transformation of rapid cycling cabbage ( Brassica oleracea var. capitata) with Agrobacterium rhizogenes
Pierre Berthomieu; Lise Jouanin
Summary Genetically transformed cabbage (Brassica oleracea var. capitata) roots were obtained after inoculation with two engineered Agrobacterium rhizogenes strains, each harbouring a plant selectable marker gene in their T-DNA. Axenic root clones resistant to kanamycin or hygromycin B were established, most of which did not exhibit the phenotypic characteristics of Ri-transformed roots. Shoot regeneration was induced from roots after treatment
Technology Transfer Automated Retrieval System (TEKTRAN)
A simple expression assay for evaluation of gene constructs for input of traits into cotton cells (Gossypium hirsutum L.) using oncogenic binary Agrobacterium strains is presented. Explants from three commercial cotton varieties, representing diverse genotypes, exhibited tumor or root formation to ...
Abstract Sonication-assisted Agrobacterium-mediated transformation (SAAT) tremendously improves the effi-
Finer, John J.
of acetosyringone to the co-culture medium enhanced transient expression. No differences were observed when · Glycine max · Transient expression · SAAT · Transformation Introduction Agrobacterium provides one: optimization of transient expression Communicated by J. M. Widholm E. R. Santarém1 · H. N. Trick2 · J. S. Essig
Delayed Leaf Senescence in Tobacco Plants Transformed with tmr, a Gene for Cytokinin Production in Agrobacterium
Catherine M. Smart; Steven R. Scofield; Michael W. Bevan; Tristan A. Dyer
The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence. Tobacco plants were transformed with the Agrobacterium fumefaciens gene fmr, under the control of the soybean heat shock promoter HS6871. This gene encodes the enzyme iso- pentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis. After heat shock,
Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).
Mhatre, Minal
Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern. PMID:23179718
Use of Agrobacterium rhizogenes to create transgenic apple trees having an altered organogenic response to hormones
Claude Lambert; David Tepfer
The apple rootstock, M26, was genetically and phenotypically transformed using the Agrobacterium wild-type strain, A4. First, chimeric plants were obtained having transformed roots and normal aerial parts. Transformed plants were then produced through regeneration from transformed roots. Transformation was demonstrated by molecular hybridization and opine analysis. The effects of hormones on organogenesis was altered in transformants: cytokinins were required to
Agrobacterium Rhizogenes rolB and rolD Genes: Regulation and Involvement in Plant Development
Maria Maddalena Altamura
Rol genes belong to the T-DNA which is transferred by Agrobacterium rhizogenes into plant cells. Each of these genes affects plant development and is regulated by the host. In this review, after a brief\\u000a historical background, the most intriguing aspects of past and current research on rolB and rolD genes are highlighted and discussed.
Direct regeneration of transformed plants from stem fragments of potato inoculated with Agrobacterium rhizogenes
A. Dobigny; S. Tizroutine; C. Gaisne; R. Haďcour; L. Rossignol; G. Ducreux; D. Sihachakr
Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberisum L.). An overnight pretreatment of internodes with a-naphthaleneacetic acid prior to bacterial infection was found to strongly inhibit shoot formation. On the contrary, infection with bacterial strains enhanced the frequency of shoot formation, compared with the controls, except for the strain
Agrobacterium rhizogenes-mediated hairy root induction in Taraxacum officinale and analysis of sesquiterpene lactones
A. Mahesh; R. Jeyachandran
Hairy roots were efficiently induced from leaf and petiole explants of Taraxacum officinale after infection with the Agrobacterium rhizogenes strains A4 and ATCC 15834. The highest frequency of hairy root initiation was observed after transformation of leaf explants with the A4 strain. Hairy roots developed from leaf tissue produced more biomass than non-transformed roots. A quantitative study of sesquiterpene lactones
Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices
Cornelia Mrosk; Susanne Forner; Gerd Hause; Helge Kuster; Joachim Kopka; Bettina Hause
Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with
Agrobacterium rhizogenes mediated induction of apparently untransformed roots and callus in chrysanthemum
Monique F. van Wordragen; Pieter B. F. Ouwerkerk; Hans J. M. Dons
The agropine type Agrobacterium rhizogenes strain LBA9402 induced callus and roots on stems of greenhouse grown plants and on leaf disks of in vitro grown plantlets of chrysanthemum (Dendranthema grandiflora Tzvel.). In this callus and roots no opines were detected, nor were any of the other features of the 'hairy root' syndrome observed. Experiments aimed to identify the nature of
Effect of Agrobacterium rhizogenes on phenolic content of Mentha pulegium elite clonal line for phytoremediation applications
Sarah Strycharz; Kalidas Shetty
Mentha pulegium elite clonal line MPH-4 is known to contain high levels of endogenous phenolic compounds. These compounds are secondary metabolites of the phenylpropanoid pathway that aid in several metabolic processes, including stress response and hormone production. Agrobacterium rhizogenes is a soil bacterium utilized for its ability to transfer its Rol genes via the Ri plasmid on t-DNA. A. rhizogenes
Agrobacterium rhizogenes -mediated DNA transfer to Aesculu s hippocastanum L. and the regeneration of transformed plants
S. Zdravkovi?-Kora?; Y. Muhovski; P. Druart; D. ?ali?; L. Radojevi?
Hairy roots were induced from androgenic embryos of horse chestnut ( Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 µ
Induction of Hairy Roots in Gmelina arborea Roxb. Using Agrobacterium rhizogenes
Shrutika Dhakulkar; Sujata Bhargava; T. R. Ganapathi; V. A. Bapat
A b s t r a c t Seedling tissues of Gmelina arborea, a medicinally important tree species, were infected with wild type Agrobacterium rhizogenes strain ATTCC 15834, which led to the induction of hairy roots in 32% of the explants. Transgenic nature of the hairy roots was confirmed by PCR using rolB specific primers, and subsequently by Southern analysis
Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.
Bull, S E; Owiti, J A; Niklaus, M; Beeching, J R; Gruissem, W; Vanderschuren, H
Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop. PMID:20010938
Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant
Margie M. Paz; Huixia Shou; Zibiao Guo; Zhanyuan Zhang; Anjan K. Banerjee; Kan Wang
Summary Conditions affecting Agrobacterium-mediated transformation of soybean (Glycine max (L.) Merr.), including seed vigor of explant source, selection system, and cocultivation conditions, were investigated. A negative correlation between seed sterilization duration and seed vigor, and a positive correlation between seed vigor and regenerability of explants were observed in the study, suggesting that use of high vigor seed and minimum seed
Germline transformation of Shepherd's purse (Capsella bursa-pastoris) by the 'floral dip' method as a tool for evolutionary and developmental biology.
Bartholmes, Conny; Nutt, Pia; Theissen, Günter
Capsella bursa-pastoris is an attractive model system for evolutionary and developmental biology. To facilitate future studies on gene function, the 'floral dip' method was adapted to achieve germline transformation of C. bursa-pastoris. The GFP and BASTA-resistance (BAR (r)) genes were used as markers for screening or selecting, respectively, putative transgenic C. bursa-pastoris plants and the beta-glucuronidase (GUS) gene as well as the GFP gene for monitoring transgene expression level. We tested two Agrobacterium strains, LBA4404 and GV3101, for their ability to transform C. bursa-pastoris. In contrast to Arabidopsis thaliana, for which both strains were able to transform different ecotypes, only GV3101 gave satisfactory transformation rates with C. bursa-pastoris. Furthermore, we evaluated the effects of different concentrations of sucrose and the surfactant Silwet L-77 on the efficiency to generate transgenic C. bursa-pastoris plants and identified an efficient medium containing 10% (w/v) sucrose and 0.02-0.05% (v/v) Silwet L-77. Using Southern hybridisation, we confirmed the integration of the marker gene in the plant genome and the stable heredity of the introduced genes in the next generation. PMID:18164559
RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit.
Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom; Yeong, Wee Chien; Pillai, Vilasini
The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants. PMID:24950439
BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium
J. Song; J. M. Bradeen; S. K. Naess; J. P. Helgeson; J. Jiang
Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium.
Identification and Characterization of Plant Genes Involved in Agrobacterium -Mediated Plant Transformation by Virus-Induced Gene Silencing
Ajith Anand; Zarir Vaghchhipawala; Choong-Min Ryu; Li Kang; Keri Wang; Olga del-Pozo; Gregory B. Martin; Kirankumar S. Mysore
Genetic transformation of plant cells by Agrobacterium tu- mefaciens represents a unique case of trans-kingdom sex re- quiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant trans- formation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was
A versatile Agrobacterium-mediated transient gene expression system for herbaceous plants and trees.
Zheng, Lei; Liu, Guifeng; Meng, Xiangnan; Li, Yanbang; Wang, Yucheng
Plant transient expression is a powerful method used widely for the functional characterization of genes and protein production. In comparison with stable transformation, it has the advantages of being simple, quick, economical, and effective. In the present study, we developed a novel transient gene expression system based on Agrobacterium-mediated transformation. This system is simple and convenient and allows for high transient expression levels. Hyperosmotic pretreatment of plants significantly improved the transient expression in this system. Furthermore, other factors, including acetosyringone concentration, cocultivation time, and Agrobacterium cell density, significantly influenced transient expression efficiency. The results showed that this method is suitable for use with herbaceous plants (such as tobacco and Arabidopsis) and trees (such as birch, poplar, tamarisk, cork, willow, and aralia), suggesting that it may be applied widely in plant transient expression studies. PMID:22610523
pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation
Roger P. Hellens; E. Anne Edwards; Nicola R. Leyland; Samantha Bean; Philip M. Mullineaux
Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders
Factors Affecting the Agrobacterium -Mediated Transient Transformation of the Wetland Monocot, Typha latifolia
Rangaraj Nandakumar; Li Chen; Suzanne M. D. Rogers
An Agrobacterium-mediated transformation system, using transient transformation assays, was used to evaluate conditions influencing transformation for the wetland monocot Typha latifolia. These studies were aimed at the long-term objective of evaluating candidate genes for phytoremediation. The binary plasmid vector pCAMBIA1301\\/EHA105, containing the ß-glucuronidase coding sequence, was used in combination with factors known to affect transformation. These included callus age at
Genetic transformation of cauliflower ( Brassica oleracea L. var. Botrytis ) by Agrobacterium rhizogenes
Chantal David; Jacques Tempé
Cauliflower plantlets were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on cauliflower hypocotyls by agropine-type strains. Fewer roots were obtained with mannopine-type strains. In vitro cultures were established both from normal and hairy roots. Plant regeneration occured spontaneously from normal and transformed roots. Regenerated plants contained the same opines (if present) as root cultures. Some mannopine-positive
Agrobacterium-mediated transformation of `Alpine' Fragaria vesca, and transmission of transgenes to R1 progeny
K. M. Haymes; T. M. Davis
Agrobacterium-mediated transformation was used to stably introduce ?-glucuronidase (gus) and neomycin phosphotransferase (nptII) marker genes into `Alpine' Fragaria vesca FRA 197, a diploid (2n = 2x = 14) strawberry. R0 generation transformants derived from a single clump of kanamycin-resistant\\u000a callus were vegetatively propagated. The presence of the gus and nptII genes in five clonal R0 runner plants was confirmed by
Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes
Jie LiuEn; En Tao Wang; Da Wei Ren; Wen Xin Chen
Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this\\u000a strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and
An Agrobacterium-mediated transient gene expression system for intact leaves
Jyoti Kapila; Riet De Rycke; Marc Van Montagu; Geert Angenon
An efficient and reproducible Agrobacterium-mediated transient gene expression system for intact leaf tissue was developed. A high level of transient expression was observed when bacteria, which were pretreated in vir gene-inducing conditions, were infiltrated into complete leaf tissue. Histochemical ?-glucuronidase assays showed large transgene-expressing sectors comprising of up to 90% of the leaf area. As a consequence of infiltration, the
Introduction of hygromycin resistance in Lotus spp. through Agrobacterium rhizogenes transformation
Francesco Damiani; Elena Nenz; Francesco Paolocci; Sergio Arcioni
The speciesLotus corniculatus andL. tenuis were transformed with anAgrobacterium rhizogenes binary vector, conferring resistance to the antibiotic hygromycin. Transgenic plants recovered from both species were tested for the ability of leaf-derived calluses to grow in a hygromycin-supplemented medium. Molecular analysis showed the integration of the Ri T-DNA and of the gene for hygromycin resistance, with a high frequency of co-transformation.
Plant regeneration from hairy roots induced by infection with Agrobacterium rhizogenes in Crotalaria juncea L
A. Ohara; Y. Akasaka; H. Daimon; M. Mii
Hairy roots were induced from leaf segments of Crotalaria juncea, which is used as a green manure crop antagonistic to nematodes, by infection with a mikimopine type wild strain of Agrobacterium rhizogenes A13 (MAFF02-10266). These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog\\u000a (1\\/2MS) medium without phytohormones. The adventitious shoots were induced from 30% of
Purification of recombinant GFP produced by Agrobacterium -mediated transient expression in Nicotiana excelsior
Y. R. Sindarovska; Y. V. Sheludko; I. M. Gerasymenko; M. A. Bannikova; N. V. Kuchuk
Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient\\u000a protocol of Agrobacterium-mediated transient expression in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification scheme has been developed and includes\\u000a ammonium sulfate precipitation and Q-sepharose anion-exchange chromatography. It results in the obtaining of a
Agrobacterium rhizogenes rol genes induce productivity-related phenotypical modifications in "creeping-rooted" alfalfa types
Giovanna Frugis; Sofia Caretto; Luigi Santini; Domenico Mariotti
Summary \\u000aAgrobacterium rhizogenes rol genes were transferred individually or in combination into the forage legume Medicago sativa L. (alfalfa). Kanamycin resistant, neomycin phosphotransferase II positive plants showed the presence of the rol inserts in their genome. Phenotypical evaluation of transgenic populations indicated significant morphological alterations of the root system, stem number per plant and plant structure. A possible utilization of
Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation
R. Baranski; E. Klocke; G. Schumann
Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively\\u000a in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on\\u000a GFP fluorescence with a
Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA
I. Capone; L. Spanň; M. Cardarelli; D. Bellincampi; A. Petit; P. Costantino
Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol
Agrobacterium rhizogenes rol genes induce productivity-related phenotypical modifications in "creeping-rooted" alfalfa types.
