- Research article
- Open Access
Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens
© Morales-Cruz et al. 2015
- Received: 6 February 2015
- Accepted: 6 May 2015
- Published: 19 June 2015
Trunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development.
The integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution.
This study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management.
- Comparative genomics
- Computational Analysis of gene Family Evolution (CAFE)
- Secondary metabolism
Fungal species that cause trunk diseases of grapevine and the symptoms associated with each disease
V-shaped wood cankers in spurs, cordons and/or trunk (Fig. 1a). Stunted shoots with shortened internodes and small, chlorotic, cupped tattered leaves, showing marginal necrosis and dead interveinal tissue (Fig. 1b). Also, causes general canopy symptoms of shoot dieback and dead spurs.
Dip. seriata, N. parvum
V- or irregular-shaped wood cankers in canes, spurs, cordons and/or trunk (Fig. 1a). Also, causes general canopy symptoms of shoot dieback and dead spurs. Can kill buds on infected canes.
V- or irregular-shaped wood cankers in canes, spurs, cordons and/or trunk (Fig. 1a). Also, causes general canopy symptoms of shoot dieback and dead spurs.
Can kill buds on infected canes.
Phomopsis cane and leaf spot is thought to be a separate disease, but is also caused by Dia. ampelina. The pathogen directly attacks all green tissues of the vine, causing necrotic lesions on the leaves, green stems, and fruit.
T. minima, P. chlamydospora, F. mediterranea, S. hirsutum
Esca disease complex
Black spots (aka “measles”) on berries (Fig. 1c).
White-rotted wood, due to secondary infection by basidiomycetes F. mediterranea and S. hirsutum, sometimes co-occurs in the trunks of vines also infected by the Esca pathogens T. minima and P. chlamydospora (Fig. 1f). However, these fungi are not responsible for fruit or leaf symptoms of Esca.
When colonizing the wood, trunk pathogens are thought to rely on their ability to enzymatically digest the plant cell walls and/or produce toxins to overcome the host’s preformed and inducible defenses [7, 11–14]. Indeed, the chemical structures of secreted toxins, as well as of the products of cell wall degrading activities, have been described for some trunk pathogens [14–16]. For example, degradation of cellulose and xyloglucans, and secretion of oxidases that might participate in the breakdown of wall-bound lignin, were detected in wood colonized by the Eutypa dieback pathogen Eutypa lata. E. lata is the only trunk pathogen that has been categorized as a wood-decay fungus, specifically a soft-rot, which is the type of wood decay caused by Ascomycetes . Metabolites with phytotoxic properties, such as naphtalenone pentaketides, polyphenols, and extracellular polysaccharides, have been found in the secretomes of Eutypa dieback, Esca and Botryosphaeria pathogens . Although this knowledge is valuable to infer the hypothetical proteins involved in pathogenesis and virulence, there are no documented candidate sequences for any of the potential virulence functions associated with trunk diseases to date. Only scarce molecular genetic information is available for trunk pathogens, compared to other better-studied pathogens of grape (e.g., powdery mildew fungus Erysiphe necator [17, 18], Pierce’s disease bacterium Xylella fastidiosa ). Given that multiple trunk pathogens often co-occur in mixed infections in the vineyard, coupled with the fact that they are taxonomically unrelated, there is a limited understanding of the mechanisms that each trunk pathogen employs to first colonize wood and then cause symptoms [20, 21].
Assembly statistics of the grapevine trunk pathogen genomes analyzed
Assembly size (Mb)
Scaffold N50 length (Kb)
Scaffold L50 count
Gene space completeness2 (%)
Dia. ampelina (DA912)
Dip. seriata (DS831)
P. chlamydospora (UCR-PC4)
E. lata (UCR-EL1)
T. minima (UCR-PA7)
F. mediterranea (MF3/22)
S. hirsutum (FP-91666)
Modifications of gene family size, as a result of the differential duplication and deletion of chromosomal regions, have been shown to provide selective advantages and contribute to adaptation in a variety of organisms, including fungi [31, 32]. Gene duplication can be advantageous by increasing the amounts of protein synthesized  or by promoting evolutionary novelty of one of the duplicated genes through subfunctionalization or neofunctionalization . In the case of fungal pathogens, variations in gene family size have been associated with the evolution of virulence functions and host adaptation [31, 34–36]. Differential expansion of gene families involved in host cell wall degradation, transport functions, and melanin biosynthesis has been found in pathogenic fungal lineages . Adaptive gene family expansion has also been associated with the shift in host preference from plants to animals in the Onygenales (Ascomycota; Eurotiomycetes) fungi .
In this study we employed a stochastic birth and death model to discover gene families that have undergone significant expansion/contraction during the evolution of the trunk pathogens. We first generated a time-calibrated phylogeny using a subset of conserved single-copy protein-coding genes  and time of origin estimates from fossil records . The resultant tree was then used to identify those gene families whose size significantly diverged from an estimated random birth-death rate expectation. We identified 90 gene families expanded in the ascomycete trunk pathogens. These gene families were also significantly enriched in putative virulence factors, including cell wall degrading enzymes and genes involved in secondary metabolism. We then applied phylogenetically-aware principal component analysis to detect differences and similarities in the repertoires of putative virulence factors from the significantly expanded gene families.
