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Pectins are major components of the primary plant cell wall, which functions as the primary barrier against pathogens. Pectin methylesterases (PMEs) catalyze the demethylesterification of the homogalacturonan domains of pectin in the plant cell wall. Their activity is regulated by PME inhibitors (PMEIs). Here, we provide evidence that the pectin methylesterase-inhibiting protein GhPMEI3 from cotton (Gossypium hirsutum) functions in plant responses to infection by the fungus Verticillium dahliae. GhPMEI3 interacts with PMEs and regulates the expression of a specific fungal polygalacturonase (VdPG1). Ectopic expression of GhPMEI3 increased pectin methyl esterification and limited fungal disease in cotton, while also modulating root elongation. Enzymatic analyses revealed that GhPMEI3 efficiently inhibited the activity of cotton GhPME2/GhPME31. Experiments using transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the GhPMEI3 gene under the control of the CaMV 35S promoter revealed that GhPMEI3 inhibits the endogenous PME activity in vitro. Moreover, the enhanced resistance to V. dahliae was associated with altered VdPG1 expression. Virus-induced silencing of GhPMEI3 resulted in increased susceptibility to V. dahliae. Further, we investigated the interaction between GhPMEI3 and GhPME2/GhPME31 using inhibition assays and molecular docking simulations. The peculiar structural features of GhPMEI3 were responsible for the formation of a 1:1 stoichiometric complex with GhPME2/GhPME31. Together, these results suggest that GhPMEI3 enhances resistance to Verticillium wilt. Moreover, GhPMEI3-GhPMEs interactions would be needed before drawing the correlation between structure-function and are crucial for plant development against the ever-evolving fungal pathogens.
Plant cell walls are highly heterogeneous extracellular structures that contain three major classes of polysaccharides—cellulose, hemicellulose, and pectin—as well as phenolic compounds and cell wall proteins (Dedeurwaerder et al., 2009; Nguyen et al., 2016). Pectin is the most complex of these polysaccharides; it is located in the primary cell wall and constitutes the principal component of the middle lamella (Johansson et al., 2002). It is composed of three fractions of galacturonans: homogalacturonan (HG), rhamnogalacturonan-I, and minor amounts of rhamnogalacturonan-II (Caffall and Mohnen, 2009). HG is a chain composed of α-1,4-linked-d-GalUA units; it is synthesized in the Golgi and then secreted to the cell wall in a highly methyl esterified (up to 80%) form (Sterling et al., 2001). There, pectin methylesterases (PMEs; EC 22.214.171.124), which are cell wall enzymes, deesterify HG, releasing methanol and protons (Dedeurwaerder et al., 2009).
In cotton (Gossypium hirsutum), PMEs are encoded by a large multigene family. Based on their structure, PMEs have been classified into two enzyme types, both of which possess a conserved PME domain (Pfam 01095; Sénéchal et al., 2015). PMEs that contain a PRO region at the N-terminal end of the catalytic domain have been designated as type I (Micheli, 2001). The PRO region shares similarity with the PME inhibitor domain (Pfam 04043; Pelloux et al., 2007) that is presumed to be cleaved from the mature catalytic portion of the protein during secretion (Dorokhov et al., 2006; Dedeurwaerder et al., 2009). The PRO domain mediates the retention of PMEs in the Golgi compartment, and regulates the enzymatic activity of PME through a posttranslational mechanism (Wolf et al., 2009; Sénéchal et al., 2015). PMEs lacking the PRO region are type II enzymes and are produced by bacteria, fungi, Physcomitrella patens, and higher plants (Dedeurwaerder et al., 2009).
Plant PMEs play an important role in preparing the substrate for processing by polygalacturonases and pectate lyases during cell wall metabolism (Johansson et al., 2002). Plant PMEs are typically multigene-encoded isoenzymes that are involved in plant defense against pathogens (Raiola et al., 2011; Bellincampi et al., 2014; Bethke et al., 2014; Lionetti et al., 2017). It is thought that plant PMEs remove methylesters in a block-wise fashion (single-chain mechanism) generating continuous, deesterified GalUA residue domains (Willats et al., 2001). However, PMEs also exist in bacteria and in fungal pathogens. There, the PME mode of action for removing methylesters is random (multiple-chain mechanism; Kohn et al., 1983; Limberg et al., 2000), except for the fungal PME from Trichoderma reesei (Markoviě and Kohn, 1984).
Moreover, the three-dimensional structures of the plant and microbial PMEs are very similar, as the proteins are all composed of right-handed β-helices. A cleft in the surface of the β-helix structure constitutes the active region of the protein. In plant PMEs, the active site cavity is relatively shallow, with no obvious steric barriers, while the active site cavity of the Erwinia chrysanthemi PME is relatively deep (Jenkins et al., 2001). Since PMEs contain neither an α/β hydrolase fold nor a catalytic Ser-His-Asp triad, they are considered a new type of hydrolases. They appear to be carboxylate hydrolases containing two Asp residues in the active site (Jenkins et al., 2001; Johansson et al., 2002).
PME inhibitors (PMEIs) belong to a large multigene-encoded protein family, PF04043, that includes invertase inhibitors (INHs). PMEIs harbor four conserved Cys residues engaged in disulfide bridge formation, and an up-and-down four-helical bundle fold, which is similar to INHs (Di Matteo et al., 2005; Lionetti et al., 2015). PME activity is efficiently regulated by endogenous PMEIs. PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable complex with 1:1 stoichiometry (Lionetti et al., 2014). A high-resolution three-dimensional structure of a PME-PMEI complex revealed that PMEI covers the pectin-binding cleft of PME and conceals the putative catalytic sites. Thus, it prevents the substrate from approaching the cleft (Di Matteo et al., 2005; Hothorn et al., 2010).
It is thought that most of the important interacting residues are conserved in plant PMEs, but not in fungal and bacterial enzymes; hence, PMEIs are ineffective against microbial enzymes (Di Matteo et al., 2005). Nevertheless, the PMEI from pepper (Capsicum annuum) exhibits antifungal activity against Fusarium oxysporum f. sp. matthiole, Alternaria brassicicola, and Botrytis cinerea (An et al., 2008). Virus-induced gene silencing of CaPMEI1 results in enhanced susceptibility to Xanthomonas campestris pv vesicatoria (An et al., 2008). The effectiveness of PMEIs in controlling endogenous PME activity was first demonstrated in planta by overexpressing AtPMEI-1 and AtPMEI-2 in Arabidopsis. The disease symptoms caused by B. cinerea and Pectobacterium carotovorum were considerably reduced in transgenic plants (Lionetti et al., 2007). Later, kiwi (Actinidia deliciosa) PMEI was shown to limit fungal infections caused by Bipolaris sorokiniana, F. graminearum, and Claviceps purpurea in durum wheat (Triticum aestivum; Volpi et al., 2011, 2013). Moreover, PMEIs counteract the action of plant PMEs and affect the susceptibility of plants to viruses. For example, Tobacco mosaic virus symptoms are reduced, and its systemic movement is limited in tobacco (Nicotiana tabacum) that heterologously expresses kiwi PMEI. Moreover, the overexpression of AtPMEI-2 in Arabidopsis results in a substantial reduction in its susceptibility to the Turnip vein-clearing virus (Lionetti et al., 2014).
PMEI was first discovered in the kiwi fruit (Balestrieri et al., 1990), and later detected in other plants, including Arabidopsis, pepper, and tomato (Solanum lycopersicum; Raiola et al., 2004; An et al., 2008; Reca et al., 2012). Disulfide bond formation and ionic interactions may be important for PMEI stability. In particular, subtle pH changes in the microenvironment greatly impact the stability of partially buried ionic interactions (Bonavita et al., 2016). For instance, the interaction between the tomato PME and the kiwi PMEI grows rapidly weaker with an increase in pH, and no complex is observed at pH 8.0 (Jolie et al., 2010). Further, at a pH >7.0, the inhibition of banana (Musa nana), carrot (Daucus carota), and strawberry (Fragaria × ananassa) PMEs by the kiwi PMEI is also reduced (D'Avino et al., 2003). Since the enzymatic activity of PME is regulated by pH, the stability of the PMEI-PME interaction is also affected by pH (Denès et al., 2000).
Cotton is an important cash crop worldwide, and is widely cultivated for the economic value of its fibers (Xu et al., 2011). The cotton fiber is an ideal model for plant cell elongation and cell wall biogenesis studies, because of its highly elongated structure (Kim et al., 2001). Cotton Verticillium wilt is caused by Verticillium dahliae, a soil-borne plant pathogenic fungus. The disease is difficult to control in cotton as the hyphae reside in the vascular (xylem) tissues of the plant. Moreover, this pathogen can overwinter as a mycelium within perennial hosts or in tubers, bulbs, or seeds, as well as in other propagative organs of the plant. Moreover, resting structures known as microsclerotia support the durable vegetative resting structures of Verticillium, which survive in the soil for many years (Fradin and Thomma, 2006). Therefore, Verticillium wilt leads to severe cotton yield loss each year and represents a major concern for cotton producers (Gao et al., 2011). However, the physiology of plant resistance against Verticillium is largely unexplored. Moreover, even less is known about the crucial fungal components involved in its pathogenicity. Historically, the roles of cell wall degrading enzymes, such as endopolygalacturonase, have attracted much attention (Fradin and Thomma, 2006). Recently, the Ve locus has been bred in various tomato cultivars and was shown to be responsible for resistance to V. dahliae (Kawchuk et al., 2001). Nevertheless, not much is known about the genetic and molecular mechanisms that underlie cotton resistance to Verticillium infection.
In the current study, we investigated whether cotton GhPMEI3 is active against GhPME2 (a type I enzyme) and GhPME31 (a type II enzyme). To this end, we expressed and purified the three proteins and performed in vitro inhibition studies. Enzyme inhibition assays and a PMEI-PME interaction analysis indicated that GhPMEI3 very efficiently inhibits these cotton PMEs. Furthermore, heterologous expression of GhPMEI3 in Arabidopsis lowered pectin levels in the cell wall, markedly enhancing the plant's resistance to the V. dahliae fungal pathogen. In silico protein modeling, docking, and electrostatic charge distribution analyses were also performed to clarify the structural basis of the PMEI-PME interaction. The current study highlights the regulation of the PMEI-PME interaction and pectin deesterification in plants. Since fungal pathogens are unable to utilize methyl esterified pectin, and this modification reduces the ability of other cell wall-degrading enzymes to hydrolyze pectin, the PMEI-PME interaction enhances plant resistance to pathogens.