Frugis, G; Caretto, S; Santini, L; Mariotti, D
Agrobacterium rhizogenes rol genes were transferred individually or in combination into the forage legume Medicago sativa L. (alfalfa). Kanamycin resistant, neomycin phosphotransferase II positive plants showed the presence of the rol inserts in their genome. Phenotypical evaluation of transgenic populations indicated significant morphological alterations of the root system, stem number per plant and plant structure. A possible utilization of these transgenics in breeding programs of the so-called "creeping-rooted" alfalfa strains is discussed. PMID:24185517
Expression of Agrobacterium rhizogenes auxin biosynthesis genes in transgenic tobacco plants
Valérie Gaudin; Lise Jouanin
Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate
Agrobacterium rhizogenes -mediated transformation of Taraxacum platycarpum and changes of morphological characters
M. H. Lee; E. S. Yoon; J. H. Jeong; Y. E. Choi
Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5±3.5%) as compared to stem (32.7±4.8%) or cotyledon (16.2±5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed
Regeneration of foreign genes co-transformed plants of Medicago sativa L by Agrobacterium rhizogenes
Deyang Lü; Xueyuan Cao; Shunxue Tang; Xia Tian
Gene encoding sulphur amino acid-rich protein (HNP) androl genes were transferred intoMedicago sativa L (alfalfa) mediated byAgrobacterium tumafeciens. Regeneration of transgenic plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues\\u000a were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency\\u000a in alfalfa. Production of co-transformed plants with
Horticultural characterization of Angelonia salicariifolia plants transformed with wild-type strains of Agrobacterium rhizogenes
Y. Koike; Y. Hoshino; M. Mii; M. Nakano
Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia ( Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS)
Regeneration of transgenic papaya plants via somatic embryogenesis induced by Agrobacterium rhizogenes
José Luis Cabrera-Ponce; Ariadne Vegas-Garcia; Luis Herrera-Estrella
AnAgrobacterium rhizogenes-mediated procedure for transformation of papaya (Carica papaya) was developed. Transgenic plants were obtained from somatic embryos that spontaneously formed at the base of transformed\\u000a roots, induced from leaf discs infected withA. rhizogenes. Transformation was monitored by autonomous growth of roots and somatic embryos, resistance to kanamycin, ?-glucuronidase\\u000a activity (GUS), and Southern hybridization analysis. Over one-third of the infected
Molecular basis for novel root phenotypes induced by Agrobacterium rhizogenes A4 on cucumber
Joëlle Amselem; Mark Tepfer
We have used the wild-type Agrobacterium rhizogenes strain A4 to induce roots on cucumber stem explants. Cultures of transformed roots obtained that were capable of hormone-autonomous growth could be grouped in three phenotypic classes. Of particular interest were extremely thick roots of a type not previously described. Characterization of the transferred DNA and of the expression of the corresponding genes
Regeneration of foreign genes co-transformed plants of Medicago sativa L by Agrobacterium rhizogenes.
Lü, D; Cao, X; Tang, S; Tian, X
Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of transgenic plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties. PMID:18726342
Cytosolic localization in transgenic plants of the rolC peptide from Agrobacterium rhizogenes.
Estruch, J J; Parets-Soler, A; Schmülling, T; Spena, A
The rolC gene of Agrobacterium rhizogenes codes for a peptide with an apparent molecular weight of approximately 20 kDa. Immunolocalization of the rolC peptide, in leaves of transgenic plants which are genetic mosaics for the expression of the rolC gene, is restricted to the phenotypically altered sectors. Subcellular fractionation of homogenates from 35S-rolC transgenic leaves shows the cytosolic localization of the rolC peptide. PMID:1884008
Agrobacterium rhizogenes -mediated transformation and plant regeneration of four Gentiana species
Ivana Mom?ilovi?; Dragoljub Grubiši?; Milan Koji?; Mirjana Neškovi?
Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium\\u000a rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species.\\u000a Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism.\\u000a Spontaneous bud initiation
Transformation of opium poppy ( Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724
Kayo Yoshimatsu; Koichiro Shimomura
Summary Transformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots
Effect of Agrobacterium rhizogenes T-DNA on alkaloid production in Solanaceae plants
E. Moyano; S. Fornalé; J. Palazón; R. M. Cusidó; M. Bonfill; C. Morales; M. T. Pińol
Inoculation of leaf sections of tobacco, Duboisia hybrid and Datura metel Solanaceae plants with A4 strain of Agrobacterium rhizogenes, induced transformed roots with the capacity to produce putrescine-derived alkaloids. In general, the hairy roots obtained showed two morphologies: typical hairy roots with high capacity to produce alkaloids and callus-like roots with faster growth capacity and lower alkaloid production. The aux1
Electroporation stimulates tranformation of freshly isolated cell suspension protoplasts of Solanum dulcamara by Agrobacterium
P. K. Chand; E. L. Rech; T. J. Golds; J. B. Power; M. R. Davey
Freshly isolated cell suspension protoplasts ofSolanum dulcamara were mixed withAgrobacterium rhizogenes, allowed to settle for 2 h, exposed to electrical pulses and further incubated for 2h. Two pulses of 600 V cm-1 for 2 msec separated by 15 sec produced transformed colonies at relative and absolute transformation frequencies which were 3–4 and 10 fold greater than those obtained by co-cultivation
Assessment of conditions affecting Agrobacterium -mediated soybean transformation using the cotyledonary node explant
Margie M. Paz; Huixia Shou; Zibiao Guo; Zhanyuan Zhang; Anjan K. Banerjee; Kan Wang
Conditions affecting Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merr.], including seed vigor of explant source, selection system, and cocultivation conditions, were investigated. A\\u000a negative correlation between seed sterilization duration and seed vigor, and a positive correlation between seed vigor and\\u000a regenerability of explants were observed in the study, suggesting that use of high vigor seed and minimum seed sterilization
High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)
NASA Technical Reports Server (NTRS)
Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)
Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.
From: Methods in Molecular Biology, vol. 344: Agrobacterium Protocols, 2/e, volume 2 Edited by: Kan Wang Humana Press Inc., Totowa, NJ
Citovsky, Vitaly
, as vectors for introducing recombinant DNA of interest into plant cells both for transient (2,3) and for sta, and Vitaly Citovsky Summary Agrobacterium most likely can transform virtually all known plant species, and experi- mental protocols for Agrobacterium-mediated genetic transformation of yet more plant species
Production of hairy root cultures and transgenic plants by Agrobacterium rhizogenes-mediated transformation.
Christey, Mary C; Braun, Robert H
Agrobacterium rhizogenes-mediated transformation results in the development of hairy roots at the site of infection. The production of hairy roots involves cocultivation of explants with A. rhizogenes and the subsequent selection of hairy roots on hormone-free medium. Hairy roots have many applications for research including secondary product production and for the study of biochemical pathways. In addition, transgenic plants regenerated from hairy roots often show an altered phenotype due to the presence of the rol genes. In this chapter we describe how to produce and grow hairy root cultures, how to regenerate shoots from these hairy roots, and how to conduct molecular analysis of these cultures. PMID:15310912
Genetic transformation of Ajuga multiflora bunge with Agrobacterium rhizogenes and 20-hydroxyecdysone production in hairy roots
Ok Tae Kim; M. Manickavasagm; Young Jun Kim; Mei Ran Jin; Kwang Soo Kim; Nak Sul Seong; Baik Hwang I
An efficient transformation system forAjuga multiflora Bunge was established by usingAgrobacterium rhizogenes strain A4. After inoculation with the bacteria, we obtained a number of hairy-root clones from micro-calli of the explant\\u000a petioles. One fast-growing line showed the highest production of 20-hydroxyecdysone (20-HE). PCR amplification of rooting\\u000a locus (rol) genes revealed that the left hand-transferred DNA of the root-inducing plasmid was
Stable Agrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection
Alvarez, José M.; Ordás, Ricardo J.
An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and ?-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15?mgL?1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600?nm) of 0.3 for 72?h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth. PMID:24376383
rol genes of Agrobacterium rhizogenes cucumopine strain: sequence, effects and pattern of expression.
Serino, G; Clerot, D; Brevet, J; Costantino, P; Cardarelli, M
By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA--which we refer to as rol alpha, beta and gamma--were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5' non-coding region of rol alpha and rol beta was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol beta in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol alpha-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter. PMID:7948887
Gene overexpression and gene silencing in Birch using an Agrobacterium-mediated transient expression system.
Zhang, Yan; Wang, Yucheng; Wang, Chao
As transient expression systems are effective methods for the functional characterization of genes, a transient gene expression and silencing system was developed for Betula platyphylla Suk (Chinese Birch). Firstly, the cinnamoyl-CoA reductase (CCR) gene and its promoter were isolated from Chinese Birch. The vectors for overexpression of CCR and RNAi-based silence of CCR were constructed and transformed into Agrobacterium, respectively. Overexpression and silence of the CCR gene were respectively, performed on Birch seedlings using an Agrobacterium-mediated transient expression system. The expression levels of CCR were determined using real-time PCR. The results showed that the transcripts of CCR notably increased in the Birch plants transformed with the CCR overexpression construct, and notably decreased in plants transformed with the silencing construct when compared with nontransgenic plants. These studies confirmed that this transient genetic transformation system works well on Birch plants, and can be used for the functional characterization of genes and protein production in Birch. PMID:22203479
Enhanced anthocyanin synthesis in foliage plant Caladium bicolor.