Genome sequencing and gene prediction of Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora
Gene models of the core eukaryotic genes reconstructed using CEGMA were used to train Augustus  for ab initio gene discovery. A total of 10,801, 9,398, and 6,986 complete protein-coding genes were identified in the genomes of Dia. ampelina, Dip. seriata, and P. chlamydospora, respectively (Table 2). A similar number of protein-coding genes (7,279) was previously reported for a European isolate of P. chlamydospora . Statistics of exon, intron, and intergenic space sizes are reported in Table S4 (Additional file 5) and Figure S2 (Additional file 6). Overall, genes appear to be evenly distributed on the scaffolds of the three genomes, with no evident clustering in gene-rich islands (Additional file 7: Figure S2). Most of the predicted protein coding genes in the three assemblies (94.77 ± 0.88 %) displayed similarity with other ascomycete sequences in the NCBI non-redundant database (BLASTP; e-value < 10−3), indicating that the large majority of the predicted genes are bona fide protein-coding genes.
Transcriptome sequencing and improvement of the protein-coding gene models in the Togninia minima genome
A hybrid gene prediction approach, using both ab initio and RNA-seq based gene discovery, was performed to improve the previously reported gene models of T. minima (isolate UCR-PA7 ). RNA-seq libraries were prepared from mRNA extracted from T. minima colonies growing on different carbon sources to maximize the number of detectable expressed genes (see Methods for details). Ninety-nine percent of the 366 million paired-end RNA-seq reads were assembled de novo into 59,610 contigs using Trinity (Additional file 7: Figure S3A; Additional file 8: Table S5 ). Open reading frames extracted from the assembled transcripts were used to train Augustus for ab initio prediction and, together with 159,358 Uniprot ascomycete curated proteins, were used for evidence-based prediction using the Maker pipeline . As a result, 11,591 complete protein-coding genes were obtained (Additional file 7: Figure S3B; Additional file 9: Table S6), a larger number compared to the 8,926 genes described previously . The integration of RNA-seq with Augustus and Maker gene discovery did not only increase the number of protein-coding genes, but also improved the predicted gene structures, evidenced by wider alignment coverage (Additional file 7: Figure S3C) and greater percent identity when aligned to the proteomes of other ascomycetes (Additional file 7: Figure S3D).
Annotation of virulence functions in the predicted proteomes
Databases and methods used to annotate protein-coding genes in the 10 genomes analyzed
Name of Database
Protein homology annotation
e-value < 10-3
Conserved domain annotation
e-value ≤ 1e-3
CAZYmes Analysis Toolkit (CAT)
CAZyme family annotation
Secondary metabolite clusters
Fungal peroxidase family annotation
e-value ≤ 1e-5
Presence and location of signal peptide cleavage sites
The Cytochrome P450 Homepage
Cytochrome P450 monooxygenase family annotation
e-value ≤ 1e-5
Transporter Classification Database
Classification of Membrane Transport Proteins
e-value ≤ 1e-5
Pathogen-Host Interaction (PHI) database
Experimentally tested genes for pathogen-host interaction
e-value ≤ 1e-5
Number of protein-coding genes annotated in each functional category
CAZymes are proteins with predicted catalytic and carbohydrate-binding domains involved in the degradation, modification, or creation of glycosidic bonds . Because secreted CAZymes can participate in the disassembly of plant cell walls during colonization by pathogens, CAZy annotation, together with prediction of protein secretion, has been used extensively for the identification and classification of cell wall degrading enzymes of plant pathogens [18, 37, 48, 49]. On average 36.6 ± 1.7 % of the putative secreted peptides in the genomes of the eight trunk pathogens were similar to proteins in the CAZy database, indicating a complex repertoire of cell wall degrading functions (Additional file 12: Figure S5; Additional file 13: Table S8; ). Glycoside hydrolases (GHs) represented the largest superfamily, ranging from 76 genes in P. chlamydospora up to 195 genes in E. lata. GH subfamilies involved in the degradation of cellulose and hemicellulose were the most abundant in all genomes (Fig. 3; Additional file 13: Table S8), and included endo-β-1,4-cellulases (GH5), β-glucosidases (GH3), xyloglucan transglucosylase/hydrolases (GH16), and β-xylosidases (GH43) [37, 49, 51]. The highest numbers of GH16 were found in E. lata, followed by the Botryosphaeriaceous fungi Dip. seriata and N. parvum. Similarly, E. lata and N. parvum were the species with the highest numbers of GH43 and GH5, respectively. Compared to all other trunk pathogens, a greater number of proteins with cellulose-binding domains (CBM1) was found in the E. lata genome.
Auxiliary activity (AA) CAZymes are enzymes with redox activity that participate in conjunction with other enzymes in the deconstruction of lignocellulosic material . AA3 genes were particularly abundant in N. parvum (21 genes) and S. hirsutum (20 genes), whereas the largest number of AA7 genes was found in E. lata (19 genes). Among the ascomycete trunk pathogens, AA1 genes were particularly abundant in N. parvum (12 genes) and Dip. seriata (9 genes). Large numbers of genes encoding AA9 were found exclusively in E. lata (24 genes) and Dia. ampelina (20 genes; Fig. 3).
