{'fraction_non_alphanumeric': 0.041995231982395013, 'fraction_numerical': 0.02948835503392628, 'mean_word_length': 5.1148239515586456, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Defence transcriptome assembly and pathogenesis related gene family analysis in Pinus tecunumanii (low elevation)

Background Fusarium circinatum is a pressing threat to the cultivation of many economically important pine tree species. Efforts to develop effective disease management strategies can be aided by investigating the molecular mechanisms involved in the host-pathogen interaction between F. circinatum and pine species. Pinus tecunumanii and Pinus patula are two closely related tropical pine species that differ widely in their resistance to F. circinatum challenge, being resistant and susceptible respectively, providing the potential for a useful pathosystem to investigate the molecular responses underlying resistance to F. circinatum. However, no genomic resources are available for P. tecunumanii. Pathogenesis-related proteins are classes of proteins that play important roles in plant-microbe interactions, e.g. chitinases; proteins that break down the major structural component of fungal cell walls. Generating a reference sequence for P. tecunumanii and characterizing pathogenesis related gene families in these two pine species is an important step towards unravelling the pine-F. circinatum interaction. Results Eight reference based and 12 de novo assembled transcriptomes were produced, for juvenile shoot tissue from both species. EvidentialGene pipeline redundancy reduction, expression filtering, protein clustering and taxonomic filtering produced a 50 Mb shoot transcriptome consisting of 28,621 contigs for P. tecunumanii and a 72 Mb shoot transcriptome consisting of 52,735 contigs for P. patula. Predicted protein sequences encoded by the assembled transcriptomes were clustered with reference proteomes from 92 other species to identify pathogenesis related gene families in P. patula, P. tecunumanii and other pine species. Conclusions The P. tecunumanii transcriptome is the first gene catalogue for the species, representing an important resource for studying resistance to the pitch canker pathogen, F. circinatum. This study also constitutes, to our knowledge, the largest index of gymnosperm PR-genes to date. Electronic supplementary material The online version of this article (10.1186/s12864-018-5015-0) contains supplementary material, which is available to authorized users.


Background
The pitch canker fungus Fusarium circinatum Nirenberg and O'Donnell [1] has resulted in losses in pine plantations, seed orchards and nurseries worldwide [2,3]. With a host range of more than 60 Pinus spp. [2], many of which are commercially important, this pathogen poses a significant threat to both forestry and conservation. The wide range of inter-and intraspecific variation in susceptibility of Pinus spp. to F. circinatum [4,5] holds the potential for effective disease management through genetic resistance.
Development of more resistant families and genotypes for susceptible Pinus spp. [6] as well as selection and generation of resistant hybrids [7] have shown promise for long-term management of F. circinatum. Breeding and selection approaches, however, are time consuming and use of resistant genotypes could select for novel pathotypes [2]. Knowledge of the molecular mechanisms underlying resistance could expedite development of resistant genotypes and improve the effectiveness of genetic resistance.
The majority of information related to plant-pathogen interactions originate from studies on model plant species such as Arabidopsis thaliana and Nicotiana spp. [8]. Comparatively few studies have investigated these interactions in trees, and even less in gymnosperms. The main barrier to studying defence responses in non-model organisms in the past was the need for a reference genome. Among plants, particularly gymnosperms, genome size and complexity hindered sequencing and assembly of a reference genome. The availability of next-generation sequencing technology has circumvented this barrier for non-model organisms by enabling transcriptome assembly from high-throughput RNA sequencing (RNA-seq) data [9,10].
While recent transcriptomic studies investigated the Pinus-F. circinatum interaction [11,12], both studies focussed on susceptible species. Knowledge of defence mechanisms in the resistant interaction is lacking. The low elevation provenance (LE) of Tecun Uman Pine (P. tecunumanii Eguiluz & J. P. Perry) is an economically important F. circinatum resistant [4] Pinus species that has shown promise in hybridisation trials with P. patula, making it a good candidate for use as a model of resistance.