Li, S J; Deng, X M; Mao, H Z; Hong, Y
A protocol was developed for Agrobacterium-mediated genetic transformation of monocotyledon foliage plant Caladium bicolor cv. Jackie Suthers using leaf disc and petiole as the explants. The explants were inoculated with Agrobacterium strain LBA4404 harboring a binary vector with the maize anthocyanin regulatory gene Lc under the control of the cauliflower mosaic virus promoter. Callus formation was induced in MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (6-BA), 0.1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/l sucrose and kanamycin 50 mg/l for selection. Resistant calli were induced for shoot generation in MS medium with 2 mg/l 6-BA and 0.2 mg/l alpha-naphthaleneacetic acid. As much as 10% of the explants gave rise to kanamycin-resistant shoots with our procedure. Transformed plants had enhanced anthocyanin accumulation in the roots, leaves and stems (epidermis and vascular bundles). Integration of the transgene into the host genome was confirmed by genomic Southern blot hybridization, and RNA blot hybridization analysis indicated that the expression of the transgene correlated with anthocyanin accumulation. This investigation illustrates the utility of anthocyanin regulatory genes in the genetic manipulation of the color of foliage plants. It also supports the premise that the Lc gene can be used as a powerful non-destructive cell autonomous visual marker in a wide variety of plants, as exemplified by the perfect symmetrical half-green/half-red plant presumably derived from the symmetrical division of one transgenic and one non-transgenic precursor meristematic cell. PMID:15372198
Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology.
Ishizaki, Kimitsune; Chiyoda, Shota; Yamato, Katsuyuki T; Kohchi, Takayuki
Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants. PMID:18535011
Influences of Agrobacterium rhizogenes strains, plant genotypes, and tissue types on the induction of transgenic hairy roots in Vitis species
Technology Transfer Automated Retrieval System (TEKTRAN)
Agrobacterium rhizogenes-mediated induction of transgenic hairy roots was previously demonstrated in Vitis vinifera L. and a few other Vitis species. In this study, 13 Vitis species, including V. aestivalis, V. afghanistan, V. champinii, V. doaniana, V. flexuosa, V. labrusca, V. nesbittiana, V. pal...
[Transformation of embryogenic Calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) mediated by agrobacterium].
Li, Da-Xu; Zhang, Jie; Zhao, Jian; Zhang, Yi; Li, Li; Liu, Su-Jun; Chen, Fei; Yang, Zhi-Rong
Formation of embryogenic calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) was induced from mature seeds as explants, and proliferated on MS medium containing 2,4-D 5.0 mg/L and KT 0.05 mg/L. An effective and stable callus regeneration system was established by optimizing the culture conditions (Tables 1, 2 and Fig.2). After the calli were subcultured 8 weeks, selected the whitish-yellow-coloured compact nodular calli that transformed with plasmid pCAMBIA1304 carrying hygromycin resistance gene (hptII) and Pseudomonas pseudoalcaligenes insecticidal protein gene (ppIP), which was mediated by an Agrobacterium strain EHA105. Resistant plants were obtained after hygromycin selection (Figs.3, 4). Some important factors that affect the transformation efficiency were studied, which included selection pressure, time of embryogenic calli proliferation, OD value of Agrobacterium suspension, temperature, medium and time of co-cultivation, and concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformation plant regeneration. This research is the first successful genetic transformation of Elymus sibiricus L. cv. Chuancao No.2 mediated by Agrobacterium. PMID:16477130
Pleiotropic effect of the insertion of the Agrobacterium rhizogenes rolD gene in tomato ( Lycopersicon esculentum Mill.)
P. Bettini; S. Michelotti; D. Bindi; R. Giannini; M. Capuana; M. Buiatti
The Agrobacterium rhizogenes rolD gene, coding for an ornithine cyclodeaminase involved in the biosynthesis of proline from ornithine, has been inserted in Lycopersicon esculentum cv Tondino with the aim of studying its effects on plant morphological characters including pathogen defense response. The analysis of plants transgenic for rolD did not show major morphological modifications. First generation transgenic plants however were
Agrobacterium-mediated transformation of two Serbian potato cultivars (Solanum tuberosum L. cv. Dragacevka and cv. Jelica)
Technology Transfer Automated Retrieval System (TEKTRAN)
An efficient protocol for Agrobacterium-mediated transformation of Serbian potato cultivars Dragacevka and Jelica, enabling the introduction of oryzacystatin genes OCI and OCII, was established. Starting with leaf explants a two-stage transformation protocol combining procedures of Webb and Wenzler...
Arabidopsis VIRE2 INTERACTING PROTEIN2 Is Required for Agrobacterium T-DNA Integration in Plants W
Citovsky, Vitaly
transformation but not for transient trans- formation. Assays based upon a promoter-trap vectorArabidopsis VIRE2 INTERACTING PROTEIN2 Is Required for Agrobacterium T-DNA Integration in Plants W,a Vitaly Citovsky,b and Kirankumar S. Mysorea,1 a Plant Biology Division, Samuel Roberts Noble Foundation
Efficient production of Agrobacterium rhizogenes -transformed roots and composite plants for studying gene expression in coffee roots
E. Alpizar; E. Dechamp; S. Espeout; M. Royer; A. C. Lecouls; M. Nicole; B. Bertrand; P. Lashermes; H. Etienne
The possibility of rapid validation and functional analysis of nematode resistance genes is a common objective for numerous species and particularly for woody species. In this aim, we developed an Agrobacterium rhizogenes-mediated transformation protocol for Coffea arabica enabling efficient and rapid regeneration of transformed roots from the hypocotyls of germinated zygotic embryos, and the subsequent production of composite plants. The
Resurrection of an ancestral gene: functional and evolutionary analyses of the Ng rol genes transferred from Agrobacterium to Nicotiana
Seishiro Aoki
The Ng rol genes, which have high similarity in sequence to the rol genes of Agrobacterium rhizogenes, are present in the genome of untransformed plants of Nicotiana glauca. It is thought that bacterial infection resulted in the transfer of the Ng rol genes to plants early in the evolution of the genus Nicotiana, since several species in this genus contain
The use of Agrobacterium rhizogenes transformed roots to obtain transgenic shoots of the apple rootstock Jork 9
Nathalie Pawlicki-Jullian; Monika Sedira; Margareta Welander
The apple rootstock Jork 9 was transformed using four different Agrobacterium rhizogenes virulent strains. The mannopine strain 8196 gave the best results in the production of chimeric plants compared to two agropine strains (A4 and 15834) and one cucumopine strain. Shoot regeneration was performed on both untransformed and transformed roots. Optimum combination and concentration of thidiazuron (TDZ) and a-naphtaleneacetic acid
Variation in structure and plant regeneration of Agrobacterium rhizogenes transformed and control roots of the potato cv. Bintje
M. P. Ottaviani; J. H. N. Schel; Ch. H. Hänisch ten Cate
Agrobacterium rhizogenes transformed and control roots of the tetraploid potato cv. Bintje were compared. Transformed roots were obtained after infection by A. rhizogenes 15834 or 1855. Both in leaf and stem segments, more roots were formed at the basal side of the segments, indicative for a polarity in root formation. As compared to control roots the transformed roots are characterized
Root induction on several Solanaceae species by Agrobacterium rhizogenes and the determination of root tropane alkaloid content
Eva Knopp; André Strauss; Walter Wehrli
Using Agrobacterium rhizogenes, roots were induced on explants of 24 different Solanaceae species and established as in vitro cultures. Some of the root clones produced tropane alkaloids at levels similar to roots of the corresponding intact plants and maintained these levels over several passages.
[Expression of Mortierella isabellina delta6-fatty acid desaturase gene in gamma-linolenic acid production in transgenic tobacco].