P450s have a broad spectrum of functions in fungi, from housekeeping activities, such as synthesis of essential membrane lipids, to synthesis of secondary metabolites, and detoxification of xenobiotic compounds [18, 52, 53]. P450 families were classified in clans according to . Differences in the number of genes belonging to the different P450 classes were observed among species, particularly between the Ascomycetes and Basidiomycetes (Fig. 3 and Additional file 13: Table S8). For example, CYP65s, CYP531s and CYP58s were abundant in the genomes of the ascomycete trunk pathogens (CYP65s: 19.33 ± 13.06; CYP531s: 10.50 ± 7.06; CYP58s: 7.33 ± 3.78), but were not detected in the genomes of the two Basidiomycetes. Conversely, the genomes of F. mediterranea and S. hirsutum presented large numbers of genes encoding CYP5150s (10 and 25, respectively) and CYP5139s (8 and 15, respectively), which were mostly absent from the ascomycete trunk pathogens (Fig. 3). Also, CYP533 was the clan with the highest number in the Basidiomycetes (67.5 ± 31.82), whereas only a few were detected in the ascomycete trunk pathogens (2.0 ± 1.41) (Fig. 3).
Fungal peroxidases are oxidoreductases involved in numerous and diverse processes, such as lignin breakdown and detoxification of reactive oxygen species produced by the host, which may be associated with virulence [55, 56]. The number of peroxidases identified in the trunk pathogens, based on similarity with proteins in the Fungal Peroxidase database (fPoxDB), ranged from 33 in P. chlamydospora to 57 in Dia. ampelina (Table 3; Additional file 13: Table S8). Potential class II peroxidases (PODs) were found only in the Basidiomycete white rotters, consistent with the hypothesis that these enzymes evolved after the divergence between Ascomycetes and Basidiomycetes .
In plant pathogens, cellular transporters are responsible not only for export of compounds involved in pathogenesis and virulence, but they also may play an essential role in protection against plant defense compounds (e.g., secondary metabolites) during pathogenesis, possibly by exporting host-derived antimicrobial compounds out of the cell [57–59]. In this study, the Transporter Classification Database (Table 3) was used to annotate cellular transporters (Additional file 13: Table S8). The electrochemical potential-driven transporters were the class with the highest number of genes across all species (42.68 ± 8.23 % of all transporters), followed by primary active transporters (27.41 ± 4.28 %). MgtE (TCDB code 1.A.26.1.1) involved in the transport of Mg2+ and Co2+, PPI (TCDB code 3.A.20.1.1) related to the import of proteins to the peroxisomal lumen, and the Major Facilitator Superfamily (MFS, TCBD code 2.A.1.14.11) were the most abundant transporter families in all trunk pathogens.
Annotation of secondary metabolism gene clusters
Estimation of gene family expansion and contraction
To compute the sizes of protein families, the 109,595 proteins of the 10 fungal genomes were clustered into gene families based on sequence similarities (BLASTP; e-value < 1e−6) using the TRIBE-MCL algorithm (Additional file 10: Table S7; ). Using as input the gene family sizes and the clock-calibrated phylogenetic tree, CAFE identified 114 gene families (9,488 genes) across all fungal species with significantly higher-than-expected rate of gains/losses (P ≤ 0.01, Additional file 16: Table S9). Mean gene gains and losses estimated by CAFE for each branch of the phylogenetic trees are shown in Fig. 5a. Among these significantly-expanded gene families, 90 (7,569 genes) were expanded in the ascomycete trunk pathogens, whereas 37 (3,101 genes) were expanded in the two basidiomycetes S. hirsutum and F. mediterranea. Seventy-two (6,126 genes) and 19 (1,388 genes) gene families were exclusively expanded in the Ascomycete trunk pathogens and in the Basidiomycetes, respectively. Seven gene families (641 genes) were significantly expanded in B. cinerea (Fig. 5b). CAFE analysis did not detect any gene family exclusively expanded in S. cerevisiae. A list of the genes from the 90 gene families significantly expanded in the ascomycete trunk pathogens, with all annotations carried out in this work, gene family groupings, is provided in Table S10 (Additional file 17).
A hypergeometric test was performed to determine if specific functional categories were significantly overrepresented in the 90 families that were significantly expanded in the ascomycete trunk pathogens (Additional file 18: Table S11). Thirteen of the 37 functional categories were found to be significantly enriched in these 90 families (P < 0.01; Fig. 5c; Additional file 18: Table S11). Enriched categories included cell wall degradation, secondary metabolism, protein catabolism, oxidative processes and cellular defense, all of which have been often related to fungal virulence (Fig. 5c). CAZYmes, transporters, and P450s represented the largest functional groups that were overrepresented in the expanded families (Fig. 5c). Expanded CAZyme families were particularly abundant in E. lata and N. parvum, whereas most of the expanded families of transporters in the trunk pathogens were found in the T. minima genome. P450s were the only category enriched in the expanded families of all species, including B. cinerea (Additional file 18: Table S11). Genes of the Fe(II)-dependent oxygenase superfamily (2OG-Fell_Oxy), as well as CAZYmes and peptidases, were enriched in the expanded families of all ascomycete trunk pathogens and the basidiomycetes. Among the 72 families expanded exclusively in the ascomycete trunk pathogens, there was a significant enrichment in genes associated with secondary metabolism, such as ketoacyl-synthases and fumarylacetoacetate (FAA) hydrolases (Additional file 18: Table S11). Further evidence in support of a differential expansion of families associated with potential virulence processes comes from our finding that 27.07 % of the 7,569 genes in the expanded gene families in the ascomycete trunk pathogens shared homology with proteins in the PHI-base database (hypergeometric test: P < 0.0001).