Studies on model plants have shown that molecular defence mechanisms consist of complex, multi-level processes [8]. In short, pathogen recognition occurs either through membrane binding of bound recognition receptors (PRRs) to pathogen−/damage−/microbe-associated molecular patterns (PAMPs, DAMPs, MAMPs) or through interaction between host resistance (R) genes and pathogen secreted effector proteins [13][14][15]. Pathogen perception results in activation of signal transduction cascades that initiate various local and systemic host defence responses including: reactive oxygen species (ROS) generation, cell wall modification, phytohormone defence pathways, defence-related protein expression and induction of systemic acquired resistance (SAR) [16][17][18][19][20][21]. An important group of markers for SAR are the pathogenesis-related (PR) proteins, a group of proteins identified due to their induction during biotic stress and direct antimicrobial activity of some PR gene family members [21][22][23]. There are currently 17 described PR gene families, classified by amino acid sequence and enzymatic activity, numbered in order of their description [22,23]. Despite being the first family identified, a potential mode of action for the PR-1 proteins in plant defence was only recently described [24]. PR-1 proteins were shown to bind and sequester sterols, directly inhibiting sterol-auxotrophic pathogens as well as sterol-prototrophic pathogens with compromised sterol biosynthesis [24]. The PR-9 family are peroxidases that could be involved in cell wall reinforcement through catalysing lignification [25]. This could result in enhanced resistance against multiple pathogens [22,23]. There are also two putative novel PR gene families, the PR-18 carbohydrate oxidases identified from Helianthus annuus and Lactuca sativa [26], and the PR-19 anti-microbial peptides identified from Macadamia integrifolia and Pinus sylvestris [27,28].
This study aimed to produce a reference sequence for P. tecunumanii transcriptome profiling and a comparable reference for P. patula, as a resource for further investigation of the Pinus-F. circinatum host pathogen interaction, and to use the generated resources to identify the PR-gene families within these species. High quality reference transcriptomes were assembled, including the first gene catalogue for P. tecunumanii to date. These references were used to identify 639 and 785 PR gene candidates in P. tecunumanii and P. patula respectively.

Plant material and inoculation trial
Four month old low elevation (LE) Pinus tecunumanii seedlings, representing four open pollinated families, were sourced from SAPPI (Shaw Research Centre, South Africa) and 4 month old P. patula seedlings from a single open pollinated family were sourced from Mondi Forests (Trahar Technology Centre, South Africa). Seedlings were maintained in the Forestry and Agricultural Biotechnology Institute (FABI) Fusarium screening greenhouse at the University of Pretoria experimental unit. Pathogen challenge was performed as described in Visser et al. [12]. Briefly, seedlings were inoculated with F. circinatum isolate FCC3579, harvested from cultures on ½ strength potato dextrose agar (½ PDA; Merck) washed with 15% (v/v) sterile glycerol and diluted to 5 × 10 4 spores/mL, or mock-inoculated with 15% (v/v) sterile glycerol, at 6 months old by clipping the apical bud and depositing 10 μL inoculum on the wound. Tissue was harvested from seedlings at three and 7 days post inoculation (dpi) for three biological replicates (BR) per group. The top 1 cm of shoot tissue, from the point of inoculation, from 16 seedlings was pooled for each biological replicate. Harvested tissue was flash frozen in liquid nitrogen and stored at − 80°C until use. Disease progression was monitored by measuring stem length and stem discolouration from the point of inoculation over 6 weeks to calculate the percentage live stem ([stem length (mm)-stem discolouration (mm)]/stem length (mm)). The difference in mean percentage live stem between inoculated and mock-inoculated plants at each time point was analysed using a Kruskal-Wallis rank sum test (p < 0.05). Re-isolation of the pathogen was performed by placing tissue harvested at 14 dpi on ½ PDA and observing culture morphology after 7 days.

RNA isolation and sequencing
Total RNA was extracted from homogenised samples, ground in a mortar and pestle, using the Plant/Fungi RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manufacturer's instructions, with the inclusion of acid washed glass beads during lysis to improve cell break down. Extracted samples were assessed using a Bio-Rad Experion™ automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA) to determine sample concentration as well as ensure sample integrity (RNA Integrity Number > 7.0) and purity (absence of genomic DNA).