Li, Ming-Chun; Liu, Li; Hu, Guo-Wu; Xing, Lai-Jun
Gamma-linolenic acid (GLA, C18:3delta6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18:2delta9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose ( Oenotheera spp.), borage (Borago officinalis) and etc. delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking Xba I and Bgl II restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains delta6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28 degrees C, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco ( Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100 microg/mL kanamycin. Plants were maintained in axionic culture under controlled conditions. Total nucleic acids were extracted and purified from anti-kanamycin transgenic tobacco and were analysed by PCR. 48 out of 80 transgenic plants were positive, in other words, transformation efficiency is 60% . This shows that Mortierella isabellina D6D gene is transformed into tobacco. Genomic DNA from PCR positive transgenic tobacco plants was digested with Hind III restriction enzyme and fractionated by agarose gel electrophoresis. Southern blotting was performed with strandard procedures for vacuum transfer of nucleic acids to nylon membrane. The probe was delta6-fatty acid desaturase gene from M. isabellina, which was labeled with DIG-dUTP via random-primed labeling. Hybridization and immumological detection were carried out the kit of DIG detection. The result shows single hybridizing bands in each of the transgenic tobacco plants DNA, but no hybridization was observed to non-transgenic tobacco. This indicates that delta6-fatty acid desaturase gene is integrated into the genome of transgenic tobacco. To provide further evidence that the introduction of the M. isabellina cDNA into the tobacco genome was responsible for the novel desaturation products, total RNA was isolated from GLA-positive transgenic tobacco plants via both PCR and Southern blotting and separated by electrophoresis through 1% formaldehyde agarose gel. Northern blotting including probe labeling, hybridization and detection was the same as Southern blotting in operation approach. A positive hybridization signal of identical mobility was obtained from RNA isolated from the transgenic tobacco plants, but not from the control tobacco plant. At last, total fatty acids extracted from the positive transgenic tobacco were analyzed by gas chromatography (GC) of methyl esters to confirm the transgenic tobacco containing a functional delta6-fatty acid desaturase gene. The result shows that two peaks were observed in the chromatogram of FAMes. GLA and octadecatetraenoic acid (OTA, C18:4delta188.8.131.52) respectively have 19.7% and 3.5% of the total fatty
Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus
D. Luth; G. A. Moore
Factors influencing transformation frequencies using the Agrobacterium-mediated protocol developed for Citrus seedling internodal stem segments in this laboratory were evaluated, with particular emphasis on decreasing the numbers of\\u000a ``escape'' shoots produced. Although the use of a wild-type ``shooty'' Agrobacterium strain allowed relatively high frequencies of ?-glucuronidase positive (GUS+) shoots to be produced, none of the shoots were free of wild-type
Cre\\/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre
Annette C. Vergunst; Paul J. J. Hooykaas
The Cre\\/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene. To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector
Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay
I. J. Puddephat; H. T. Robinson; T. M. Fenning; D. J. Barbara; A. Morton; D. A. C. Pink
In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers TL sequences carrying the rol genes
Transport of gamma-butyrobetaine in an Agrobacterium species isolated from soil.
Nobile, S; Deshusses, J
An Agrobacterium sp. isolated from soil by selective growth on gamma-butyrobetaine (gamma-trimethylaminobutyrate) as the sole source of both carbon and nitrogen has been shown to possess an inducible transport system for this growth substrate. This transport system has a Kt of 0.5 microM and a maximal velocity of 3.8 nmol/min per mg (dry weight). The influx of gamma-butyrobetaine is optimal at pH 8.5 and operates against a concentration gradient. The transport system shows a high specificity for trimethylamine carboxylic acid molecules of defined chain length. gamma-Butyrobetaine uptake was significantly reduced in osmotically shocked cells and a gamma-butyrobetaine binding activity was detected in the crude shock fluid. This suggests a transport mechanism involving a periplasmic gamma-butyrobetaine binding protein. PMID:3782024
Salicortin: a repeat-attack new-mechanism-based Agrobacterium faecalis beta-glucosidase inhibitor.
Zhu, J; Withers, S G; Reichardt, P B; Treadwell, E; Clausen, T P
Salicortin, a natural product abundant in most members of the Salicaceae family, is a mechanism-based inactivator of Agrobacterium faecalis beta-glucosidase. Inactivation is delayed in the presence of competitive inhibitors, thereby demonstrating the requirement for an enzyme-bound salicortin before inactivation. Product studies suggest that inactivation proceeds via a quinone methide intermediate formed by the fragmentation of the aglycone of salicortin while it is bound to the enzyme. Tryptic digest and HPLC/MS studies confirm the role of quinone methide attack and also show that the enzyme undergoes multiple modifications. In addition, when the inactivation was run in the presence of a mutant inactive form of the enzyme, HPLC/MS analyses clearly showed no modification of the mutant enzyme, demonstrating that the quinone methide does not exist in free solution and suggesting that inactivation is active-site directed. PMID:9601065
Salicortin: a repeat-attack new-mechanism-based Agrobacterium faecalis beta-glucosidase inhibitor.
Zhu, J; Withers, S G; Reichardt, P B; Treadwell, E; Clausen, T P
Salicortin, a natural product abundant in most members of the Salicaceae family, is a mechanism-based inactivator of Agrobacterium faecalis beta-glucosidase. Inactivation is delayed in the presence of competitive inhibitors, thereby demonstrating the requirement for an enzyme-bound salicortin before inactivation. Product studies suggest that inactivation proceeds via a quinone methide intermediate formed by the fragmentation of the aglycone of salicortin while it is bound to the enzyme. Tryptic digest and HPLC/MS studies confirm the role of quinone methide attack and also show that the enzyme undergoes multiple modifications. In addition, when the inactivation was run in the presence of a mutant inactive form of the enzyme, HPLC/MS analyses clearly showed no modification of the mutant enzyme, demonstrating that the quinone methide does not exist in free solution and suggesting that inactivation is active-site directed. PMID:9601065
Agrobacterium-mediated transformation of chickpea with alpha-amylase inhibitor gene for insect resistance.
Ignacimuthu, S; Prakash, S
Chickpea is the world's third most important pulse crop and India produces 75% of the world's supply. Chickpea seeds are attacked by Callosobruchus maculatus and C. chinensis which cause extensive damage. The alpha-amylase inhibitor gene isolated from Phaseolus vulgaris seeds was introduced into chickpea cultivar K850 through Agrobacterium-mediated transformation. A total of 288 kanamycin resistant plants were regenerated. Only 0.3% of these were true transformants. Polymerase chain reaction (PCR) analysis and Southern hybridization confirmed the presence of 4.9 kb alpha-amylase inhibitor gene in the transformed plants. Western blot confirmed the presence of alpha-amylase inhibitor protein. The results of bioassay study revealed a significant reduction in the survival rate of bruchid weevil C. maculatus reared on transgenic chickpea seeds. All the transgenic plants exhibited a segregation ratio of 3:1. PMID:17006016
Screening of plant cell culture collection for efficient host species for Agrobacterium-mediated transient expression.
Sindarovska, Y R; Golovach, I S; Belokurova, V B; Gerasymenko, I M; Sheludko, Y V; Kuchuk, N V
Agrobacterium-mediated transient expression is an approach for short-time expression of heterologous genes in plant systems. During the last decade transient expression was regarded as a potent protocol for high scale production of foreign proteins in plants including pharmaceutically valuable proteins. In vitro grown plant cell cultures represent a suitable system for accumulation of heterologous proteins under controlled conditions. Since host characteristics may strongly influence transient expression efficiency, we performed screening of undifferentiated cell cultures for transient expression ability using GUS as a reporter. Analysis of 248 plant species belonging to 49 families from the National Germplasm Bank of the World Flora of the Institute of Cell Biology and Genetic Engineering (Kyiv, Ukraine) allowed for selection of about 50 plant species exhibiting detectable beta-glucuronidase activity. PMID:25181853
Genetic transformation of Glaphimia glauca by Agrobacterium rhizogenes and the production of norfriedelanes.