Bioinformatics Analysis of GhPMEI3, GhPME2, and GhPME31
The inferred domain architecture of GhPMEI3 was characterized using multiple alignment and bioinformatics approaches. GhPMEI3 was predicted to be encoded by a 720-bp open reading frame encoding a 240-amino acid protein with no signal peptide. The theoretical Mr of GhPMEI3 is 25.96 kD (that of the purified protein was 43.96 kD, with the protein linked to an 18-kD Trx tag, S tag, and His tag), with a pI of 5.07. The Mrs of GhPME2 and GhPME31 are 82.96 kD and 61.53 kD (including a 26-kD GST-tag from the vector), with isoelectric points of 9.23 and 7.52, respectively.
To functionally characterize GhPMEI3, multiple sequence alignment with various PMEIs was performed (Fig. 1). Similarly to other PMEIs, the structure of GhPMEI3 is mainly helical, with four long antiparallel helices (α1–4) arranged in a classic up-and-down four-helical bundle (Fig. 1; Di Matteo et al., 2005). GhPMEI3 has four Cys residues that form two disulfide bridges, one of which connects the α2 and α3 helices, stabilizing the interior of the bundle. The other disulfide bridge connects helices αa and αb in the N-terminal region (Di Matteo et al., 2005). PMEIs belong to the PF04043 family (Pfam database, http://pfam.xfam.org/) and share several structural properties with INH; however, their target enzymes are not related (Di Matteo et al., 2005; Lionetti et al., 2015).
GhPME2 is predicted to encode a 25-amino acid signal peptide (SignalP 4.1 server), while GhPME31 does not encode a signal peptide (Fig. 2). GhPME2 and GhPME31 show similarity at the primary structure level with Arabidopsis PMEs (AtPME38 and AtPME31). GhPME2 contains an N-terminal extension PRO region and shares similarity with PMEIs, as well as a PME catalytic domain; however, GhPME31 contains only the PME catalytic domain (Fig. 2).
A phylogenetic tree for the amino acid sequences of GhPMEI3, GhPME2, and GhPME31 was generated, using sequences of proteins that had been functionally characterized in plants and deposited in the NCBI database (Fig. 3). To construct the phylogenetic tree, the neighbor-joining approach was employed. The phylogenetic analysis indicated that the inferred GhPMEI3 protein shared 63% identity with the PMEI from Vitis vinifera, while GhPME2 and GhPME31 shared 100% and 92% identity with PME2 and PME31, respectively, from the cacao tree (Theobroma cacao).
Expression of GhPMEI3, GhPME2, and GhPME31 in Response to Stress
GhPMEI3 expression may possibly be induced as part of the immune response against V. dahliae. Therefore, the expression of GhPMEI3, GhPME2, and GhPME31 was evaluated following fungal infection in the presence of the methyl jasmonate (MeJA) and ethylene (ET) defense hormones, under hydrogen peroxide (H2O2) stress, and in response to oligogalacturonide (OG) treatment. Specific primers were used to assess the transcription of these genes via RT-qPCR. The expression of all three genes was up-regulated in response to the different stress factors (Fig. 4). The expression of GhPMEI3 and GhPME31 peaked 1 d postinoculation with Vd991 (a virulent strain of V. dahliae), while the expression of GhPME2 peaked 3 d postinoculation (Fig. 4A). GhPME31, GhPME2, and GhPMEI3 reached their maximum expression 0.5, 12, and 24 h following treatment with MeJA, respectively, but the expression of GhPMEI3 and GhPME31 dropped to near-basal levels after 9 h (Fig. 4B). Upon ET treatment, GhPME2 and GhPME31 were up-regulated after 0.5 h; the expression of GhPME2 continued to increase until it reached a maximum at 30 h, while the expression of GhPME31 decreased; the expression of GhPMEI3 increased slowly until peaking at 30 h (Fig. 4C). The expression of GhPMEI3, GhPME2, and GhPME31 in response to H2O2 showed different trends. The levels of GhPMEI3 and GhPME31 remained unchanged, while the expression of GhPME2 increased at 0.5 h, after which it plateaued, and finally reached a maximum at 24 h (Fig. 4D). GhPMEI3 expression is induced by exogenous OGs, an elicitor involved in various defense responses (Lionetti et al., 2017). Accordingly, the incubation of cotton seedlings with OGs caused a sustained increase in GhPMEI3 expression and a transient increase in GhPME2 expression, but did not affect GhPME31 levels. This increase in expression was detectable as early as 1.5 h after treatment, and basal transcript levels were observed after 24 h (Fig. 4E). These results were consistent with a previous report that showed that AtPMEI expression is strictly regulated by JA and ET (Lionetti et al., 2017). In that study, after induction with B. cinerea, JA and ET positively regulated AtPMEI10 and AtPMEI11 expression, while ET negatively regulated AtPMEI12 expression.
Pectin-derived OGs are constituents of the cell wall. Modifications of pectins, such as pectin methyl esterification, may alter the plant defense response. The degree of pectin methyl esterification (DM) can influence the type of OGs present, in turn affecting plant resistance to pathogens, on account of their elicitor activity (Lionetti et al., 2007). Moreover, differences in methylester distribution are hypothesized to affect the type of OG elicitor released during plant-pathogen interactions (Wiethölter et al., 2003). Thus, it is possible that GhPMEI3 may affect the DM that is required for eliciting a defense response in the cotton plant.
Purification and Characterization of Recombinant GhPMEI3, GhPME2, and GhPME31 Proteins
Recombinant GhPMEI3, GhPME2, and GhPME31 proteins were produced in the Escherichia coli BL21 DE3 strain (see "Materials and Methods"). The molecular masses of the three purified proteins, as determined by SDS-PAGE, were close to the values inferred from their gene sequences (Fig. 5A). The identity of the purified GhPMEI3 protein was further confirmed by western blotting, with its His6-tag recognized by a His-tag antibody. Moreover, the identities of GhPME2 and GhPME31 proteins were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Fig. 5, B and C). Mascot search results revealed that the two proteins share a high amino acid sequence similarity with two pectinesterases: PE = 2 (SV = 1; Mascot score 33,384; 7 peptide matches; accession no. R9QQU5; Fig. 5B) and PE = 4 (SV = 1; Mascot score 18,432; 6 peptide matches; accession no. A0A0D2RVP9), respectively (Fig. 5C). Hence, the two purified proteins were confirmed as GhPME2 and GhPME31.
Inhibition of the PME and Antifungal Activity of GhPMEI3
Purified GhPME2 and GhPME31 proteins were used to characterize the PME-inhibiting capacity of GhPMEI3. Thus, 1 mU of PMEs was incubated for 30 min in the presence of various amounts (0.5–4 μg) of purified GhPMEI3 (Fig. 5D). As shown, 0.5 μg of purified GhPMEI3 inhibited both GhPME2 and GhPME31 (∼10% inhibitory activity). The inhibitory activity was 40% when 2 μg of purified GhPMEI3 was used in the assay, and reached 80% when 4 μg of purified GhPMEI3 was used. Further, biochemical assays revealed that GhPMEI3 exhibits a similar inhibitory activity toward GhPME2 and GhPME31 (Fig. 5E).
The purified GhPMEI3 inhibited the growth and development of fungal pathogens in vitro. At 50 μg/mL, it inhibited the mycelial growth of V. dahliae, F. oxysporum f. sp. vasinfectum, and B. cinerea (Fig. 6, A–C). Moreover, it inhibited spore germination of F. oxysporum f. sp. vasinfectum (Fig. 6, D–F) and V. dahliae (Fig. 6, G–I), at a concentration of 250 μg/mL.
Altered Growth of Transgenic Arabidopsis Plants and Enhanced Resistance to V. dahliae
The growth and development of transformed plants grown on solid Murashige and Skoog medium were clearly different from the wild type, especially with respect to root length and the number of lateral roots. A significant 50% increase in the root area and 13% increase in root length were observed in transgenic plants when compared to wild-type plants (Fig. 7, A–C). The elongation zone of the root cells in transgenic Arabidopsis plants appeared obviously elongated when compared with the wild-type plants (Fig. 7, D–G). It has been established that methyl esterification of pectin is highest during the cell expansion stage and decreases in the cell elongation phase (Goldberg, 1984), playing a key role in plant growth. In transgenic Arabidopsis plants, the activities of PMEs are specifically inhibited by PMEIs, and the plants develop longer roots when grown in a vertical position, which is primarily attributed to lengthening in the cell elongation phase (Lionetti et al., 2007). Further, the fine-tuning of the DM of the homogalacturonan backbone seems crucial for plant growth and development (Hocq et al., 2017). Consistently, our observations suggested that an increased pectin DM elongated the cell expansion phase and positively affected plant growth (Supplemental Fig. S1).
The resistance of transgenic Arabidopsis plants to V. dahliae was next investigated. Leaves from the wild-type plant and two transgenic lines were inoculated with V. dahliae conidia, and the lesion areas were monitored 5 d postinoculation. Substantial differences were observed in the areas of expanding lesions in transformed lines and wild-type plants. Compared with the wild type, the lesion areas were reduced in lines 3 and 5 (Fig. 8, A and B). Proteins from wild-type and transgenic plants were extracted to examine the inhibition of GhPME2 and GhPME31 in these different contexts. In vitro assays revealed that proteins from transgenic plants clearly inhibited the activity of GhPME2 and GhPME31 (Fig. 8C). To test whether the reduced disease symptoms were associated with the overexpression of GhPMEI3, the relative VdPG1 levels in wild-type and transgenic plants were analyzed following inoculation with V. dahliae. Gene transcription was significantly higher in the wild-type plants than in transgenic plants. No VdPG1 expression was detected in noninoculated plants (Fig. 8D). In addition, plant phenotypes correlated with the degree of V. dahliae colonization. The fungal biomass was determined by RT-qPCR in each transgenic line and normalized to the biomass, with respect to the wild-type plants, to determine the GhPMEI3-mediated biomass reduction. The accumulation of fungal biomass was substantially reduced in the GhPMEI3 transgenic lines in comparison with nontransgenic plants (Fig. 8E).