Inoculated and mock-inoculated samples, for both time points, were sent to Novogene (Novogene Corporation Inc., Chula Vista, CA, USA) for strand specific RNA-Sequencing on an Illumina HiSeq2500 (Illumina, San Diego, CA, USA). Samples were sequenced in three sets for each species, to optimise read length for transcriptome assembly (Additional file 1: Table S1). Sample set one consisted of a single sample of pooled RNA from all six three dpi samples (500 bp insert, PE250 sequencing). Sample set two consisted of the six samples (3 BR inoculated, 3 BR mock-inoculated) for three dpi (300 bp insert, PE125 sequencing). Sample set three consisted of the six samples (3 BR inoculated, 3 BR mock-inoculated) for seven dpi (300 bp insert, PE150 sequencing).

Transcriptome assembly and annotation
RNA-sequencing libraries obtained from Novogene were assessed using FastQC [29]. Preliminary transcriptome assembly was performed using Trinity v2.2.0 [30], on two datasets (Additional file 1: Table S1). Reads were quality trimmed and filtered using trimmomatic v0.32 (Additional file 1: Table S2) [31]. For each species, all 13 trimmed and filtered libraries were combined to produce a full dataset. In silico read normalisation, to a maximum coverage of 100, was performed using Trinity on the full dataset to produce a normalised dataset. Both datasets were used for transcriptome assembly. Twenty strand specific preliminary assemblies were produced, with a minimum contig length of 350, using two different: k-mer sizes (25 & 31), stringencies for de Bruijn graph construction, and algorithms for transcript reconstruction (Additional file 1: Table S2). The normalised dataset for both species was mapped against the P. taeda v1.01 draft genome assembly using GSNAP 2016-11-07 (Genomic Short Read Alignment Program; Additional file 1: Table S2) [32] for genome guided assembly. Preliminary assemblies were filtered to obtain the longest isoform per locus, an assembly code (Additional file 1: Table S2) added to the transcript identifiers, and combined to form a highly redundant assembly. Redundancy was reduced using the Eviden-tialGene [33] tr2aacds pipeline v2016.07.11. Assembly statistics were calculated using Transrate v1.0.3 [34].
Primary transcripts from the tr2aacds pipeline were annotated using the eukaryotic non-model transcriptome annotation pipeline v0.7.3.2 (EnTAP) [35]. In short, the normalised dataset was mapped to the transcripts using Bowtie2 v2.3.0 [36]. Transcript expression was calculated using RSEM v1.3.0 (RNA-Seq by Expectation-Maximum) [37] and transcripts with an FPKM < 1 (Fragments Per Kilobase of transcript per Million mapped reads) were discarded. Frame prediction was performed on transcripts with detectable expression using GeneMarkS-T v5.1 March 2014 [38], transcripts without a predicted reading frame were discarded, and predicted proteins clustered to 90% identity using Usearch v9.0.2132 [39]. Diamond v0.8.31 [40] was used to obtain protein BLAST alignments for predicted protein sequences of the remaining transcripts against: the RefSeq Complete Protein database, the UniProt-KB/Swissprot database, and the Arabidopsis thaliana proteome; using a minimum query coverage of 80%, a minimum target coverage of 60% and a minimum e-value of 1e-4. Only the best hits across all three databases were retained. Functional annotation, Gene Ontology (GO) annotation relative to the full GO database and orthologous group assignment, was performed using EggNOG 4.5 [41]. Unannotated sequences were discarded as erroneously assembled transcripts. Non-pine origin sequences were removed based on best hit taxonomy (discarding sequences with best hits from archaea, fungi, insects, bacteria, viruses and vertebrates), for sequences with BLASTp alignments, and orthologous group taxonomic scope (discarding sequences not associated with Viridiplantae, Eukaryota or Ancestor lineages), for sequences without BLASTp alignments, to produce the P. tecunumanii (Pnte_v1.0) and P. patula (Pipt_v2.0) draft transcriptome assemblies. For ease of comparison, GO terms were normalised to level two of the classification tree. Putative TAIR10 identifiers were assigned to transcripts based on best hits to the A. thaliana proteome. Completeness and contiguity of the assembly was determined using BUSCO v3.0 (Benchmarking of Single-Copy Orthologs; [42]) against the eukaryote_odb9 (303 BUSCOs) and embryophyta_odb9 (1440 BUSCOs) lineages and compared to 26 other gymnosperm assemblies (Additional file 1: Table S3) obtained from the Tree-Genes database [43].