Náder, Blanca L; Cardoso Taketa, Alexandre T; Iturriaga, Gabriel; Pereda-Miranda, Rogelio; Villarreal, Ma Luisa
Transformed root cultures of Galphimia glauca (Malpighiaceae) were established by infecting cotyledons and hypocotyls with Agrobacterium rhizogenes ATCC 15 834. Cotyledon-derived cell lines were grown in liquid B5 nutrient medium without phytohormones and have shown the typical hairy roots phenotype over two years of continuous subculturing. PCR analysis was used to confirm the integration of rol A and rol C genes into the plant genome. The transformed cultures synthesized three major norfriedelanes, the new glaucacetalins A-C (1-3), which were secreted into the nutrient medium. The structural elucidation of these in vitro produced metabolites was performed by the application of high resolution NMR techniques that proved them to be triterpenoids related to the known galphimines, the sedative principles of this plant species. These results suggest the possibility of further biotechnological exploration of sedative friedelane biosynthesis by in vitro plant organ cultures. PMID:15643554
Natural plant genetic engineer Agrobacterium rhizogenes: role of T-DNA in plant secondary metabolism.
Chandra, Sheela
Agrobacterium rhizogenes is a natural plant genetic engineer. It is a gram-negative soil bacterium that induces hairy root formation. Success has been obtained in exploring the molecular mechanisms of transferred DNA (T-DNA) transfer, interaction with host plant proteins, plant defense signaling and integration to plant genome for successful plant genetic transformation. T-DNA and corresponding expression of rol genes alter morphology and plant host secondary metabolism. During transformation, there is a differential loss of a few T-DNA genes. Loss of a few ORFs drastically affect the growth and morphological patterns of hairy roots, expression pattern of biosynthetic pathway genes and accumulation of specific secondary metabolites. PMID:22048847
Natural genetic transformation by agrobacterium rhizogenes . Annual flowering in two biennials, belgian endive and carrot
Limami; Sun; Douat; Helgeson; Tepfer
Genetic transformation of Belgian endive (Cichorium intybus) and carrot (Daucus carota) by Agrobacterium rhizogenes resulted in a transformed phenotype, including annual flowering. Back-crossing of transformed (R1) endive plants produced a line that retained annual flowering in the absence of the other traits associated with A. rhizogenes transformation. Annualism was correlated with the segregation of a truncated transferred DNA (T-DNA) insertion. During vegetative growth, carbohydrate reserves accumulated normally in these annuals, and they were properly mobilized prior to anthesis. The effects of individual root-inducing left-hand T-DNA genes on flowering were tested in carrot, in which rolC (root locus) was the primary promoter of annualism and rolD caused extreme dwarfism. We discuss the possible adaptive significance of this attenuation of the phenotypic effects of root-inducing left-hand T-DNA. PMID:9765539
Agrobacterium rhizogenes-mediated transformation and regeneration of the Apocynum venetum.
Jia, Haiyan; Zhao, Bing; Wang, Xiaodong; Wang, Yuchun
A system for the Agrobacterium rhizogenes-mediated transformation and plant regeneration of A. venetum has been developed. The highest transformation frequency was 100%, achieved by using strain LBA9402 with root explants. The highest density of hairy roots reached 22 when root explants transformed by R1000 cultured in the dark. Adventitious shoots were obtained from profusely branched, fast-growing (type PBF) hairy roots, and the adventitious shoot induction frequency was 20%. Regenerated shoots rooted easily on hormone-free 1/2 MS solid medium in 2 weeks. Approximately 1/3 regenerated plants derived from hairy roots exhibited prolific roots with shortened internodes. Whereas other regenerated plants showed another phenotype: long intemodes, strong stems, and fleshy blades. However, all regenerated plants displayed a relatively fast development procedure and stronger than the aseptic seedlings. Polymerase chain reaction (PCR) analyses confirmed the hairy root lines and regenerated plants were induced by A. rhizogenes. PMID:19149183
Sequential monitoring of transgene expression following Agrobacterium-mediated transformation of rice.
Saika, Hiroaki; Nonaka, Satoko; Osakabe, Keishi; Toki, Seiichi
Although Agrobacterium-mediated transformation technology is now used widely in rice, many varieties of indica-type rice are still recalcitrant to Agrobacterium-mediated transformation. It was reported recently that T-DNA integration into the rice genome could be the limiting step in this method. Here, we attempted to establish an efficient sequential monitoring system for stable transformation events by visualizing stable transgene expression using a non-destructive and highly sensitive visible marker. Our results demonstrate that click beetle luciferase (ELuc) is an excellent marker allowing the observation of transformed cells in rice callus, exhibiting a sensitivity >30-fold higher than that of firefly luciferase. Since we have previously shown that green fluorescent protein (GFP) is a useful visual marker with which to follow transient and/or stable expression of transgenes in rice, we constructed an enhancer trap vector using both the gfbsd2 (GFP fused to the N-terminus of blasticidin S deaminase) and eluc genes. In this vector, the eluc gene is under the control of the Cauliflower mosaic virus 35S minimal promoter, while the gfbsd2 gene is under the control of the full-length rice elongation factor gene promoter. Observation of transformed callus under a dissecting microscope demonstrated that the level of ELuc luminescence reflected exclusively stable transgene expression, and that both transient and stable expression could be monitored by the level of GFP fluorescence. Moreover, we show that our system enables sequential quantification of transgene expression via differential measurement of ELuc luminescence and GFP fluorescence. PMID:23026817
AgarTrap: a simplified Agrobacterium-mediated transformation method for sporelings of the liverwort Marchantia polymorpha L.