Silencing of GhPMEI3 Increases the Susceptibility of Cotton to V. dahliae
Wild-type cotton plants and cotton plants in which GhPMEI3 was silenced with virus-induced gene silencing (VIGS) were treated with Czapek medium as a control, or V. dahliae. Two weeks later, the disease progression following V. dahliae infection was monitored based on the degree of stunting. In the control check (CK) treated plants, little stunting was observed, while the silenced plants inoculated with V. dahliae exhibited evident and consistent stunting (Fig. 9, A–C). The transcript levels of GhPMEI3, GhPME2, and GhPME31 in silenced plants were determined by RT-qPCR. In the completely and partially silenced cotton plants, the transcript levels were considerably higher than those in the CK plants (Fig. 9D). Moreover, the possible contribution of GhPMEI3 in controlling GhPME2 and GhPME31 activity in silenced cotton plants during V. dahliae infection was evaluated. The GhPME2 and GhPME31 activity levels were determined in CK and in GhP1, GhP2, and GhP3 silenced plants after V. dahliae inoculation, using biochemical assays (Lionetti et al., 2015). The increase in PME activity observed in silenced plants was higher than in CK plants (Fig. 9E). This demonstrated that GhPMEI3 functioned as an inhibitor to control the activity of PMEs during fungal infection. To further investigate the correlation between PMEI, PMEs, and PG, the expression of VdPG1 in CK and silenced plants infected with V. dahliae was monitored. VdPG1 transcript levels were higher in silenced plants than in CK plants, and no significant differences were observed between the three silenced plants (Fig. 9F). To confirm the observed phenotypes, the fungal biomass was determined by RT-qPCR in each silenced plant and normalized to the biomass of CK plants. This analysis revealed that the basal defense against V. dahliae was compromised in the GhP1 line, with the GhP2 and GhP3 lines showing levels of susceptibility that were similar to CK plants (Fig. 9G).
Docking Analysis of GhPMEI3 with GhPME2 and GhPME31
PMEIs contain four long helices (α1–4), packed in an antiparallel orientation, in a classic up-and-down helical bundle (Di Matteo et al., 2005). As previously reported, GhPME2 and GhPME31 form a 1:1 stoichiometric complex with GhPMEI3, with GhPMEI3 covering most of the shallow cleft of the enzyme where the ligand-binding site is located. Docking analyses revealed that GhPMEI3 interacts with GhPME2 and GhPME31 in the putative active site region via its helices. Furthermore, the helices of GhPMEI3 were positioned perpendicularly to the β-helix in either GhPME (Fig. 10, A and B).
Electrostatic Surface Potentials of the Individual GhPMEI3, GhPME2, and GhPME31 Proteins, and Protein Complexes
The electrostatic surface potential of the GhPMEI3, GhPME2, and GhPME31 proteins was then predicted to analyze their surface charge distributions (Fig. 11, A–C). The mode of assembly of the two protein complexes, that is GhPMEI3-GhPME2 and GhPMEI3-GhPME31, is presented in Figure 11, D and E, respectively. The surface charge distributions over the individual proteins clearly differed, particularly for GhPME2 and GhPME31, over the concave interaction site. However, the surface potential of the two protein complexes was similar, as was the mode of binding.
In this study, we provided insights into how PMEI modulates the activity of PME through the formation of the PME-PMEI complex. We chose GhPMEI3 as a possible pathogen resistance-associated PMEI, and GhPME2 and GhPME31 as representatives of two different types of PMEs. The alignment of the GhPMEI3 sequence with sequences of functionally characterized PMEIs showed that GhPMEI3 has two INH sequences (Fig. 1). The structure of an INH from tobacco (Nt-CIF) has been previously elucidated (Hothorn et al., 2004a). KwPMEI and Nt-CIF are strikingly similar from a structural point of view, but recognize different target enzymes, as key amino acids that are involved in the formation of intermolecular H-bonds with PMEs are only conserved in PMEIs. Therefore, the structural view of the PMEI-PME complex provided insights into the specific binding between GhPMEI3 and GhPME2/ GhPME31. Since the highly conserved INHs lack the PKF motif in the α3 helix, GhPMEI3 may be unable to participate in interactions with INHs, but might participate in the formation of enzyme-inhibitor complexes (Lionetti et al., 2015). Many PME proteins are predicted to contain an N-terminal PRO region, which shares moderate homology with PMEIs (Wolf et al., 2009). The PRO region is considered to be crucial for protein targeting to the endoplasmic reticulum (Micheli, 2001), and is thought to play an auto-inhibitory role in PMEs during secretion to the apoplast (Giovane et al., 2004; Lionetti et al., 2007). Consistent with the PME classification, GhPME2 was characterized as a type-I PME and GhPME31 as a type-II PME (Fig. 2).
Previous studies suggested that PMEI can markedly inhibit the activity of plant PMEs and that pectin methyl esterification can play a role in plant resistance to bacterial and fungal pathogens (An et al., 2008; Volpi et al., 2011). The ability of GhPMEI3 proteins to inhibit the activities of cotton GhPME2 and GhPME31 was tested in enzymatic assays (Fig. 5, B and C). In addition, GhPMEI3 exhibited antifungal activity against V. dahliae, F. oxysporum f. sp. vasinfectum, and B. cinerea in vitro (Fig. 6, A–C), suggesting that GhPMEI3 might directly interfere with the plant pathogen during infection. It was initially thought that PMEIs are unable to inhibit the PMEs of fungal pathogens (Giovane et al., 2004; Di Matteo et al., 2005). However, more recently, the overexpression of AtPMEI-1 or AtPMEI-2 inhibitors in Arabidopsis was shown to increase the DM of the pectin cell wall and reduce plant susceptibility to fungal and bacterial necrotrophs (Lionetti et al., 2007; Raiola et al., 2011). In addition, plant resistance to fungal pathogens is compromised in PMEI mutants where PMEI expression is impaired (Lionetti et al., 2017). In agreement with these previous findings, GhPMEI3 was able to restrict fungal mycelial growth. Hence, it may function to reinforce the cell wall barrier and promote the antifungal resistance in cotton.
To investigate the role of GhPMEI3 in plant growth and plant pathogen interactions, we heterologously expressed GhPMEI3 in Arabidopsis. GhPMEI3-expressing transgenic plants displayed reduced activities of GhPME2 and GhPME31, and alterations in the pectin DM and in the plant growth response (Fig. 7). In addition, the transformed plants showed a reduced susceptibility to V. dahliae (Fig. 8, A and B), since the methyl esterification of pectin may be associated with pectin-degrading enzymes, such as endo-polygalacturonase (Lionetti et al., 2007). Thus, it is likely that the higher DM of pectins in transgenic plants hampered the activity of VdPG1, produced by V. dahliae, thus further delaying the colonization of the fungus and increasing resistance to pathogens (Fig. 8, D and E). Recently, a significant reduction in disease symptoms caused by B. sorokiniana and F. graminearum was reported in transgenic wheat (Volpi et al., 2011). Further, the ability of PMEI to restrict the progression of Tobacco mosaic virus infection in tobacco and Turnip vein-clearing virus infection in Arabidopsis was recently demonstrated (Tundo et al., 2016). PMEIs in tobacco and transgenic Arabidopsis plants not only affect the PMEs already present in the plant, but also inhibit the viral or fungal induction of PME activity. Therefore, they further prevent the pathogen from exploiting the host's susceptibility factors that are required for infection (Lionetti et al., 2014). The enhanced resistance of these transgenic plants to viral and fungal pathogens is attributed to the methyl esterification of pectin, which obstructs the growth of these pathogens and reduces the capacity of their PGs to hydrolyze methyl-esterified pectin (Tundo et al., 2016).
In the current study, virus-induced gene silencing was used to investigate the effects of GhPMEI3 loss of function in cotton. The experiments revealed that GhPMEI3 silencing compromised cotton resistance to V. dahliae (Fig. 9, A–C). These observations were similar to those in a previous study with PMEI mutants (Lionetti et al., 2017). In addition, following inoculation with V. dahliae, the relative expression of GhPME2 and GhPME31, and also of VdPG1, was higher in the GhPMEI3-silenced plants than in the wild type. This suggested that GhPMEI3 controls pectin methyl esterification through PME during V. dahliae infection. Further, the comparison of CK and GhPMEI3-silenced plants revealed a significant reduction of DM in the latter (Supplemental Fig. S2).
Plant pathogens produce cell wall-degrading enzymes, such as polygalacturonase and pectin lyase, during infection (An et al., 2008). Among these, PGs play a primary role, particularly because they act as virulence factors during plant–pathogen interactions (Volpi et al., 2011), for example B. cinerea and tomato (ten Have et al., 1998), and Alternaria citri and citrus (Isshiki et al., 2001). Previous studies have demonstrated that highly methyl esterified pectins are less susceptible to the action of PGs from B. sorokiniana and F. graminearum and to other fungal PGs than pectins with lower DM (Limberg et al., 2000; Volpi et al., 2011). This suggested that the increased level of pectin methyl esterification in transgenic plants impairs the ability of the fungus to colonize host tissues and hampers the activity of endopolygalacturonases produced by fungal pathogens (Lionetti et al., 2007; Volpi et al., 2011). The observation that VdPG1 was expressed at a relatively low level in lines 3 and 5 further indicates that pectin methyl esterification, associated with PMEI overexpression in plants, results in a structure that is inaccessible to PGs secreted by fungal pathogens, which hydrolyze pectin during infection and colonization of host tissues.
PMEI and PME form a 1:1 stoichiometric complex and the inhibitor masks the putative active site of the enzyme. The four-helix bundle of GhPMEI3 is perpendicular to the parallel PME ligand-binding cleft structure, and three helices (α2, α3, and α4, but not α1) interact with the enzyme in the proximity of the shallow cleft (Fig. 10, A and B). Masking of the pectin-binding cleft of PMEs masks the substrate-binding and catalytic sites, further preventing the access of the substrate (Di Matteo et al., 2005; Sénéchal et al., 2015). By exposing its N-terminal α-hairpin, PMEI interacts with the C-terminal helix at the PME surface (Hothorn et al., 2004b). The N-terminal region of AtPMEI1 was proposed to be important for the interaction with PMEs. This region may play a crucial role in the structural stability of PMEIs, but is not extensively involved in the formation of the PMEI-PME complex (Hothorn et al., 2004a, 2004b). According to previous docking studies, GhPMEI3 binds in an open conformation at the N terminus and interacts with the C terminus of GhPMEs, which is consistent with a report proposing that PMEI may easily bind to homologous type-I PMEs (Micheli, 2001). It is likely that the putative binding site of the inhibitor on a bacterial enzyme is much deeper than in plant PMEs, thus leading to steric hindrance that prevents their interaction with PMEIs (Di Matteo et al., 2005). The residues that are crucial for the PMEI-PME interaction are deemed to be conserved in plant PMEs, but not in the fungal PMEs, which might provide another possible explanation for the lack of inhibition (Di Matteo et al., 2005).