Phylogenetically informed principal-components analyses of potential virulence factors in the expanded gene families of ascomycete trunk pathogens
PCAs showed that species associated with similar symptoms presented more similar repertoires within the expanded gene families, which often did not correlate with the phylogenetic relationships between species. For example, the two Esca pathogens, T. minima and P. chlamydospora, were consistently grouped together in all four biplots (Figs. 5–8), suggesting that the two species possess similar virulence repertoires. This is in spite of the fact that T. minima is more closely related phylogenetically to Dia. ampelina, and P. chlamydospora to the two Botryosphaeriaceae N. parvum and Dip. seriata (Fig. 5a). Nonetheless, these two unrelated Esca pathogens cause similar leaf and fruit symptoms, which are unique compared to symptoms of the other trunk pathogens. Expansions of gene families associated with sugar transport were mostly responsible for the tight clustering of the two Esca pathogens in the biplot based on Pfam annotations (Fig. 6). All four PCAs revealed similar repertoires within the expanded gene families for E. lata, Dia. ampelina, and N. parvum, which were consistently separated in the biplots, on the other side of the Y-axis (PC2), from Dip. seriata, P. chlamydospora, and T. minima. Indeed, E. lata, Dia. ampelina, and N. parvum cause similar wood symptoms (v-shaped to irregular-shaped wood cankers), and they all cause shoot dieback and dead spurs. Expansions of families annotated with Pfam Glycoside hydrolase 61 (equal to CAZy AA9), DUF1996, proteinase inhibitor I9 and Peptidase 38 were mostly responsible for the clustering of E. lata and Dia. ampelina (Fig. 6). Similarities between these two species, particularly in larger numbers of AA CAZymes, mainly AA9s (Fig. 7), genes associated with polyketide synthesis (t1PKS) (Fig. 8b), and P450s genes, primarily CYP54 and CYP531 (Fig. 8c), resulted in their clustering. Clustering of N. parvum and Dip. seriata was observed when gene counts based on Pfam annotations and CAZYme homologies were analyzed. N. parvum showed a distinct expansion of AA3 CAZymes (Fig. 7) and genes encoding secondary metabolic functions, with 49 genes (NPRS + NPRS-t1PKS) compared to 16 ± 7.21 in E. lata, Dia. ampelina and Dip. seriata, and only 11 ± 1.41 in the two Esca pathogens (Fig. 8). A similar pattern was observed when counts of genes coding for P450s were used as input for phylogenetic PCA (Fig. 8): while PC1 clearly separated the Esca pathogens from the rest, PC2 separated N. parvum, Dia. ampelina, and E. lata.
In this study we describe the draft genome sequences of three grapevine trunk pathogens, causal agents of Phomopsis dieback, Botryosphaeria dieback and Esca. This genomic information, together with the previously-released draft genome sequences of other important ascomycete trunk pathogens [23–25] and two basidiomycetes associated with Esca , provide the genomic resources necessary to begin analyzing the complex repertories of potential virulence profiles of these destructive fungi . All genomes in this study showed a comparable degree of completeness in relation to both genome size estimates, based on k-mer distribution and representation of conserved genes . Genome sizes, as well as number of protein-coding genes and repetitive DNA content, were similar to those of other common plant pathogens with a necrotrophic life style, such as B. cinerea  Sclerotinia sclerotiorum  and Colletotrichum spp. . As observed in , P. chlamydospora has a relatively smaller genome (and gene content) compared to the other species analyzed. The application of third generation sequencing technologies will help improve these draft assemblies that despite their estimated completeness suffer from limitations due to the use of short reads, which in addition to fragmentary assemblies may include chimeric contigs, erroneous copy numbers and collapsed repetitive regions [75, 76]. We cannot rule out that some protein-coding genes may be missing from the final transcriptomes predicted from the shotgun-sequenced genomes because of (i) inaccessibility of certain genomic regions to Illumina sequencing, (ii) incomplete assemblies, and (iii) possible errors in the ab initio gene discovery [76, 77]. Further studies of in planta gene expression using RNA-seq may refine the predicted gene models . The effectiveness of integrating transcriptome sequencing with comparative and ab initio approaches for gene prediction is evidenced in this work by the significant improvement of the predicted genes in the genome of T. minima.