Tsuboyama, Shoko; Kodama, Yutaka
The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha. PMID:24259681
Identification of T-DNA in the root-inducing plasmid of the agropine type Agrobacterium rhizogenes 1855
L. Spano; M. Pomponi; P. Costantino; G. M. S. Slogteren; J. Tempé
The region of the virulence plasmid of the agropine type Agrobacterium rhizogenes 1855 (pRi-1855) which is transferred to plant cells upon infection (T-DNA) has been identified by means of Southern blot hybridizations with the T-DNA of the mannopine type A. rhizogenes 8196. The presence in the plant genome of the pRi-1855 sequences thus identified is demonstrated in carrot roots derived
Auxin regulates the promoter of the root-inducing rolB gene of Agrobacterium rhizogenes in transgenic tobacco
Christophe Maurel; Jean Brevet; Hélčne Barbier-Brygoo; Jean Guern; Jacques Tempé
The regulation in tobacco of the rolB and rolC promoters of Agrobacterium rhizogenes pRi 1855 TL-DNA was studied by using the ß-glucuronidase (GUS) reporter system in transgenic plants. A 20- to 100-fold increase of GUS activity was selectively induced by auxin in rolB-GUS transformed mesophyll protoplasts, whereas this auxin-dependent increase was only 5-fold in rolC-GUS protoplasts. Moreover, both gene fusions
Precise Incubation Period for the Agrobacterium mediated Transformation Efficiency in Potato (Solanum tuberosum L.) cvs. Cardinal and Atlas
S. R. Sarker; M. Hossain; F. Shirin
Precise incubation period facilitates significant response to Agrobacterium mediated genetic transformation of potato cultivars Atlas and Cardinal. The protocol yielded an average transformation rate of 58.4% for internodal explants compared to 38% for leaf explants in Atlas while 47.6% for intenodal explants compared to 27.6% for leaf explants in potato cultivar Cardinal. Highest survival rates and transient GUS activity was
Promoters of the rolA, B, and C Genes of Agrobacterium rhizogenes Are Differentially Regulated in Transgenic Plants
Thomas Schmiilling; Jeff Schell; Angelo Spena
Chimeric genes containing the 8-glucuronidase reporter gene under the control of the rolA, B, and C promoters of Agrobacterium rhizogenes are expressed in a regulated manner in transgenic plants. The intergenic region separating the rolB and C genes represents a bidirectional promoter. This bidirectional promoter regulates transcription for both genes in a similar fashion in aerial organs of the plants,
Modification of phenotype in Belgian endive ( Cichorium intybus ) through genetic transformation by Agrobacterium rhizogenes : conversion from biennial to annual flowering
Li-Yan Sun; Gérard Touraud; Constance Charbonnier; David Tepfer
Belgian endive (Cichorium intybus) was genetically transformed usingAgrobacterium rhizogenes to insert wild type root-inducing, leftward, transferred DNA (Ri TL-DNA) into the nuclear genome. Transformed root cultures gave rise to plants (R0 generation) having the transformed phenotype described for other species, including increased branching, sterility, annual\\u000a flowering and wrinkled leaves. Transformation circumvented the need for vernalization in order to flower, but
Horizontal gene transfer: regulated expression of a tobacco homologue of the Agrobacterium rhizogenes rolC gene
Alain D. Meyer; Takanari Ichikawa; Frederick Meins
A tobacco homologue (trolC) of the rolC gene of the Agrobacterium rhizogenes Ri-plasmid was cloned and sequenced from Nicotiana tabacum L. cv. Havana 425. The coding region of trolC is similar in sequence (69–87% for DNA and 5489% for the deduced amino acid sequence) to rolC genes of the agropine, mannopine, and mikimopine strains of Ri-plasmids and the N. glauca
Hairy root induction and plant regeneration of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao via Agrobacterium rhizogenes -mediated transformation
Y. Q. Zhou; H. Y. Duan; C. E. Zhou; J. J. Li; F. P. Gu; F. Wang; Z. Y. Zhang; Z. M. Gao
The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from\\u000a hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The
Genetic transformation of Harpagophytum procumbens by Agrobacterium rhizogenes : iridoid and phenylethanoid glycoside accumulation in hairy root cultures
Renata Gr?bkowska; Aleksandra Królicka; Wojciech Mielicki; Marzena Wielanek; Halina Wysoki?ska
A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types\\u000a of explants and acetosyringone (AS) (200 ?M). The highest frequency of transformation (over 50% of explants forming roots\\u000a at the infected sites after 6 weeks of culture on Lloyd and McCown
The non-conserved region of cucumopine-type Agrobacterium rhizogenes T-DNA is responsible for hairy root induction
M. C. Failla; F. Maimone; A. De Paolis; P. Costantino; M. Cardarelli
The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part [1,2]. We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains. We show here that the
Expression and inheritance of inserted markers in binary vector carrying Agrobacterium rhizogenes -transformed potato ( Solanum tuberosum L.)
R. G. F. Visser; A. Hesseling-Meinders; E. Jacobsen; H. Nijdam; B. Witholt; W. J. Feenstra
Transgenic shoots were regenerated from eight diploid potato hairy root clones obtained by transformation with Agrobacterium rhizogenes harboring next to its wild-type Ri-plasmid a binary vector containing the neomycin phosphotransferase and the ß-glucuronidase genes. The plants exhibited the typical hairy root phenotype. Of the plants isolated, 58% were tetraploid and 38% were diploid. Flowering and tuberization was much better in
Agrobacterium mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence
Indra Sandal; Uksha Saini; Benoît Lacroix; Amita Bhattacharya; Paramvir Singh Ahuja; Vitaly Citovsky
Tea is one of the major crops in Asia and Africa, and its improvement by genetic modification is important for economy of\\u000a many tea-producing regions. Although somatic embryos derived from cotyledon explants have been transformed with Agrobacterium, the leaves of several commercially important tea cultivars have remained recalcitrant to transformation, largely due to\\u000a bactericidal effect of polyphenols that are exuded
Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA.
Capone, I; Spanň, L; Cardarelli, M; Bellincampi, D; Petit, A; Costantino, P
Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol genes A, B and C as a single insert (rolA + B + C) and by rolB alone provided an extended segment beyond its 5' non-coding region was included in the construction. A depression of rooting capability was caused by the inclusion of rolC together with rolB (rolB + C). In all cases co-inoculation with the Agrobacterium carrying TR-DNA-borne auxin genes was necessary for root induction since none of the rol constructions was in itself capable of eliciting any response; an exceeding majority of these roots were however shown to contain rol genes but no TR-DNA. Rooting was also elicited if rol constructions were co-inoculated with a strain carrying TL-DNA genes 13 and 14 (ORF13 + 14) instead of the TR-DNA strain. These roots were shown to contain both rol genes and ORF13 + 14. Striking differences in growth properties were shown by roots containing different complements of TL-DNA genes. Typical hairy root traits, high growth rate, branching and, most noticeably, absence of geotropism, were shown by roots containing rolB alone, while roots with rolA + B + C were geotropic as normal carrot roots. Hairy root traits were conferred to rolA + B + C roots by the concomitant presence of ORF13 + 14 and by the addition of auxin to the culture medium. A model is presented which attempts to rationalize the growth patterns by assigning interplaying roles to the various TL-DNA genes involved. PMID:2562759
Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H(+)-pyrophosphatase gene (SbVPPase) from Sorghum bicolor.
Anjaneyulu, Ediga; Reddy, Palle Surender; Sunita, Merla Srilakshmi; Kishor, Polavarapu B Kavi; Meriga, Balaji
A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80-90% homology at the nucleotide and 85-95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50-70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25-100% in transgenics, while malondialdehyde (MDA) showed a 2-4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5-2.5-fold higher Na(+) and 0.4-0.8-fold higher K(+) levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress. PMID:24877670
Plastid marker gene excision in greenhouse-grown tobacco by agrobacterium-delivered Cre recombinase.
Tungsuchat-Huang, Tarinee; Maliga, Pal
Uniform transformation of the thousands of plastid genome (ptDNA) copies in a cell is driven by selection for plastid markers. When each of the plastid genome copies is uniformly altered, the marker gene is no longer needed. Plastid markers have been efficiently excised by site-specific recombinases expressed from nuclear genes either by transforming tissue culture cells or introducing the genes by pollination. Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the bar (au) gene, confers a visual aurea leaf phenotype; thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The bar (au) marker-free plastids are transmitted through seed to the progeny. PCR and DNA gel blot (Southern) protocols to confirm transgene integration and plastid marker excision are also provided herein. PMID:24599855
D-glucosaminate dehydratase from Agrobacterium radiobacter. Physicochemical and enzymological properties.
Iwamoto, R; Imanaga, Y; Soda, K
The bacterial distribution of D-glucosaminate dehydratase [EC 220.127.116.11] was investigated and Agrobacterium radiobacter (IAM 1526) was found to have the highest enzyme activity. The enzyme was formed inducibly in a glycerol-urea medium by D-glucosamine, D-galactosamine, and D-glucosamine, but not by D-mannosamine. The enzyme purified from the cells grown in the glucosamine-glycerol-urea medium was shown to be homogeneous by ultracentrifugation. The molecular weight was determined to be about 66,000 by the sedimentation equilibrium method, and 72,800 by the gel permeation chromatography low-angle light scattering method. The pH optimum is 8.3-9.0. The enzyme catalyzed the dehydration of D-glucosaminate (relative activity: 100, Km: 2.8 mM), D-galactosaminate (31.5, 5.0 mM), D-mannosaminate (17.5, 29 mM), D-threonine (5.1, 4.8 mM), D-serine (3.2, 0.026 mM), and L-serine (1.1, ND), but not L-threonine. The reverse reaction does not occur. The enzyme is inhibited by typical inhibitors of pyridoxal 5'-phosphate enzymes, such as L'penicillamine, and also by carbonyl reagents, thiol reagents, divalent metals, and several D-amino acids and D-amino sugars. PMID:7068563
Effective Immobilization of Agrobacterium sp. IFO 13140 Cells in Loofa Sponge for Curdlan Biosynthesis.