Taken together, the inhibition of cotton PMEs by GhPMEI3 might explain how the enzymatic activity of PME is coordinated within the cell wall. In Figure 12, we propose a model of PMEI involvement in plant immunity against fungal pathogens. Above all, PME is required for the release of MeOH, H+, and pectin-COO− during infection. MeOH, as a damage-associated molecular pattern (DAMP)-like alarm signal, down-regulates the expression of the pathogen-related GhPMEI3. Moreover, H+ ions affect the pH of the plant cell wall and promote the hydrolysis of VdPG1 to homogalacturonan. Pectin-COO−, which contains exposed negative charge groups, can bind cations; Al3+ affects the methylation status of pectin in the root cell walls and regulates PME activity. OGs are oligomers of α-1,4-linked galacturonosyl residues that can be generated by a partial hydrolysis of polygalacturonic acid or released from plant cell walls through partial degradation of homogalacturonan; they are recognized as DAMPs, leading to the activation of the plant immune response (Nothnagel et al., 1983; Ferrari et al., 2013). The degree and pattern of pectin methyl esterification influences the release and type of OGs (Bethke et al., 2014). Moreover, polygalacturonase-inhibiting proteins (PGIPs) interact with PG and lead to the accumulation of OGs. OGs can be detected by members of the wall-associated kinase family and are responsible for the constitutive activation of pathogen-related defense responses and other signal transduction pathways in plant cells, including reactive oxygen species production, ET production, and callose deposition (Brutus et al., 2010; Ferrari et al., 2013; Bethke et al., 2014). Finally, PMEI may directly inhibit the activity of fungal PMEs and fungal PGs. However, it is possible that GhPMEI3 interacts with plant GhPME2 and GhPME31, leading to pectin methyl esterification and influencing the physicochemical properties of the cell wall. Ultimately, this impairs the ability of V. dahliae to grow on methyl esterified pectin and reduces the capacity of VdPG1 to hydrolyze methyl esterified pectin, thus protecting the plant cell wall from degradation. The current study provides mechanistic insights into the regulation of enzyme activity by their inhibitors and the interaction between PMEI and PME using molecular modeling. Broadening our knowledge of how plants regulate cell wall metabolism is of key importance for improving plant resistance to fungal pathogens.
Plant Growth and Preparation of Fungal Pathogens
Seeds of the state cotton (Gossypium hirsutum), isolate 2006001 (original strain no. GK44), were provided by the Cotton Research Institute, Chinese Academy of Agricultural Sciences. Plump seeds were selected and sterilized using a 4% (v/v) NaClO solution, 75% (v/v) ethanol, and then rinsed with sterile water. The seeds were planted in a mixture of vermiculite and nutrient soil (1:2, w/w), and cultured in a growth chamber with a light intensity of 150 μmol/m2/s and a photoperiod of 16 h/8 h.
Wild-type Arabidopsis (Arabidopsis thaliana, Col-0) and transgenic Arabidopsis plants were grown in a controlled environmental chamber maintained at 22°C, 70% relative humidity, with a 16-h photoperiod. For vertical growth, the seeds were sterilized and germinated for 12 d on agar plates containing 0.8% (w/v) agar and 1% (w/v) Suc, with a 16-h photoperiod, in a vertical position (Lionetti et al., 2007). Root lengths and areas were measured using a root scanner (Expression 11000XL; Epson). Root elongation zones were observed under a microscope and photographed under a 4×/0.25 numerical aperture objective using a Nikon eclipse Ti microscope.
Virulent strains of Verticillium dahliae Vd991, Fusarium oxysporum f. sp. matthiole, Alternaria brassicicola, and Botrytis cinerea were prepared from cultures grown for 5 d at 25°C in Czapek liquid medium; the experiments were performed using conidial suspensions of 106 conidia/mL.
Sequence and Phylogenetic Analyses
A homology search using sequences deposited in the NCBI database was conducted to verify that the obtained sequences encode GhPMEI3, GhPME2, and GhPME31. The SMART software (http://smart.embl-heidelberg.de/) was used for domain structure prediction and the sequence alignment was prepared using the ESPript program (Robert and Gouet, 2014). The phylogenetic analysis was carried out in MEGA 5.1 (Bednarek et al., 2009).
Determining the Effects of Exposure to Fungal Pathogens and Mediators of Stress Responses on Gene Expression in Plants Using RT-qPCR
Cotton seedlings were cultured in a growth chamber at a day/night temperature of 25°C/22°C. The seedlings were infected with V. dahliae and the leaves were sprayed with 10 μL/L ET, 50 μm MeJA (Sigma-Aldrich), or 1% (v/v) H2O2 (Tapia et al., 2005). For OG treatment, the seeds were placed in a culture flask containing MS medium with 0.5% (w/v) Suc and incubated for 10 d; then, the medium was replaced with fresh medium containing 100 μg/mL OGs with a degree of polymerization of 10 to 15, and the incubation was continued under the same conditions before harvesting (Ferrari et al., 2003).
Total RNA was extracted from the cotton complete stool using an RNA extraction kit, according to the manufacturer's instructions (RA103-01; Biomed, Enzo Life Sciences). The isolated RNA was treated with RNase-free DNase I (Sigma-Aldrich) and reverse-transcribed into cDNA using the Fast Quant cDNA reverse kit (Tiangen Biotech), as per manufacturers' instructions.
Specific primers for the detection of the GhPMEI3, GhPME2, and GhPME31 transcripts and primers for the detection of the endogenous control (EF1ɑ from cotton) are listed in Supplemental Table S1. The expression levels of target genes (GhPMEI3, GhPME2, and GhPME31) relative to the reference gene (EF1ɑ) were analyzed using the comparative CT (2−ΔΔCT) method. The RT-qPCR conditions were as follows: an initial denaturation step of 30 s at 95°C, followed by 40 cycles of denaturation for 5 s at 95°C and annealing for 34 s at 60°C.
Purification of the Recombinant GhPMEI3, GhPME2, and GhPME31 Proteins, and Testing the Inhibition of PME Activity
The GhPMEI3, GhPME2, and GhPME31 open reading frames were amplified using the primers listed in Supplemental Table S1, and cloned into the PET-32a or pGEX-4T-1 vectors for the production of fusion proteins harboring a His6-tag or a GST-tag, respectively (Supplemental Fig. S3). The transformants were grown in LB medium at 37°C, with shaking at 200 rpm. At OD600 = 0.6–1.2, isopropyl-β-d-1-thiogalactopyranoside was added to a final concentration of 1.0 mm. The cultures were grown at 28°C for an additional 4 h with shaking at 200 rpm, and the cells were harvested by centrifugation at 10,000g for 15 min. The pellets were resuspended in PBS and the cells were disrupted using an ultrasonic cell breaker. The proteins were purified by precipitation under denaturing and refolding conditions, as previously described (Wang et al., 2013). Recombinant GhPMEI3 was purified using a His6-tagged protein purification kit (CW BIO), and GhPME2 and GhPME31 were purified using a GST purification kit (Takara). The thrombin cleavage capture kit (Novagen) was used to remove the tags. The purified GhPMEI3, GhPME2, and GhPME31 proteins were resolved by SDS-PAGE and their concentrations determined using the Bradford method (Bradford, 1976), with bovine serum albumin as a standard.
The resolved GhPMEI3 proteins were blotted onto a polyvinylidene difluoride immobilon-p membrane (Millipore) following the manufacturer's protocol (100 V for 30–60 min). The membranes were blocked in 1× TBS-Tween containing 5% (w/v) fat-free skimmed milk for 2 h at room temperature. The membranes were then incubated in a solution of anti-His6-tag monoclonal antibody (1:10,000, diluted in a blocking solution; Proteintech Group) at 4°C, overnight. They were then washed five times (10 min each) with 1× TBS-Tween with shaking. Finally, the membranes were washed with peroxidase-conjugated affinipure goat antimouse IgG (1:8,000, diluted in the blocking solution; Origene). The signal was detected using the ECL system (LI-COR Biosciences). The GhPME2 and GhPME31 protein spots were excised from the 2D gel and digested with trypsin, as reported previously (Granvogl et al., 2007). The MS analysis was carried out according to the method described by Prabhu (Prabhu et al., 2015).
The PME activity (mU or nmol/min) was determined using an alcohol oxidase-coupled microassay, as described previously (Klavons and Bennett, 1986). The reaction solution contained 90% methyl esterified Citrus pectin (Sigma-Aldrich P9561; 0.8 mg/mL) and 0.005 U of Pichia alcohol oxidase (Sigma-Aldrich A2404) in 98 μL of 50 mm phosphate (Na) buffer at pH 8.0 (Dedeurwaerder et al., 2009). The reaction was initiated by the addition of 2 μL of diluted extract and stopped by transferring the reaction to a plate containing 100 μL of the developing solution. The plate was incubated at 67°C for 15 min, and sample absorbance was measured at 420 nm using a UV spectrophotometer (TU-1900). The amount of MeOH released by PME was calculated by comparing the OD420 values to the standards (containing 0.3 mm methanol; Dedeurwaerder et al., 2009). The PME enzyme assays using NADH were performed as previously described (Grsic-Rausch and Rausch, 2004). As a negative control, the same reaction was performed with heat-denatured (95°C, 5 min) PME or water instead of PME.
Determination of the in Vitro Antifungal Activity of GhPMEI3
The antifungal activity of GhPMEI3 was determined as previously reported (An et al., 2008). The plant pathogens V. dahliae, F. oxysporum f. sp. vasinfectum, and B. cinerea were used in the current study. Briefly, the fungi were incubated on potato dextrose agar at 25°C for 1 week; they were then transferred to 24-well tissue culture plates (TCP20-24; Bio Basic) containing 4× concentrated potato dextrose broth (100 μL) and 0, 50, 100, or 250 μg/mL of GhPMEI3. The spore suspensions were cultured for an additional 4 to 7 d. Spore germination and hyphal growth were examined as previously described (An et al., 2008). Briefly, the spore suspensions were placed on glass slides and incubated for 12 h at 25°C; spore germination and the hyphae were then photographed under a Nikon eclipse Ti microscope using a 4×/0.25 numerical aperture objective.