Functional annotations of the genomes of the 8 trunk pathogens confirmed the complex array of virulence factors that these organisms may utilize during colonization of grapevines. We observed remarkable variation in the number of genes assigned to specific functional categories among the trunk pathogens, which in some instances (and with statistical significance) reflected lineage-specific, gene family expansions and contractions. Gene family expansions result from the retention in a fungal population of duplicated genes, which provide adaptive advantage [35, 79]. Gene duplications can arise from events of genome or chromosomal duplications, TE retroposition, or unequal crossing-over . Gene duplication can lead to functional diversification or increase in protein synthesis, which can play a role in fungal adaptive divergence ). An increase in the number of paralogous genes in families associated with virulence and nutrient uptake has been described in the case of obligate parasites [35, 82–86]. Larger family sizes of virulence factors were also found in species with broader host ranges, compared to host-specialized pathogens in the Metarhizium genus . Copy number variations (CNVs) within species have also been described not only as a mechanism underlying host adaptation [88–90], but also in the development of resistance to antifungal compounds in fungi  (e.g., fungicide resistance in Erysiphe necator ). The extent and frequency of adaptive CNV in trunk pathogen populations has not been investigated. Nonetheless, as whole-genome sequences of more isolates of each trunk pathogen species become available, comparative approaches can be applied to scan the genomes for CNV loci and determine whether they encompass any of the genes in the significantly-expanded families we identified.
Because natural selection is the major force behind the differences in gene family size between species , focusing on families with faster rates of gene gain can help identify functions that may be associated with host adaptation, pathogenicity, or virulence . Among the ascomycete trunk pathogens, we identified 90 gene families that have undergone significant expansion. The expanded families in the ascomycete trunk pathogens were enriched in genes that, at least based on in silico annotations, are expected to contribute to virulence and nutrient uptake. The overrepresentation of PHI genes, as well as of secreted CAZymes, P450s, and genes involved in secondary metabolism, supports the role of gene duplication and consequent gene family expansion in the evolution of trunk pathogens. Furthermore, results of phylogenetic PCAs of the sizes of expanded families highlighted similarities between pathogens that did not correspond to their phylogenetic relationships. Instead, pathogens were grouped more often according to similarities in disease symptoms, which suggests there is convergent evolution.
The predicted secretomes of all trunk pathogens encompassed functions that can potentially target all components of primary and secondary plant cell walls (Fig. 3). Unlike pathogens that thrive on pectin-rich tissue, such as B. cinerea [49, 91], which possesses high numbers of pectolytic enzymes, overall the ascomycete trunk pathogens showed a wider array of enzymes that target cellulose and hemicellulose, such as endo-β-1,4-cellulases (GH5), β-glucosidases (GH3), xyloglucan transglucosylase/hydrolases (GH16), and β-xylosidases (GH43). As these are wood-colonizing fungi, we might expect their genomes to include a range of genes encoding for wood-degrading enzymes, especially E. lata, which is a known soft-rot fungus, and N. parvum, which colonizes grapevine wood more rapidly than most trunk pathogens . In agreement with the observation that glucose-rich polymers are degraded in wood colonized by E. lata , we found significant expansion of genes coding for CAZymes containing the CBM1 domain, a carbohydrate-binding module that strongly binds to crystalline cellulose and that is required for full activity of fungal cellulases. E. lata is a soft-rot fungus and has a similar gene family expansion pattern to the white-rot basidiomycete Phanerochaete chrysosporium, in which expansion of CBM1s was also found . Indeed, cellulose-degrading systems of some soft-fungi are as advanced as those of typical white-rot fungi . In contrast to E. lata, P. chlamydospora underwent the least amount of expansion in secreted CAZymes, which is consistent with past findings of no detection in vitro of xylanase or cellulases and no visible degradation of lignified cell walls in wood colonized by the latter .
The predicted secretomes of the ascomycete trunk pathogens were also rich in auxiliary enzymes (AAs), which catalyze oxidative processes that facilitate the enzymatic disassembly by other CAZymes of recalcitrant plant cell wall components, including lignin . Soft-rot fungi degrade lignin, but to a lesser degree even than brown-rot fungi; they can degrade enough lignin to access other cell wall components that are more efficiently degraded (Worrall et al., 1997). The expansion of AA7s was common in all trunk pathogens, with the exception of P. chlamydospora. AA7s are gluco-oligosaccharide oxidases that can oxidize a variety of carbohydrates and can contribute to lignin degradation acting in conjunction with peroxidases . AA3s were expanded the most in N. parvum, and AA9s in E. lata and Dia. ampelina, suggesting that specific oxidative processes are associated with these different dieback pathogens. Indeed, the AA3s -- extracellular hemoflavoenzymes and known components of the secretomes of lignocellulose-degrading fungi  -- are involved in degradation of cellulose, hemicellulose, and lignin . AA9s are copper-dependent lytic polysaccharide monooxygenases, previously classified as GH61, and are commonly found in genomes of fungal wood decayers. AA9s enhance the breakdown of lignocellulosic material in combination with cellulolytic enzymes by catalyzing the oxidative cleavage of cellulose, which increases substrate accessibility to other CAZymes [50, 97]. N. parvum together with Dip. seriata also showed expansion of AA1 genes that encode multicopper oxidases, including laccases, which could also participate in lignin breakdown .