Results and discussion
F. circinatum disease progression on P. tecunumanii and P. patula Seedlings of P. tecunmanii and P. patula were inoculated with F. circinatum and the decline in percentage green stem monitored over the course of 6 weeks (Fig. 1). Stem discolouration, at the point of inoculation, was visible on all seedlings at 7 days post inoculation with no discernible difference in lesion colour or length between inoculated and mock-inoculated seedlings. By 14 dpi a clear difference in lesion colouration was visible between treatment groups for both species, with inoculated seedlings displaying purple lesions, and a significant difference in lesion length was observed between P. patula treatment groups (p < 0.05). Initial mortality of P. patula seedlings was observed at 21 dpi. By 42 dpi chlorosis prevented further measurements of P. patula seedlings. While a significant difference in percentage green stem was observed for P. tecunumanii seedlings after 14 dpi, this was due to more wound discolouration on mock-inoculated relative to inoculated seedlings and all seedling had more than 98% live stem. The difference in stem discolouration between P. tecunumanii and P. patula is therefore consistent with the classification of P. tecunumanii as a resistant host [4].

Transcriptome assembly
Illumina sequencing and subsequent filtering by Novogene produced ca. 530-and ca. 570 million clean read pairs for P. tecunumanii (LE) and P. patula, respectively (Additional file 1: Table S1). Following read trimming and filtering, the P. tecunumanii (LE) full dataset contained a total of ca. 870 million reads (81.6% of the clean reads) representing ca. 120 Gb of sequence; the P. patula full dataset contained ca. 950 million reads (83.5% of clean reads) representing ca. 130 Gb of sequence. Normalisation to 100X read coverage retained ca. 54 million reads (6.2% of the full dataset) in the normalised dataset for P. tecunumanii (LE) and ca. 77 million reads (8.1% of the full dataset) in the normalised dataset for P. patula.
Four de novo assemblies, with two different k-mer lengths and stringencies, were constructed using the full dataset (Fig. 2). The normalised dataset was used to construct eight de novo and eight genome guided assemblies; using two different k-mer lengths, stringencies and reconstruction algorithms. Preliminary assembly generated 20 transcriptomes containing a total of 3,023,703 transcripts for P. tecunumanii (LE) and 5,868,982 transcripts for P. patula, after filtering assemblies to only retain the longest isoform per gene (Fig. 2).
Redundancy across the preliminary transcripts was reduced based on coding potential using the tr2aacds pipeline from EvidentialGene. Following open reading frame (ORF) prediction, 36.2% and 37.4% of input sequences were classified as non-redundant, for P. tecunumanii (LE) and P. patula respectively, of which 29.6% and 37.4% were retained as informative after substring dereplication using CD-HIT-EST v4.6.1 [48,49]. Removal of low quality transcripts resulted in 182,681 sequences grouped into 91,552 loci for P. tecunumanii (LE), defined as the PnteEviGene assembly, and 542,459 sequences grouped into 325,974 loci for P. patula, defined as the PiptEviGene assembly (Fig. 2). When compared to the P. tecunmanii (LE) Trinity assemblies the PnteEvi-Gene assembly showed a better ratio of assembled transcripts to overall assembly length as reflected by its high N50 and mean contig length, which was greater than any of the input assemblies, indicating that the Eviden-tialGene pipeline successfully selected the longest assembled isoforms from among the Trinity assemblies. For P. patula on the other hand, comparison of the EviGene assembly to the Trinity assemblies showed less of an improvement. Still, the PiptEviGene assembly, similar to the PnteEviGene assembly, had a higher average transcript length and N50 than any of the Trinity assemblies.