Extraction of Alcohol-Insoluble Residues and Determination of DM
The alcohol-insoluble residues were extracted as described elsewhere (Lionetti et al., 2017). Briefly, wild-type, transgenic, or silenced plants (uninfected, mock, and V. dahliae-treated leaves of 6-week-old plants) were collected and homogenized. They were washed twice with 70% (v/v) ethanol preheated to 70°C, vortex-mixed, and centrifuged at 14,000g for 10 min to collect the precipitate. The precipitate was resuspended in a chloroform:methanol mixture (1:1), shaken at room temperature for 30 min, and collected at 14,000g; it was then resuspended in 1 mL of 80% (v/v) acetone, twice, and the supernatant was discarded after centrifugation. The pellet that contained the alcohol-insoluble residues was dried overnight in a fume hood at room temperature, and starch was removed by α-amylase treatment (Lionetti et al., 2017).
The DM of pectin in transgenic and silenced plants was determined using the alcohol oxidase-coupled assay (Klavons and Bennett, 1986). The reaction solution contained 90% methyl esterified Citrus pectin (0.8 mg/mL; Sigma-Aldrich P9561) and 0.005 U of Pichia alcohol oxidase (Sigma-Aldrich A2404) in 98 μL of 50 mm sodium phosphate buffer (pH 8). The mixture was preincubated for 3 min at 30°C; then, 2 μL of the diluted extract was added and the incubation was continued at 30°C for 15 min; 100 μL of the developing solution was added to stop the reaction (Klavons and Bennett, 1986; Dedeurwaerder et al., 2009). The experiment was performed after preincubating the proteins at 30°C for 3 h, and the reaction was stopped by boiling at 100°C for 10 min; 0.03 U of alcohol oxidase was added, and the mixture was incubated for 15 min at room temperature with shaking. This was followed by the addition of 100 μL of the developing solution (Lionetti et al., 2015). The mixture was then incubated at 67°C for 15 min, and OD420 was determined using a TU-1900 spectrophotometer. Standards containing 0.3 mm methanol were included in each batch of assays. The amount of methanol released by PME was quantified based on the measured OD420 values (Dedeurwaerder et al., 2009).
Extraction of Total Proteins and Determination of PME Activity in Transgenic Plants Inoculated with Vd991
Total protein extraction from transgenic Arabidopsis specimens was performed as previously described (Lionetti et al., 2014). Briefly, 2 mL of extraction buffer (1 m NaCl, 20 mm sodium acetate, 0.02% [w/v] sodium azide, and protease inhibitor, 1:100, pH 5.5) was added per 1 g of plant tissue. The samples were shaken at 4°C for 1.5 h, and centrifuged for 15 min at 15,000g; the supernatant was then collected. Protein concentrations were determined using the Bradford method, with bovine serum albumin as a standard. The proteins were resolved using SDS-PAGE, loading 5 μg of protein extract per lane. The proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Prabhu et al., 2015).
Arabidopsis plants were inoculated with V. dahliae by leaf dipping (5 × 106 conidia/mL). Plants that were mock-inoculated with Czapek-Dox medium were used as a control (Lionetti et al., 2007). The disease symptoms were quantified with ImageJ software. The fragments were inserted into pCAMBIA-1304 vector with NcoI as restriction site (Supplemental Fig. S4), and the GhPMEI3 transgenic lines were selected (Supplemental Fig. S5). The PME activity was detected using the radical gel diffusion assay in wild-type and transgenic GhPMEI3 Arabidopsis plants after inoculation with Vd991 (Downie et al., 1998; Lionetti et al., 2007). Briefly, gels were prepared using 0.1% of 81% methyl esterified lime pectin, 1% (w/v) agarose, 12.5 mm citric acid, and 50 mm Na2HPO4 (pH 7.0). Wells (9-mm diameter) were punched, and protein samples were loaded into each well. The plates were then incubated at 30°C for 16 h; the gels were stained with 0.05% (w/v) ruthenium red for 45 min, and washed thoroughly with distilled water. The diameter of the red-stained areas was indicative of the hydrolysis of esterified pectin in the gels.
VIGS and Determination of PME Activity in Plants Inoculated with Vd991
VIGS vectors were constructed and Agrobacterium tumefaciens-mediated gene silencing in cotton was performed as previously described (Gao et al., 2011). GhCLA1, GhPMEI3, GhPME2, and GhPME31 cDNAs were PCR-amplified using the primers listed in Supplemental Table S1. The amplified fragments were cloned into pTRV1 and pTRV2 vectors (a kind gift from Professor Yule Liu, Tsinghua University, China) to construct the pTRV1-pTRV2:GhCLA1, pTRV1-pTRV2:GhPMEI3, pTRV1-pTRV2:GhPME2, and pTRV1-pTRV2:GhPME31 vectors, accordingly. These vector derivatives were used to transform A. tumefaciens GV3101 strain. The cultures and cells were handled as described earlier (Gao et al., 2011). Cultures of bacteria harboring pTRV1 and pTRV2 vectors were mixed in a 1:1 ratio, and the cotyledons of 2-week-old cotton seedlings were infiltrated with a needleless syringe. Small holes were punched on the underside of the cotyledon to facilitate the infiltration. The assays were performed with at least six plants for each constructed vector, and the experiments were repeated at least three times.
V. dahliae Vd991 was grown on potato dextrose agar at 25°C for 7 d. Spore suspensions were prepared at 1 × 106 conidia/mL in a solution containing 0.001% (v/v) Tween 20 (Gao et al., 2011). Both the control and VIGS-silenced plants were stem-inoculated using a syringe needle ∼1 cm below the cotyledons (Bolek et al., 2005).
In Planta Fungal Biomass Assessment and PG Expression Analysis
V. dahliae biomass was assessed as previously described (Zambounis et al., 2007; Ellendorff et al., 2009). For Verticillium inoculations, 2- to 3-week-old wild-type and single insert transgenic Arabidopsis lines were uprooted. The roots were cleaned with water and then dipped in a suspension of 106 conidia/mL for 3 min (Fradin et al., 2011). Wild-type and GhPMEI3-silenced cotton were inoculated with Verticillium as previously described (Gao et al., 2011). The control plants were treated with potato dextrose broth without the conidia. Arabidopsis samples were collected on day 21 after treatment, and cotton samples were collected 18 d after the inoculation. To estimate the amount of Verticillium biomass, DNA was extracted from 100 mg of liquid nitrogen-ground sample powder and further analyzed by RT-qPCR. The primer sequences (ITS1-F/ST-Ve1-R and FOV-3F/FOV-4R) are listed in Supplemental Table S1. To evaluate the expression of PG-encoding genes in Arabidopsis and cotton tissues, total RNA was extracted from whole plants and then reverse-transcribed into cDNA (Tiangen Biotech). The RT-qPCR reactions were performed as described above, with primers listed in Supplemental Table S1.
Homology Modeling and Protein Docking
Three-dimensional structure homology modeling of GhPMEI3, GhPME2, and GhPME31 was performed using Phyre2 software in the normal modeling mode (Kelley et al., 2015). The structure of kiwi PMEI (PDB ID 1XG2, chain B) was used as a template to construct a homology model of GhPMEI3, based on score and rank; the models of GhPME2 and GhPME31 were built using the complex (PDB ID 1XG2.1.A) and carrot PME (PDB ID 1GQ8) as structural templates with high scores (Supplemental Fig. S6). The postrefinement and visualization of the structural models were carried out as previously described (Sénéchal et al., 2015; Liu et al., 2017; Supplemental Table S2). The interactions of GhPMEI3 with GhPME2, and GhPMEI3 with GhPME31 were assessed by docking analysis using the PatchDock (Schneidman-Duhovny) molecular docking algorithm, based on the shape complementarity principles (Liu et al., 2017). Model data of the top 20 best models were then refined and minimized in FiberDock; the highest-scoring ranked models were selected as the most probable prediction candidates.
Electrostatic Potential Distribution Analysis
The DeepView-Swiss-PdbViewer (Guex and Peitsch, 1997) was employed to compute the distributions of the electrostatic potentials for GhPMEI3, GhPME2, and GhPME31, and for the GhPMEI3-GhPME2 and GhPMEI3-GhPME31 complexes. The surface charge was visualized using UCSF Chimera v. 1.10.1 software (Pettersen et al., 2004).
Accession Numbers
Sequence data for the proteins analyzed in the current article may be accessed in the GenBank library under the following accession numbers: KY933672, for GhPMEI3; KY933673, for GhPME2; and KY933674, for GhPME31.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. GhPMEI3 regulates the level of pectin methyl esterification during fungal infection.
Supplemental Figure S2. Determination of the DM in CK and GhPMEI3-silenced cotton lines.
Supplemental Figure S3. Schematic representation of PET-32a-GhPMEI3 and pGEX-4T-1-GhPME2/GhPME31 bacterial expression plasmids.
Supplemental Figure S4. Construction of the overexpression vector pCAMBIA1304-GhPMEI3.
Supplemental Figure S5. Transcript level determination in T1-transgenic Arabidopsis.
Supplemental Figure S6. Homology modeling of GhPMEI3, GhPME2, and GhPME31.
Supplemental Table S1. Primers used in RT-PCR and RT-qPCR.
Supplemental Table S2. Refinement of GhPMEI3-GhPME2 and GhPMEI3- GhPME31 complexes.
We thank Professor Yule Liu (Tsinghua University, China) for the kind gift of pTRV1 and pTRV2 vectors for the gene silencing experiments.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yuxia Hou (houyuxia{at}cau.edu.cn).
Y.H., F.L., and N.L. conceived and designed the experiments; N.L., Y.S., and Y.P. executed the experiments; N.L. analyzed the data; N.L. wrote the manuscript; X.Z., P.W., and X.L. contributed reagents, materials, and analysis tools.
↵1 This work was sponsored by the "Seven Crop Breeding" National Major Project (grant no. 2016YFD0101006), the Genetically Modified Organism Breeding Major Project (grant no. 2018ZX08005001-002), and the State Key Laboratory of Cotton Biology Open Fund (grant no. CB2017B03).
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- Received October 3, 2017.
- Accepted January 9, 2018.
- Published January 23, 2018.


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This morning's Gospel reading is Luke 23:35–43:
The rulers sneered at Jesus and said,
"He saved others, let him save himself if he is the chosen one, the Christ of God." Even the soldiers jeered at him. As they approached to offer him wine they called out, "If you are King of the Jews, save yourself." Above him there was an inscription that read, "This is the King of the Jews."