Secondary metabolites with phytotoxic activity (i.e. toxins) are integral components of the battery of virulence factors of grapevine trunk pathogens [15, 61, 98–102]. Although toxins secreted by some grapevine trunk pathogens have been chemically characterized and tested for virulence, none of the genes involved in their synthesis have been identified to date. In fungi, genes involved in the synthesis and transport of secondary metabolites are typically clustered together with the gene coding for the key biosynthetic enzyme [63, 103]. Large numbers of secondary metabolic clusters were observed in the ascomycete trunk pathogens, mostly associate with the synthesis of (i) polyketides and fatty acid-derived compounds (PKS), (ii) terpenes (TS), and (iii) non-ribosomal peptides and amino acid-derived compounds (NRPS). While NRPS may be responsible for the synthesis of toxic polypeptides [15, 101], clusters centered around the key enzyme polyketide synthase may participate to the production of naphtelenone pentaketide toxins found in T. minima, P. chlamydospora, Dip. seriata and N. parvum . A remarkable expansion of genes associated with non-ribosomal peptides and amino acid-derived compounds was found in N. parvum, while the greatest expansions of families involved in polyketide synthesis (t1PKS) were found in genomes of E. lata and Dia. ampelina. The differences between the Botryosphaeria dieback pathogens N. parvum and D. seriata in gene counts of these secondary metabolite clusters reflect their different rates of wood colonization  and the more rapid rate of wood necrosis caused by the former .
Our results also showed that grapevine trunk pathogens possess a large number of P450s as found in other wood-rotting organisms [86, 106, 107]. P450s are crucial components of multiple processes ranging from the biosynthesis of secondary metabolites such as toxins and hormones [52, 108] to degradation and detoxification of antimicrobial plant defense compounds . Interestingly, basidiomycetes and ascomycete trunk pathogens show very distinctive expansions of specific CYPs. Of particular interest are the CYP65s which were more abundant in the ascomycete trunk pathogens (e.g. 34 genes in N. parvum and 34 in E. lata) compared to B. cinerea (11) and were not detected in the two basidiomycete analyzed and S. cerevisiae. CYP65s are P450s predicted to participate in pathways of secondary metabolism, including toxin biosynthesis .
As part of this study we expanded the currently available genomic resources for grapevine trunk pathogens and incorporated this information with previously released genomes in a comparative analysis to catalogue genes and gene families with putative virulence functions. The draft genomes and their annotated protein-coding genes presented in this paper provide not only the necessary references for in planta expression profiling and whole-genome re-sequencing for genetic diversity and association studies, but also the molecular information required for targeted knock-out mutations, overexpression or gene tagging as protocol for genetic transformation of some of these species are available [111, 112]. Comparisons between in planta and in vitro transcriptomes will define the expression dynamics of the proposed virulence factors during the interaction with the host. Whole genome re-sequencing of multiple isolates will determine the pattern of sequence polymorphisms  and structural variations  in pathogen populations and their association with pathogen aggressiveness and host range. The functional validation of these potential virulence factors by reverse genetic approaches will ultimately lead to a more comprehensive understanding of the mechanisms underlying the different grapevine trunk diseases, which will enable the development of more accurate diagnostic tools and novel effective control methods.
Vineyards were surveyed for general signs of trunk diseases, including low vigor, stunted canopy, missing spur positions, dead cordons, and overall decline. Samples were collected from cankered, necrotic wood parts observed in cross-sections of vine trunks and arms. Isolates were recovered from the margins of wood cankers. Five pieces of wood, approximately 2 × 5 mm, were cut from the canker margin with a flame-sterilized knife, surface-sterilized in 0.6 % sodium hypochlorite (pH adjusted to 7.2) for 30 s, rinsed twice in sterile distilled water for 30 s, and plated on potato dextrose agar (PDA, Difco, Detroit, MI) amended with tetracycline (1 mg/L). PDA plates were incubated at 25 °C in darkness for several days. Fungal colonies were further hyphal-tip purified. Dip. seriata isolate SBen831 (DS831) was recovered from diseased grapevine wood (V. vinifera) sampled in Hollister, San Benito Co., California, in June 2011. Phomopsis viticola (teleomorph: Dia. ampelina) isolate Wolf912 (DA912) was recovered from diseased grapevine wood in the upper trunk of a ‘Seeded Thompson” vine sampled in the USDA-ARS National Germplasm Repository grapevine collection located in Winters, Solano Co., California, in May 2011. P. chlamydospora isolate UCR-PC4 was recovered from diseased grapevine wood (V. vinifera cv. Flame) from the black necrotic streaks of the wood vascular system sampled in Coachella Valley, Riverside Co., California, in November 2011. Species were confirmed by aligning the internal transcribed spacer (ITS) DNA sequences with their respective type specimens (Additional file 1: Figure S1). ITS sequences were submitted to GenBank and can be found under the following accessions: KP296243 (Dip. seriata DS831), KM669964 (Dia. ampelina DA912), and KP296244 (P. chlamydospora UCR-PC4).