Martinez, Camila Ortiz; Ruiz, Suelen Pereira; Nogueira, Marcela Tiemi; Bona, Evandro; Portilho, Márcia; Matioli, Graciette
Curdlan production by Agrobacterium sp. IFO13140 immobilized on loofa sponge, alginate and loofa sponge with alginate was investigated. There was no statistically-significant difference in curdlan production when the microorganism was immobilized in different matrices. The loofa sponge was chosen because of its practical application and economy and because it provides a high stability through its continued use. The best conditions for immobilization on loofa sponge were 50 mg of cell, 200 rpm and 72 h of incubation, which provided a curdlan production 1.50-times higher than that obtained by free cells. The higher volumetric productivity was achieved by immobilized cells (0.09 g/L/h) at 150 rpm. The operating stability was evaluated, and until the fourth cycle, immobilized cells retained 87.40% of the production of the first cycle. The immobilized cells remained active after 300 days of storage at 4 °C. The results of this study demonstrate success in immobilizing cells for curdlan biosynthesis, making the process potentially suitable for industrial scale-up. Additional studies may show a possible contribution to the reduction of operating costs. PMID:25946555
Relationship between Spatial and Genetic Distance in Agrobacterium spp. in 1 Cubic Centimeter of Soil
Vogel, J.; Normand, P.; Thioulouse, J.; Nesme, X.; Grundmann, G. L.
The spatial and genetic unit of bacterial population structure is the clone. Surprisingly, very little is known about the spread of a clone (spatial distance between clonally related bacteria) and the relationship between spatial distance and genetic distance, especially at very short scale (microhabitat scale), where cell division takes place. Agrobacterium spp. Biovar 1 was chosen because it is a soil bacterial taxon easy to isolate. A total of 865 microsamples 500 ?m in diameter were sampled with spatial coordinates in 1 cm3 of undisturbed soil. The 55 isolates obtained yielded 42 ribotypes, covering three genomic species based on amplified ribosomal DNA restriction analysis (ARDRA) of the intergenic spacer 16S-23S, seven of which contained two to six isolates. These clonemates (identical ARDRA patterns) could be found in the same microsample or 1 cm apart. The genetic diversity did not change with distance, indicating the same habitat variability across the cube. The mixing of ribotypes, as assessed by the spatial position of clonemates, corresponded to an overlapping of clones. Although the population probably was in a recession stage in the cube (103 agrobacteria g?1), a high genetic diversity was maintained. In two independent microsamples (500 ?m in diameter) at the invasion stage, the average genetic diversity was at the same level as in the cube. Quantification of the microdiversity landscape will help to estimate the probability of encounter between bacteria under realistic natural conditions and to set appropriate sampling strategies for population genetic analysis. PMID:12620832
Agrobacterium rhizogenes-Mediated Transformation of the Parasitic Plant Phtheirospermum japonicum
Ishida, Juliane K.; Yoshida, Satoko; Ito, Masaki; Namba, Shigetou; Shirasu, Ken
Background Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood. Methodology/Principal Findings We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6-dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation. Conclusions We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocols described here will allow functional analysis of genes involved in plant parasitism. PMID:21991355
An protocol for genetic transformation of Catharanthus roseus by Agrobacterium rhizogenes A4.
Zhou, Mei-Liang; Zhu, Xue-Mei; Shao, Ji-Rong; Wu, Yan-Min; Tang, Yi-Xiong
Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. To study and extract catharanthine and other metabolites from C. roseus, a technique was developed for producing hairy root cultures. In this study, the Agrobacterium rhizogenes A4 was induced in the hairy roots from leaf explants, and the concentration of antibiotics (100 mg/L kanamycin) was elucidated for selection after transformation. The polymerase chain reaction amplification of rol genes results revealed that transgenic hairy roots contained rol genes from the root induced (Ri)-plasmid. Catharanthine from C. roseus hairy roots was separated and analyzed using high-performance liquid chromatography. Over-expression of CrOrca3 (octadecanoid-responsive Catharanthus AP2/ERF domain), and cytohistochemical staining methods were used to validate transgenic hairy roots from C. roseus. Hairy root culture of C. roseus is a valuable approach for future efforts in the metabolic engineering of terpenoid indole alkaloids in plants. PMID:22328251
Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters.
Lee, M H; Yoon, E S; Jeong, J H; Choi, Y E
Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5+/-3.5%) as compared to stem (32.7+/-4.8%) or cotyledon (16.2+/-5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared. The frequency of adventitious shoot formation from transformed roots was much higher (88.5+/-9.8%) than that of non-transformed roots (31.7 +/-9.5%) on hormone-free medium. Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots. The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants. Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. The differences observed may reflect the modification of morphological root characters by introduction of rol genes. PMID:14986056
An efficient protocol for genetic transformation of watercress (Nasturtium officinale) using Agrobacterium rhizogenes.
Park, Nam Il; Kim, Jae Kwang; Park, Woo Tae; Cho, Jin Woong; Lim, Yong Pyo; Park, Sang Un
Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties. To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01-0.02 ?mol/g of DW) and 4-methoxyglucobrassicin (0.06-0.18 ?mol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06-0.21 ?mol/g of DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive agent (indole-3-carbinol). PMID:21161399
Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation.
Georgiev, Milen I; Ludwig-Müller, Jutta; Alipieva, Kalina; Lippert, Annemarie
An efficient protocol for the establishment of transformed root culture of Verbascum xanthophoeniceum using sonication-assisted Agrobacterium rhizogenes-mediated transformation is reported. Only 10 days after the inoculation with A. rhizogenes ATCC 15834 and 45 s ultrasound exposure, hairy roots appeared on 75% of the Verbascum leaves. Ten hairy root lines were isolated, although only half of them were free of bacterial contamination and started growing when excised from mother explants. The transgenic nature of the most vigorously growing hairy root clones (VX1 and VX6) was confirmed by polymerase chain reaction. Under submerged cultivation both hairy root clones accumulated high biomass amounts (12.8 and 14.3 g L(-1), respectively) and significant amounts of bioactive phenylethanoid glycoside verbascoside (over 6-times more than in mother plant leaves). LC-APCI-MS analyses confirmed verbascoside accumulation in hairy root clones along with three other phenylethanoid glycosides (forsythoside B, leucosceptoside B and martynoside) and an iridoid glycoside aucubin. This is the first report on the induction of hairy roots of Verbascum plants. PMID:21184229
In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss.
Sharafi, Ali; Sohi, Haleh Hashemi; Mirzaee, Hooman; Azadi, Pejman
In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including ?-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis. PMID:25320471
Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation.
Lu, Chaofu; Kang, Jinling
Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products. PMID:17899095
An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.).
Pandey, Sonika; Mishra, Avinash; Patel, Manish Kumar; Jha, Bhavanath
Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-?M 6-benzyladenine and 2.0-?M ?-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-?M acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient ?-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed ?-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and ?-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin. PMID:23813408
The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.
Sharifi, Sara; Sattari, Taher Nejad; Zebarjadi, Alireza; Majd, Ahmad; Ghasempour, Hamidreza
We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10-14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 ?g g(-1) of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L(-1) naphthaleneacetic acid and 2 mg L(-1) 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots. PMID:24554840
The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium -mediated transformation of flax ( Linum usitatissimum )
Frédéric Lamblin; Aurélie Aimé; Christophe Hano; Isabelle Roussy; Jean-Marc Domon; Bart Van Droogenbroeck; Eric Lainé
In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants,\\u000a we have adapted the selectable marker system PMI\\/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 184.108.40.206) that converts mannose-6-phosphate, an inhibitor of glycolysis, into\\u000a fructose-6-phosphate (glycolysis
Seasonal Fluctuations and Long-Term Persistence of Pathogenic Populations of Agrobacterium spp. in Soils
Krimi, Z.; Petit, A.; Mougel, C.; Dessaux, Y.; Nesme, X.
Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence. No pathogenic Agrobacterium strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 × 105 pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer. PCR amplifications of a vir sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter. The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines. The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils. An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil. In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids. In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains. These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions. PMID:12089015