Now one of the criminals hanging there reviled Jesus, saying, "Are you not the Christ? Save yourself and us." The other, however, rebuking him, said in reply, "Have you no fear of God, for you are subject to the same condemnation? And indeed, we have been condemned justly, for the sentence we received corresponds to our crimes, but this man has done nothing criminal." Then he said, "Jesus, remember me when you come into your kingdom." He replied to him, "Amen, I say to you, today you will be with me in Paradise."
Today the church celebrates the Solemnity of Christ the King, and closes out the liturgical year. A year ago, Pope Francis opened the liturgical year as a year of mercy, and called for the holy doors in each diocese to remain open as a symbol of the Lord's mercy and openness. We have walked through them on a couple of occasions during the past year and hoped to squeeze through them one more time, expecting the doors to close today. Instead, we had an opportunity to witness the ceremony closing the doors and completing the year-long observance.
Just before the doors closed, however, one enterprising man managed to sneak through them — even after the ceremony ended. He literally squeezed his way through the opening, dodging the closing double doors without quite touching them. In doing so, this unknown middle-aged man reminded me about the nature of God's mercy, and its timelessness — and our own relationship with Him.
Today's first two readings focus on kingdoms and power. In 2 Samuel, the tribes of Israel anoint David as king, following the will of the Lord who put him in charge of the Israelites even when Saul was still king. The Lord had at the time given David two charges, the second of which was to be "commander of Israel." The first, however, was to "shepherd my people Israel" — to care for them, to love them, and to serve them. Power serves — it is not served.
We see Paul hint at the same point in his letter to the Colossians. Paul exalts Jesus as "the firstborn of all creation," declaring that "He is before all things, and in him all things hold together." Regardless, Jesus did not come to earth to declare a material kingdom of power and might, but came to serve us and save us, "making peace by the blood of his cross through him." He came to serve rather than be served, and that service extends throughout time.
Throughout history, the concept of a servant king is all but alien to humanity. This is why the crucifixion was intended to utterly discredit Jesus. After all, a true king and Messiah would choose to save himself first, right? If Jesus could not come to His own rescue by using His power to His own benefit, then that power didn't really exist at all. "If you are the King of the Jews," the soldiers and the crowd jeer, "save yourself."
Jesus had a different mission in mind, of course. His first role was the same as David's — to shepherd the Lord's people Israel, and the rest of the world as well, and the timeline for this turns out to be all but limitless. At this point, the Lord's plan of salvation had already unfolded over at least two millennia. Israel was intended to serve as a nation of priests to convert the world to the Lord, but it shortly chose to operate as a political nation-state instead. Between David's rise to the throne and the Crucifixion, Israel would split into two, and both kingdoms would eventually collapse, be restored, and be defeated again because of the way that the Israelites and Judeans trusted more in their own devices than in the Lord.
Yet time never really ran out — not for the Israelites and Judeans, and not for any of us. Jesus comes to save fallen humanity by redirecting the Lord's people Israel to become not a nation of priests that will bring all nations to Jerusalem, but a church of priests instead that will bring the eternal Jerusalem to all nations. Rather than raise armies and crush the Romans while gaining material wealth, Jesus chooses to serve in such a way that all can be saved.
Jesus spoke of this in other terms before the Crucifixion. In his Sermon on the Mount, Jesus offers two specific beatitudes that He exemplifies in this sacrifice. "Blessed are the meek," He says. Meek in this sense does not mean powerless; it refers to a choice to refrain from using power in order to benefit others. In another part of the same sermon, Jesus exhorts people to "love their enemies," saying it is not enough to simply love one's friends. Jesus demonstrates both in His effort on the cross to complete His sacrifice to save the very people jeering Him, and the very people who nailed Jesus to the cross.
As King, did Jesus have the power to come down from the cross and crush His enemies? Of course. Does He have the authority to declare His disgust at us and toss us aside in our sinfulness? As our King, He most certainly does. But He chooses not to do so, to put aside that power in favor of mercy, in order that all may be saved. Even in the last few and worst moments of His suffering on the cross in this Gospel reading, Jesus succeeds in saving the soul of the repentant thief — a man who had already been condemned for his crimes and suffering the most ignominious execution the Romans could offer.
One might say that the repentant thief snuck in through the doors just as they were closing. But of course, that's not the case. Jesus' mercy is endless for those who seek it out. We closed the holy doors as part of our symbolic celebration of this, a finite celebration in time that reminds us of the infinite mercy of Christ. Jesus' love shepherds us through those doors, opening as we approach, calling out to us to give ourselves to the Lord by choosing to walk through them. His meekness belies His power as King, but Jesus laid that down in order to ensure that all of His flock hear His voice and be comforted by it rather than cower in fear.
We might at times feel as though the doors are closed, trapped in our own sin and shame. If we look closely enough, though, we'll see them open for us — and like that unnamed gentleman at the cathedral last night, we should not hesitate to pass through them. Our King awaits to greet us with joy and welcome us back to the flock.
"Sunday Reflection" is a regular feature, looking at the specific readings used in today's Mass in Catholic parishes around the world. The reflection represents only my own point of view, intended to help prepare myself for the Lord's day and perhaps spark a meaningful discussion. Previous Sunday Reflections from the main page can be found here. For previous Green Room entries, click here.


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To stay healthy, it's a good idea to have dental issues diagnosed before they develop into bigger conditions. These oral problems can be identified during a comprehensive dental examination with our experienced team. During your bi-annual cleanings, we always review your oral condition and screen for problematic areas. However, a comprehensive dental exam is performed on an annual basis and consists of a more thorough analysis. Dental exams at West Bridgewater Dental are performed to assess your oral health by looking for tooth decay, gum disease, and signs of oral cancer. Our team of board-certified dentists and specialists will determine your need for procedures and evaluate the health of your bite and jaw. Digital images will be used to gain a better view of any potential hidden concerns in your teeth and jawbone. If you live near West Bridgewater, MA, make an appointment for a dental exam for your family at our established, modern practice.
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Oral cancer is an abnormal group of cells in the mouth that may affect the gums, lips, jawbone, tongue, salivary glands, roof and floor of the mouth, and throat. Each dental examination at West Bridgewater Dental includes a screening for oral cancer to check for any symptoms, which may include abnormal lesions, tissue discoloration, sores, lumps, or bumps. Our skilled dentists will also palpate your neck and jaw for abnormal growths. Should any areas of concern be discovered, we may perform a biopsy or refer you to a specialist for further care.
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West Bridgewater, MA residents of every age should receive a dental exam each year. Some of the most common conditions, such as periodontal (gum) disease, often go unnoticed without proper evaluation. If oral problems are identified early, you're more likely to have greater treatment options.
The ADA (American Dental Association) suggests that children visit a dentist before turning a year old. During your visit, our dentists can talk with you about common oral concerns, including the use of pacifiers, teething, and baby bottle tooth decay. Our caring team at West Bridgewater Dental can teach you ways to clean and care for your baby's oral health so he or she develops good habits early in life. Dental examinations are also crucial for older children as their teeth, gums, and jaw develop.
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Your exam will begin with an initial assessment of your teeth, tongue, gums, throat, the roof of your mouth, inside your cheeks, and other checkpoints. This includes checking for symptoms of oral cancer in your mouth. Our team may take digital x-rays, screen for gum disease, and evaluate your neck, head, and jaw, including the bite and temporomandibular joints (TMJ). We'll then discuss any concerns discovered and review your treatment options. Any other conditions you may have and prescriptions you're taking will be reviewed. Our dentists will then determine if these issues might affect your dental health by increasing your risk for dry mouth, tooth decay, or other oral concerns. Lastly, we'll evaluate any restorations you have, such as fillings, crowns, bridges, and dentures, to assess the wear and fit.
When to schedule your next appointment at West Bridgewater Dental will be based on the results of your exam. If no oral problems are detected, then you can follow a home oral care routine with flossing and brushing every day, plus yearly dental examinations and twice-yearly professional cleanings. If our team of dentists does find a concern that needs to be treated, procedures can be planned and reviewed.
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I seldom engage these discussions and usually allow them to folow their course, however, the constant reference by blacks to the JPI as the "authority" on the issue of jail vs. college, somewhat disturbs me, on multiple levels, of which the willingness to accept without question any organizations advancement of negative statistics about black men. I digreess, my point, has anyone in the community ever wrote a letter to, sent an email, or made a phone call to the National Center for Education Statistics (Telephone: 202-502-7300) (outside of yours truly) and as I constantly say asked the right questions? Try this out for starters….. ask them what was the total number of black men enrolled in post-secondary education or colleges and universities in 2000. The answer will amaze you (the quite too often touted claim and source data for the JPI cellblocks vs. classrooms comparison ~ oh BTW, funny thing happened at the JPI website last week, the link to the study was removed, guess they're lucky I have a downloaded pdf Cellblocks vs. Classrooms on my desktop!)
…Thank you very much JPI!


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Lance recommends you:
Early Morning hot air Balloon Ride Over Luxor, Egypt
An amazing trip AND we have overcome our fear of heights. A #hotairballoon ride over #Egypt is the trip of a lifetime as you soar above Queen Hatshepsut's temple, the Valley of the Kings and much more. All this before breakfast, an #aspirational start to the day.
We woke up at about 3:30am and left the boat by 4:00am for our hot air balloon ride. We had spent a lot of time debating whether to do it because 1) none of us are morning people 2) two of us were scared to death of heights and 3) it was somewhat expensive…but we could not have been happier that we decided to go.
we could not have been happier that we decided to go
When we got to the take-off site, we had a few minutes to watch the balloons being inflated all around us. The massive pilot lights and rising balloons against the pre-dawn sky was amazing, especially in the midst of a setting that was already so spectacular.
Once in the basket, Laura, her mom, her sister, and I took our positions along with a Spanish couple who didn't speak English (and seemed miserable) and several staff members from the balloon company.
Although it was clear our balloon was going up, it didn't even seem like we were moving. It was an unusual sensation to feel like we were hovering in mid-air, but it was great for me and my sister-in-law who both have a fear of heights. We had been pretty worried, but we ended up loving every minute of it.
We had been pretty worried, but we ended up loving every minute of it
We soared over all the fabulous sites of the area, including Queen Hatshepsut's temple, the Colossi of Memnon, the Valley of the Queens, and a little of the Valley of the Kings.