Axenic cultures of isolated DA912, DS831 and UCR-PA7 were grown in PDA (Difco) media. Around 100 mg of mycelia were collected in a 2 ml micro-centrifuge tube and frozen in liquid nitrogen, mycelia were ground while still frozen. DNA extraction was done with a modification of the Cetyltrimethylammonium Bromide (CTAB) protocol , beginning with 500 μl of extraction buffer. Concentration and purity of the DNA was measured with Nanodrop 2000c Spectrophotometer (Thermo Scientific). Integrity was checked with an agarose gel.
Genome sequencing, assembly, and gene prediction
DNA libraries were prepared as described in . Genomic DNA was fragmented with the Bioruptor NGS (Diagenode, USA) in 6–9 cycles of 15 s / 90s (on/off). Libraries were prepared using the KAPA KK8201 library preparation kit (KAPA Biosystems, USA) with approximately 1 μg of fragmented DNA as input. NEXTflex primers were used as barcodes (BIOO Scientific, TX, USA) and size selection was done with the Pippin Prep platform (Sage Science, USA). Final libraries were evaluated for quantity and quality with the High Sensitivity kit in a Bioanalyzer 2100 (Agilent Technologies, CA, USA). Sequencing was carried out on an Illumina HiSeq2500 machine at the DNA Technologies Core at UC Davis. Paired-end reads of 150 bp in length were generated and a median sequencing coverage of 50X, 79X, and 107X was achieved for Dia. ampelina, Dip. seriata, and P. chlamydospora, respectively. Quality trimming (Q > 20) and adapter contamination removal were carried out using sickle (v1.210; ) and scythe (version 0.991; ), respectively. Assembly of high quality reads was performed using CLC Workbench 6.1. Assembly parameters were optimized to achieve maximal assembly completeness of the gene space estimated using the Core Eukaryotic Genes Mapping Approach (CEGMA) analysis . Contigs with similarity to non-fungal sequences in the complete NCBI nt collection were considered contaminants and were discarded. The assemblathon_stats.pl script  was used to compute basic assembly metrics. Genome sizes were calculated from DNA k-mer count distributions . Gene models of the core eukaryotic genes reconstructed using CEGMA were used to train Augustus (v. 2.5.5, ) for ab initio gene prediction using default parameters. Predicted Incomplete coding sequences were discarded.
Transposable elements annotation
Transposable elements were annotated using both ab initio and reference based computational approaches as described in . Ab initio prediction was carried out using RepeatModeler (v1.0.7; ). RepeatModeler output (i.e. all identified repeats with the exception of TEs marked as “unknown”) were added to the RepeatMasker database and used as reference for homology-based discovery of interspersed repeats and low complexity DNA sequences using RepeatMasker (v4.0.2; ).
Transcriptome sequencing, de novo assembly, and gene prediction (T. minima)
In March of 2013 pruned-dormant grapevine canes from V. vinifera cv. Cabernet Sauvignon and V. vinifera cv. Merlot plants were collected. The canes were cut into 1 cm long fragments, dried at 60 °C for 24 h to reduce water content and ground to fine particles using a Wiley Mini-Mill (Thomas Scientific, NJ, USA). The ground wood (2 % w/v) was mixed with agar (1.5 % w/v) . T. minima UCR-PA7 was propagated at 25 °C in PDA and the minimal media with wood described above. The fungus was grown for at least two cycles in the same medium before the actual experiment to avoid a carryover of signals from other media.
Mycelia were collected in a 2 ml micro-centrifuge tube in a sterile environment and were quickly frozen in liquid nitrogen. A stainless-steel bead of 5 mm was added to each tube and ground in the TissueLyser II (Qiagen, CA, USA) at 30 Hz for 30 s with the Teflon adapters pre-cooled to avoid sample thawing. One milliliter of TRIzol reagent (Ambion, USA) was added to the ground mycelia; extraction of total RNA was done following manufacture’s protocol. The RNA was cleaned up with the RNeasy Plant Mini Kit (Qiagen) including the on-column DNase enzymatic treatment. Purity was measured with the Nanodrop 2000 spectophotometer (Thermo Scientific), quantity with the RNA kit of the Qubit 2.0 Fluorometer (Life Technologies, CA, USA) and integrity in an agarose gel.
Libraries were prepared using the Illumina TruSeq RNA sample preparation kit v.2 (Illumina, CA, USA), following Illumina’s protocol (Low-throughput protocol) and barcoded individually. Final libraries were evaluated for quantity and quality with the High Sensitivity kit in a Bioanalyzer 2100 (Agilent Technologies, CA). Libraries were sequenced with HiSeq 2500 as 100 bp paired-ends (Illumina, CA, USA).
Scythe and sickle were used to quality filter and trim the raw reads as described above. De novo genome-guided assembly using TopHat (version 2.0.9; ) with parameters -r 100 --mate-std-dev 50 --min-intron-length 20 and Trinity (version r20131110; with jaccard_clip option on ) was performed as describe in . The perl script transcripts_to_best_scoring_ORFs.pl from Trinity was used to extract putative open reading frames from the assembled contigs. Gene structures of the CEGs identified in the genomic scaffolds by CEGMA together with the de novo assembled transcripts were used to train Augustus (v. 2.5.5 ) for ab initio gene discovery on repeat-masked scaffolds. MAKER (version 2.8; ) was then used to integrate the ab intio prediction with homology based gene prediction using the de novo assembled transcripts and 159,358 Uniprot ascomycetes curated proteins.