Annotation
Expression based filtering of the PnteEviGene and PiptE-viGene assemblies retained ca. 85% and ca. 63% of transcripts (Table 1). GeneMarkS-T successfully predicted coding regions for ca. 95% of expressed transcripts for both species, though the P. patula assembly contained a ca. 10% lower proportion of complete reading frames compared to the P. tecunumanii assembly. Protein clustering retained ca. 77% of the EvidentialGene transcripts for P. tecunumanii, while only ca. 51% of P. patula transcripts were retained, indicating that greater redundancy was retained in the P. patula assembly during CD-hit--EST clustering.
Gene Ontology (GO) terms were assigned to ca. 57% (16,197) of P. tecunumanii contigs; 11,157 contigs with biological process (BP) terms, 8086 contigs with molecular function (MF) terms, and 15,077 contigs with cellular compartment (CC) terms (Table 1). For P. patula, GO terms were assigned to ca. 64% (33,712) of contigs; 24,709 contigs with BP terms, 18,956 contigs with MF terms and 31,760 contigs with CC terms. Among the top MF terms identified for both species were hydrolase activity, transferase activity, ion binding, protein binding and two parent terms for nucleic acid binding (organic cyclic acid binding, heterocyclic compound binding), similar to what has been observed for other conifer reference sequences [11,12,50,51] (Fig. 3). The most common BP terms were indicative of rapid and extensive metabolic activity within the analysed tissue, as has been shown for the interaction between P. radiata and F. circinatum [11].
Completeness (total complete and fragmented BUS-COs), contiguity (total complete BUSCOs/total complete and fragmented BUSCOs) and redundancy (duplicated BUSCOs) of the assemblies was determined by comparison to the BUSCO eukaryote (n = 303) and embryophyte (n = 1440) lineage datasets [42] (Fig. 4). where C refers to the percentage of complete BUSCOs, S refers to the percentage of complete and single copy BUSCOs, D refers to complete and duplicated BUSCOs, F refers to fragmented BUSCOs and M refers to missing BUSCOs) showed high completeness (97.7% and 98.7%) and contiguity (99.0% and 99.0%) for both assemblies. While redundancy (38.3% and 54.1%) was also high for both assemblies, a similar trend was observed for the other gymnosperm assemblies analysed (Fig. 4) .6%],F:2.6%,M:9.7%) showed lower redundancy (9.9% and 11.6%). The completeness (89.8% and 90.3%) and contiguity (96.9% and 97.2%) of Pnte_v1.0 and Pipt_v2.0 for the embryophyte lineage was the highest amongst analysed assemblies. The high redundancy in the assemblies likely reflects assembled haplotypes [42] due to the high genetic variance present in the populations from which the data was generated, indicating that more variance was present in the P. patula seedlings compared to the P. tecunumanii (LE) seedlings.
PR genes identified in A. thaliana, B. distachyon, P. trichocarpa, O. sativa and V. vinifera [47] were used to identify putative PR orthogroups for 16 of the 17 currently classified plant PR classes, as well as the two Percentages relative to clustered GeneMarkS-T assemblies for EviGene columns and relative to total sequences for Pnte_v1.0 and Pipt_v2.0 putative novel classes (Additional file 1: Table S10). PR-15 and PR-16 were both classified as PR-16 due to their high homology and the classification of PR-15 as monocot specific [52]. Putative PR orthogroups were identified for 15 PR families in P. tecunumanii and 16 PR families in P. patula (Additional file 1: Table S11). PR-12 and -13 members were absent from both assemblies. Both of these appear to be angiosperm specific as the PR-12 orthogroup only contained sequences from the basal angiosperm A. trichopoda and dicots, while the PR-13 orthogroup only contained sequences from monocots and brassicaceae, similar to what has previously been observed [47,53]. The PR-6 family was absent from the P. tecunumanii assembly, indicative of insufficient expression for assembly rather than absence from the genome. Two putative PR-6 genes were identified in the P. patula assembly, interrogation of EggNOG annotations identified the "potato-inhibitor I family domain" found in the PR-6 type member in both sequences. The only PR classes for which putative members were present in all species were PR-2 and PR-9, although no single orthogroup was present in all species. Putative PR-1 orthologues were only absent from some of the chlorophyte species. PR-7 members were present for all viridiplantae species with PR-8, − 11 and − 18, while not identified for all species, similarly present across all viridiplantae lineages. The PR-10 and PR-17 orthogroups only contained sequences from embryophyte species and the PR-14 orthogroup only contained tracheophyte sequences. While some chlorophyte sequences were present, the PR-3, − 5, Fig. 3 Distribution of biological process and molecular function Gene Ontology (GO) terms in the assembled transcriptomes. Green -Pinus tecunumanii assembly. Blue -Pinus patula assembly and − 16 orthogroup sequences were mostly spread evenly between the embryophyte lineages. The majority of identified PR-4 and -6 sequences were identified in angiosperms. Despite being initially identified from a dicot, putative PR-19 sequences, as expected, were only identified in the lycophyte S. moellendorfii, coniferous gymnosperms, the basal angiosperm A. trichopoda and in low numbers in monocots [54].