Snorkelling in El Gouna
4x4 Safari to Coloured Canyon and Shopping in Dahab
Seeing everything from the air gave us a totally different perspective on the locations of all the landmarks and the engineering achievements it took to construct them.
We landed in a field (right by a goat herder!) only about a half-mile from where we started after a 45 minute flight. The vendor, Horus Airship and Balloon, was first rate. They paid attention to safety, used good equipment and generally ran a smooth operation.
Although we had been up for at least five hours, it was only 9am, so it was time to head to the hotel for a nap…


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What can you say about don'tblinkoryou'lldie (fka DBOYD) that hasn't already been echoed by the community? Highly skilled with sound design and composition, don'tblinkoryou'lldie is a high-energy experience that puts your body and listening skills to work simultaneously with some of the most lush, fleshed-out detail I've heard from a Game Boy. If this is your first introduction to don'tblinkoryou'lldie, buckle up and get ready to never blink again in your life.
Battle Jazz Classics II encapsulates a very solid repertoire of what you can expect from don'tblinkoryou'lldie's entire discography. Very few people manage to make their music fast-paced and intensive while managing to still make it melodic enough to be something you could expect out of a dramatic scene in a video game. Even then, out of those very few, even less manage to put in a technical flair that would seem overwhelming to even the average aspiring chipmusician. This is where don'tblinkoryou'lldie walks through a crowd towards the camera, hands off a water bottle and, keeping with the meme, says "Hold my beer."
Nothing better encapsulates that statement than songs such as 'Michiko'. Meanwhile, other tracks such as 'The Anxiety' also explore slower rhythms and melodic structures while still managing to keep the same level of energy throughout the whole album. On top of that, there are a lot of times where don'tblinkoryou'lldie fills in any potential 'empty' space with little quips from other channels or instruments to help keep things interesting to your ears and attention span.
One track that really stands apart from the rest of the album is 'Undone'; bringing out what sounds like the internal conflict of one's perception of good and evil and then having aesthetics from each perspective influence the next part of the track. Here is where I'm blown away that this track is made with one instance of LSDJ, as the arps that bring in the dramatic, evil-sounding section are so incredibly lush that it almost feels unnatural for them to cut so immediately during moments of the percussion making quick fills. You'd expect to hear the tail end of the delay and reverb, but instead you realize they're still just two dry instruments playing tricks on your ears. Then, suddenly, you're brought back to the song with some catchy d&b rhythms and a change of mood of the track and along for the ride once again.
'Battle Jazz Classics II' is as one would expect from don'tblinkoryou'lldie; it's a highly impressive piece of work on a technical and composition standpoint that still sounds ever-so-unique to the artist. It's got a lot of high energy moments and really drives home its own unique story that keeps you listening on repeat. Something I specifically enjoy about this EP is that the more you listen to it, the more you pick up details you've missed the last time around and it really makes you appreciate the level of detail that's gone into this five-track beast. You can pick up 'Battle Jazz Classics II' from Cheapbeats' bandcamp, and you'll thank yourself later for doing so.
Bandcamp | Soundcloud | Twitter | Facebook


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Colonizing Sigma Draconis – Do you know how much paperwork is involved!!!
As I peck away at my next book I have come to the realization that colonizing a distant world will be a great challenge for humanity. And it won't be because of the distance, hostile environment, or technology required. It will be because of the bureaucracy, permits, and paperwork. Building a realisitc story with realistic characters for Sigma Draconis has been fun yet daunting at the same time. Really looking forward to how the final story turns out. I'm excited to get reactions from readers.
Nearly done with Act 1 (of3) for my next work Sigma Draconis. Can't wait to write the next two acts. Those are going to be hot!!! Some good old fashioned Sci-fi especially in Act 3. Mark my words!
Thanks for taking the time to look at my first book over the weekend. All the purchases and downloads are greatly appreciated.


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by this treatment of the blackout in Queens.
Let me start out by saying that I fully recognize the energy crisis in our fair country, and I freely admit that we are a very litigious society. I do not think that New Yorkers are better than anyone else. I do not equate an 'ickle blackout' with a hurricane. My household pays extra each month for green power. We have nary a non-EnergyStar appliance in our home. We use non-chemical cleaners, have cork tile in the kitchen, reuse grocery bags when I can't fit everything into my cloth one. I care about conservation. I practice conservation. There are things I wish I could do but am constrained by the co-op rules–I can't install solar panels. Can't plant a tree to shade my a/c. Can't police other residents of the building to ensure they are using their a/cs sparingly and refraining from running major appliances in the high-energy use hours. Most certainly can't run down to Times Square and turn out the damn lights that keep it daylight until, well, daylight.
This blackout, however, is nothing like the grocery running out of your favorite brand of bread. It bears no resemblance to camping. It has nothing to do with not having your organic coffee and sauna in the morning after you do your yoga video.
The blackout means that people are sitting in brick boxes stacked atop other brick boxes with buildings on every side. Most have no cross-ventilation. Most were built in the 1970's where a/c may not have been prevalent, but fans certainly were. Many elderly people and families with small children live in Queens. The number without power at the peak was up around 25,000 customers (an estimated 100,000 people.) The blackout began on Monday, July 17th. Today, July 27th, power has been restored to everyone. And the reason for the blackout? Poorly maintained subterranean cables. A big part of the reason it was not fixed sooner? Initial Con Ed estimates were that 2500 people were without power based on complaint calls.
So let's just get all of this straight. Con Ed underestimated the initial number of customers without powerin a region historically heavily hit by outages, using cables that are fragile and aging affecting people in varying degrees from all walks of life. 90,000 additional people were living on reduced voltage meaning that elevators, air conditioners and refrigerators did not work.
You're absolutely right, guys. I don't know what I was thinking. It's exactly like that time I went to the grocery store and had to buy white bread instead of whole grain. Seriously, fuck you and your high horse.
Hard as I try, I'm still rankled
July 27, 2006 by Jen
by this treatment of the blackout in Queens.
12 Responses
We've had some blackouts in my part of the country as well. Nothing as bad as Queens but in the 110/115° temps for days on end, people are dying.
The Fresno paper said the coroner can't keep up.
PG&E could give Con-Ed a run for their money on mismanagement.
Our temperatures haven't been anywhere near as high as you guys have had out in CA either. It's scary stuff.
Hopefully all of this blackout nonsense causes all of these power companies' management issues to be exposed before too many people die.
While I thought the bread comment was a bit much, I still have my doubts that litigation will solve any of these problems.
My points were made to illustrate that these problems don't happen on their own-they are created by hordes of people who demand plentiful, CHEAP power, regardless of HOW it happens. People tend to be shortsighted, and it frustrates the hell out of me.
I'm also sick to death of the constant blame game, where it's ALWAYS someone elses fault. American needs to start picking up it's britches, and taking some accountability for the problems they create.
That said, the face that your local governments have done nothing (seemingly) to help is a travesty. I don't expect Big business to help me. I expect my government to prevent people from dying.
something to think about during the next mayoral race at least…
I know I sound really callous about this, but these problems don't just happen, and people suffer so much from it….
Thordora, I have my doubts as well about litigation. What I took offense to was the belittling of the situation in the original post and in some of the subsequent comments.
I absolutely agree with you that problems don't happen on their own. I am often ashamed to live in a society full of so many over-entitled, selfish, head-in-the-sand people. I was just telling my husband this morning that it is highly unfortunate that people across the country are dying in the heat, and nobody seems to want to take a step back and try to solve the root of the problem because it takes too much thought and effort.
TThe NYC political system with its borough-based intricacies is another issue altogether. The mayor governs all of NYC, but there are also Borough Presidents, various city council members, etc. Getting anything done in NYC government is damn near impossible. It's not an excuse, but oftentimes things get lost in the mix. In the case of the blackout, the government response would likely have been different had Con Ed not initially understated the number of customers affected (2500 vs. 25,000 is a huge difference.)
I don't think it's callous to look at the overreaching problem though. It's the only way to find any answers to the whole mess. When everybody gets bogged down in the woe-is-me crap, nothing gets done.
man, I'm just glad I don't live in a huge city anymore. There is ZERO accountability it seems.
thordora, I often wish I didn't live in a big city. The issue of *who* is accountable is such a touchy one that it often seems that figuring out who is unworthy of the time or attention.
I would like to think that I am accountable for the energy I use, but it just as easily could've been me suffering in a blackout with a child as someone who runs central air at 70 degrees fahrenheit with no regard for conservation measures.
It's one of the trade-offs we make for cheap public transit, heavy educational resources, etc. I'm still not sure if it's a worthwhile one. A trick bag for sure. I would like to be able to say that my energy company is prepared for eventualities of heat waves and that my neighbors are mindful of their part in energy usage. I'm not sure there is any place in the US where that is the case.
Where I live, you can always just burn shit.
I miss Toronto, but I can't imagine being in the city with small kids. Just stuff like the ton of goldfinches feeding off thistles in my backyard-city living wouldn't provide that..
Cities need to plan better, and NOT rely on corporations. I'd be mad at government, but as Ann and I have discussed, there really is no one to "trust" anymore.
It's sad, and it's scary.
Jen, thanks for your comment on isamericaburning.
The Christian reconstructionists I'm always ranting about base their beliefs on an extreme form of Calvinism.
(Nothing like your Presbyterians).
We seem to be corresponding via comment boxes.
It's not only scary, it's sad. I get a little tired of being slammed for being Christian by people who don't know me or what I believe.
We're not all alike by any means.
on August 12, 2006 at 2:31 pm | Anonymous
Interesting site. Useful information. Bookmarked.
on August 17, 2006 at 6:30 pm | Anonymous
Your site is on top of my favourites – Great work I like it.
I jsut stumbled across your site thru a wordpress link. I really enjoyed reading your blog. Although I will come back again to spend some more time. Cheers


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The Design Digest: March 28, 2019
Welcome to my first Design Digest, a new regular roundup of the latest news and resources for modern creatives. Every Thursday, I'll be sharing with you news from the tech world, makers worth watching and design resources that rock.
Tech News You Can Use
It seems fitting to start with this insightful article from Jon Evans about social media. I recently did a post about taking a break from social, so this was timely. h/t to Jessica C.
Are you using the Social Warfare plugin on your website? Might be a good idea to catch up on the latest security issues and vulnerabilities plaguing this and another handy plugin. Pinterest is going public and there is a lot of talk about what it actually is. Is it a social platform? A visual search engine? Here are two takes, one from the New York Times and another from The Verge.