Basic annotation of all predicted protein coding sequencing was assigned based on similarity to ascomycete peptides in GenBank with Blast2GO  and to conserved domains in the Pfam database  (Additional file 10: Table S7). Databases, software and parameters used for the functional annotations are shown in Table 3. The presence of secretion signal peptides was evaluated using SignalP v.4.1 . Proteins were clustered in families based on sequence similarity using BLASTP (e-value < 10−6) followed by Markov clustering with TribeMCL .
Construction of a clock-calibrated phylogenetic tree
Twenty-six single copy conserved peptides used in  for phylogeny reconstruction and CAFE analysis were extracted for the five Ascomycete species (Aspergillus niger, Cryphonectria parasitica, Pichia stipitis, Stagonospora nodorum, and Trichoderma reesei) . S. cerevisiae orthologs (2S88C reference strain) were downloaded from http://www.yeastgenome.org. All proteins were independently examined using BLASTP (v.2.2.29+) against the proteomes of the 6 ascomycete trunk pathogens and B. cinerea. Seventeen protein families were confirmed to have exactly one top hit for all species (Additional file 19: Table S12). Nine families were excluded to avoid including potential paralogs. S. cerevesiae, S. hirsutum, and F. mediterranea orthologs were obtained from  and added to the set of orthologous peptides. Alignment of each set of orthologous peptides was carried out with MUSCLE (v. 3.8.31; ). Alignments were then concatenated (total of 17,843 positions) and Gblocks (v. 0.91; maximum number of contiguous nonconserved positions allowed = 4, minimum length of a block allowed = 10; ) was used to remove ambiguous regions, resulting in an alignment of 8422 amino acids for fifteen species.
Alignments were imported into BEAUti (v. 1.8.0) to prepare for BEAST (v. 1.8.0) analysis. Monophyletic partions of data were specified in BEAUti (Additional file 20: Text S1). The Ascomycota partition was dated using an offset of 400 MY and a log mean of 6.11 following  (Node A, Fig. 3a). The Letiomycota partition was dated using an offset of 306 MY with a log mean of 5.57, consistent with  (Node B, Fig. 3a). Six MCMC chains were launched using BEAST with length of 1,000,000 (WAG substitution model, 4 Gamma Categories + Invariant sites, Lognormal relaxed clock, Birth-death process). Resulting trees were concatenated with LogCombiner (v. 1.8.0) and loaded into TreeAnnotator (v. 1.8.0) to create a consensus tree (Additional file 21: Figure S8). Branch lengths and tree topology were consistent with previous literature . Although estimated sample size (ESS) values did not reach above 100 (Additional file 22: Table S13), the topology of recent divergence in the Dothideomycetes and Diaporthales partitions, which was not specified in BEAUti, arranged as previously described in [37, 38, 70, 71] and were confirmed by phylogenetic analysis of the same alignments using MrBayes  and PhyML . As observed in [37, 38, 70], there is weak statistical support for the taxonomic grouping of P. chlamydospora and Dip. seriata (Posterior P = 0.5145, Additional file 21: Figure S8). Our tree topology confirmed phylogenetic relationships between P. chlamydospora and Dip. seriata reported in .
Computational analysis of gene family evolution (CAFE)
The tree created with BEAST was trimmed prior to CAFE analysis by removing the five species originally used for branch length calibration. Gene numbers annotated for 8031 tribes, each containing at least one protein in no less than four species, for each of the ten remaining species, along with the tree, were used to run CAFE (v. 3.1; ). CAFE was run with default parameters optimizing the lambda parameter (option -s) to 0.000940686 with a P-value cutoff of 0.01 (option -p). Viterbi P-values were computed for each significant tribe to assess significant expansion or contraction along a specific branch.
Phylogenetic principal component analysis
The phyl.pca from the R package phytools (www.phytools.org/) was used to compute phylogenetic PCA. The clock-calibrated phylogenetic tree was used to correct for nonindependence among observations. The BEAST tree as well as matrices of Pfam, CAZyme, secondary metabolism, and P450 gene counts were used as input.
The whole genome shotgun projects have been deposited in the GenBank database [accession nos.: LAQI00000000 (Dip. seriata), LCUC00000000 (Dia. ampelina), LCWF00000000 (P. chlamydospora)]. The raw reads are available via Sequence Read Archive [accession nos.: SRR1772171 (Dip. seriata), SRR1693722 (Dia. ampelina), SRR1772173 (P. chlamydospora). RNA-sequencing data used in this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database, http://www.ncbi.nlm.nih.gov/geo (no. GSE64404).
This research was funded by the USDA, National Institute of Food and Agriculture, Specialty Crop Research Initiative (grant number 2012-51181-19954). AMC was also partially supported by a Horticulture & Agronomy graduate fellowship (UC Davis), a Horace O. Lanza Scholarship, a Wine Spectator Scholarship, and a The Pearl & Albert J. Winkler Scholarship in Viticulture.
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