Potential lineage specific chitinase gene family expansions were observed when comparing two of the identified PR-3 orthogroups (OG0000134 and OG0000642), as well as the two PR-8 orthogroups identified (Fig. 5).
When looking at the PR-3 orthogroups, both groups appear present in relatively equal amounts in gymnosperms, while OG0000134 is more prominent in angiosperms. The opposite is seen for the two PR-8 groups, with both relatively similar in angiosperms but OG0000252 more prominent in gymnosperms. Interestingly, in both cases the pattern observed for the brassicales is similar to the gymnosperms not the angiosperms.
More putative PR genes were identified in P. patula (801) relative to P. tecunumanii (646). While lower amounts of P. tecunumanii transcripts in an orthogroup is likely due to the difference in the number of transcripts assembled, the Fig. 4 Comparison of completeness, contiguity and redundancy for assembled transcriptomes (underlined) to other available gymnosperm assemblies. BUSCOs were identified for the (a) eukaryotic (n = 303) and (b) embryophyta (n = 1440) lineage datasets. The primary y-axis refers to the percentage of BUSCOs in each category. The secondary, dashed, y-axis refers to the total contig count in each assembly reverse could indicate transcripts absent from the P. patula defence response. P. patula had more transcripts for all PR gene classes identified except the PR-4 chitinase family (OG0001724), for which 3 transcripts were identified in both species (Fig. 6). The number of transcripts identified for the PR-5, PR-10, PR-11, PR-14, PR-17, PR-18 and PR-19 families, were very similar between species, with at most five transcripts more in P. patula. When comparing the four PR-5 orthogroups present in both species, OG0000039 and OG0001011 had the same amount of transcripts and OG0000062 had more P. patula transcripts, while OG0000094 had one more P. tecunumanii transcript. Of the 51 PR orthogroups containing transcripts from either species, 3 only had P. patula transcripts, 26 had more P. patula than P. tecunumanii transcripts, 14 had the same amount of transcripts and only 8 had more P. tecunumanii transcripts. Similar to the PR-5 orthogroup (OG0000094), two PR-2 (OG0000138; OG0000235) and two PR-9 (OG0000400; OG0002881) orthogroups had one more P. tecunumanii transcript. An additional two PR-9 (OG0000025; OG0000107) and one PR-1 (OG0000244) orthogroup had two more transcripts from P. tecunumanii compared to P. patula. The relative expansion observed in four of the PR-9 peroxidase orthogroups could indicate a more robust cell wall reinforcement or oxidative burst response in P. tecunumanii, however, this remains to be functionally determined.  Authors' contributions EAV performed the experimental work, data analysis, interpretation and drafted the manuscript. SN conceived the study, obtained funding to support the research, participated in study design and coordination, and assisted in biological interpretation of data and drafting of the manuscript. JLW provided technical support and computational resources for data analysis, participated in study design, and assisted with evaluation of the manuscript. AAM participated in the experimental design of the study and assisted with critical evaluation of the manuscript. All authors have read and approved the final version of the manuscript.
Ethics approval and consent to participate Not applicable. Pine seedlings used in this study were sourced from Sappi and Mondi South African commercial breeding programmes. The seedlings were kindly provided by Dr. Nicky Jones, Sappi Shaw Research Centre and Dr. Kitt Payn, Mondi Forests, Trahar Technology Centre. No field permissions were necessary to collect the plant samples.

Consent for publication
Not applicable.