Am I the only one perplexed by the MailChimp breakup with Shopify. I know a ton of makers who rely on both, so this is big news. It's messy. Looks like there's some privacy issues involved and this: Mailchimp just bought an e-commerce platform too. Never heard of Lemonstand? Me neither.
Makers To Watch
Ok, here's what you came here for. Here are some makers that you need to watch. They're doing things and doing things well!
Gray Magazine featured Fernweh Woodwork last week and I AM SO IN LOVE WITH THESE CHAIRS! I'm planning a full interview with Justin Nelson, founder of this Bend, Oregon-based studio. It's the kind of chair that makes you want to sit and contemplate life with a warm cup of Earl Gray. Discover Fernweh here.
I took my weekly dip into the Dezeen pool and found these bowls by architect Frida Escobed and textiles from Apparatus to be worthy of a second look. The bowls were made for made for tequila brand Maestro Dobel and handmade from rainbow obsidian. New York-based Apparatus does a lot of incredible work, as you can see from their website. These stunning textiles celebrate creative director Gabriel Hendifar's Persian heritage.
PT. 9 – THE BEAUTY OF SINGLE NEEDLE WORK from RALEIGH DENIM WORKSHOP
Get lost watching the Raleigh Denim Tiny Films series. It's a great way to see why handmade denim is so worth the pennies. I'm a bit obsessed with their Chore coat right now. I need this in my life. If you're looking for a great podcast, try Clever's interview with artist and designer Zoë Pawlak then check out her incredible art here. For more textiles, ikat weaving is on display over at Cool Hunting. The detailing is amazing!
Lee Coren has textile wall art! Because her vegan handmade bags and accessories are not amazing enough, Lee had to go and add these lovelies. I'll have a comprehensive review of her new goodies soon. Until then, enjoy.
Good Design
I'm a bit obsessed with non-alcoholic spirits right now. I discovered Seedlip last year and now I can add Ellie Webb's Caleño to my collection. Discovered via The Dieline, this new Juniper & Inca Berry beverage sounds like a winner and the branding by Design Bridge is fire.
Here are some freebies: Anson is a free bold geometric typeface from London's Hanson Method. Looks like a great font for posters and maybe a branding project. Here are two great free mock-ups: a stationery mock up from Free Design Resources and this poster template from my favs over at Mr Mockup.
Lastly, here's one more free font: Aintonio. Because you can never have too many handwritten scripts, can you? Have a maker to share with me? Some cool design news? Leave a link below or drop me a note at firstname.lastname@example.com


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X-Men Origins: Wolverine
The title of this movie leaves little to theimagination when it comes to the movie's premise. This is Wolverine's backstory. This is where the audience discover what made Wolverine Wolverine. And for the fnaboys, this is the opportunity to assess how well the writers have managed to tell a story they will presumably know intimately. I say "intimately" quitely deliberately as there is no way that the story of Wolverine's developement is unrelated to the puberty most readers will have been encountering while enjoying the original comics. It is probably best, if perhaps a little too much protest, that I point out now that I discount myself from the fanboydom that I imagine will have it's claws ready to pierce holes in the narrative.
The movie covers a period of about 150 years; from James Logan's sickly childhood and sibling bullying, to the memory loss and wilderness years after which we find him in the first X-Men movie. The narrative the movie follows is clearly not true to the comic book history. Cinema demands a tighter structure with a finer weave. But most of the significant events and moments of his life are present, if not quite the way Marvel originally structured them.
Following a question-raising preamble, we are luckily, from a viewer perspective, granted permission to rush through the first 140 years in an epic and suitable opening credits. As if flicking through a comic, we witness Logan's evolution as both a fighter and a brother. We get to see his humanity while witnessing his brother's incremental self-destruction. This developmental disparity triggers the first events that later prove to be Wolverine's motivation.
It will probably come as no surprise to anyone, least of all the fanboys, that there is family conflict, a love story, morality issues and a man struggling to discover and embrace his own identity sewn into the resultant story.
For the sake of personal safety, I shall end my retelling there and move on to my more judgemental interpretation.
The single most impressive aspect of this movie for me (the non-fanboy) is the casting. I think it is generally accepted that Hugh Jackman makes an acceptable Woverine. But much of the other casting has fueled internet debates for the last year or so. Liev Schreiber is perfectly cast. He is an actor who is consistently unrecognised in movies and he here finally gains the opportunity to let rip with a performance saved from hamminess by his astonishing physicality.
Underused, but almost suspiciously so, is Taylor Kitsch's Gambit. Clearly a charismatic character initially embued an alluring danger, he suffers a little because of the script in later scenes. But it is hard not to see that a franchise like X-Men would never waste the opportunity to develope such a sexy and dangerous character.
The most significant of the other roles is probably Stryker. Established by Brian Cox in X2, it was always a role that I thought suffered by it's proximity to the similar character in the Bourne trilogy. Taken on in this movie by the visually more believable Danny Huston, the character's history and hand in creation of Wolverine are far better explored.
It's worth noting some of the other supporting cast's contribution, as people like first timer, and prepostorously named, Will.I.Am are actually a pleasant surprise. Will, specifically, is stunningly understated. A far cry from his usual stage persona. Ryan Renolds, a personal favourite of mine, is massively underused. But is luckily left with enough ambiguity to his narrative end that there is plausible reason for return.
It would be unrealistic to expect a flawless summer blockbuster, especially this early on in the season. So it is with pleasure that I intend to wave a pointer at the few flaws. There are narrative failings (why does Wolverine suddenly stop aging at about 38? for example), it is over-edited during some of the fight sequences (the bumph I was handed upon entering the cinema suggests that many of the actors went to great lengths to develope their skills which are ultimately massively underused), there is a little repetition and it occassionally verges on the trite when it comes to certain narrative explanations. But these really are small criticisms of a movie which consistently surprises.
This is a big budget, big expectation, little boy's summer blockbuster. It delivers what it promises and a little bit more. Not enough to stake a claim in motion picture history, but enough to ensure some DVD sales and a sequel (or further Origins movie). There are enough in-jokes and nods of the head to please most dilligent fans, but there will inevitably be some that frown upon it, just because they can. This is a great fun movie that will only endear you to the eponymous character that little bit more.


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91.20 €
Only a fool believes filters can trick his Instagram followers. A wise man sips Man Tea for real life muscles.
Traditionally my ingredients have been used to assist with:
Hong Cha (Black Tea 22%), Lu Cha (green tea 22%), Ji Li (Bindii), Ren Shen Hua (Ginseng), Gan Cao (Licorice Root)


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Megan te Boekhorst isn't your typical 23-year-old.
She has a double major in communications and interactive art and technology under her belt. She runs a non-profit business that supports a scholarship program for Grade 12 students. And if that's not impressive enough, the New West resident also organizes and hosts goal coaching workshops, including one at River Market on April 11.
"It's a really transformative process. You get to engage in your life and what you want it to look like in 10 years," te Boekhorst told the Record. "We'll do some brainstorming, come up with a list of core values, do some meditative visioning sessions and just have fun."
The recent SFU graduate said becoming a mentor happened on a whim and slightly out of boredom. "I was kind of tired of the same routine in school, having taken six years to complete my degree, and I wanted to challenge myself," she said.
That challenge came in the form of a personal blog called "In a sequoia." Since its launch last summer, the online platform has grown from a few posts to a place where subscribers can purchase goal coaching packages. Whether it's a 90-minute Skype call or four one-on-one meetings throughout the year, te Boekhorst boasts of getting clients living a life of pure bliss, "a life that feels, to its core, right."
But the path to success hasn't always been an easy one.
The self-proclaimed bohemian feminist admits she has struggled with depression and an eating disorder over the years. As a way of ending the stigma around mental health, she launched the Vibrant Lives Scholarship Foundation earlier this month.
"When I was in high school, I was a big nerd, I was on student council and always looking for new opportunities. I know there are a lot of kids out there like me who have such potential in them, but it just needs to be tapped a bit more," she added. "The scholarship is meant for Grade 12's who are working to end that stigma. I'll accept a painting, a piece of writing, perhaps a proposal for a project, basically anything they think helps the cause."
Funds for the scholarship are being raised through Karuna, a product line te Boekhorst started specifically to support the foundation. The line currently has a natural coffee scrub on the market that retails for $28.
"Karuna will also be at Got Craft in Vancouver in May, so that's a big event I'm looking forward to," she said, adding the scrub was recently picked up by a store in Ontario. The first scholarship will be handed out in 2016, hopefully in the amount of $500 or higher, according to te Boekhorst.
Her efforts over the next month, however, will be geared towards organizing a goal coaching workshop at the Quay.
"New West is such a beautiful community and the River Market is such a gorgeous space to host it at," she said.
Find Your Goal'd runs from 1 until 3 p.m on Saturday, April 11 in the community square (second floor) at River Market. Cost is $27, but space is limited, so you're asked to arrive 15 minutes early to register. You can also visit www.inasequoia.com to sign up online.
When asked what roadblocks people have when it comes to achieving their goals, te Boekhorst said it's finding that balance.
"Too many of us are trying to achieve a definition of balance society believes in, and not what balance actually reflects for them. Find Your Goal'd is about digging deep and finding out what you truly believe in."


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Response to White Paper Educational Excellence Everywhere
10 May 2016 in Education > Education & Schools >
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The ADCS response to the Department for Education White Paper Educational Excellence Everywhere.
The response includes the ADCS perspective on the announcement made by the Secretary of State on Friday 6 May 2016 that she is not now planning to legislate to compel all schools to become academies.
Tags assigned to this article:EDUCATION 166 EAPC 77 ACADEMIES 8
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Are you looking for GED® testing locations in Wild Rose WI? The GED® test is only given in pencil and paper format. Usually in order to take the GED® examination the student must be over 17 years old. The GED® testing cost in the state of Wisconsin typically ranges from $40-$90.
The exam covers five topics: language arts (reading), language arts (writing), mathematics, science and social studies. Each section of the test awards a score between 200 and 800. Most states require students to obtain a score of 410 on each of the five topics and an overall average of 450 for the exam to pass.
Usually GED® testing sites in Wild Rose WI are offered at:
- Wild Rose Independent School District
- Wild Rose Community Colleges
- Wild Rose High Schools
- Wild Rose Adult Learning Centers