The wild side of grape genomics.

With broad genetic diversity and as a source of key agronomic traits, wild grape species (Vitis spp.) are crucial to enhance viticulture's climatic resilience and sustainability. This review discusses how recent breakthroughs in the genome assembly and analysis of wild grape species have led to discoveries on grape evolution, from wild species' adaptation to environmental stress to grape domestication. We detail how diploid chromosome-scale genomes from wild Vitis spp. have enabled the identification of candidate disease-resistance and flower sex determination genes and the creation of the first Vitis graph-based pangenome. Finally, we explore how wild grape genomics can impact grape research and viticulture, including aspects such as data sharing, the development of functional genomics tools, and the acceleration of genetic improvement.

A super-pangenome of the North American wild grape species.

BACKGROUND: Capturing the genetic diversity of wild relatives is crucial for improving crops because wild species are valuable sources of agronomic traits that are essential to enhance the sustainability and adaptability of domesticated cultivars. Genetic diversity across a genus can be captured in super-pangenomes, which provide a framework for interpreting genomic variations. RESULTS: Here we report the sequencing, assembly, and annotation of nine wild North American grape genomes, which are phased and scaffolded at chromosome scale. We generate a reference-unbiased super-pangenome using pairwise whole-genome alignment methods, revealing the extent of the genomic diversity among wild grape species from sequence to gene level. The pangenome graph captures genomic variation between haplotypes within a species and across the different species, and it accurately assesses the similarity of hybrids to their parents. The species selected to build the pangenome are a great representation of the genus, as illustrated by capturing known allelic variants in the sex-determining region and for Pierce's disease resistance loci. Using pangenome-wide association analysis, we demonstrate the utility of the super-pangenome by effectively mapping short reads from genus-wide samples and identifying loci associated with salt tolerance in natural populations of grapes. CONCLUSIONS: This study highlights how a reference-unbiased super-pangenome can reveal the genetic basis of adaptive traits from wild relatives and accelerate crop breeding research.

Multigenic resistance to Xylella fastidiosa in wild grapes (Vitis sps.) and its implications within a changing climate.

Xylella fastidiosa is a bacterium that infects crops like grapevines, coffee, almonds, citrus and olives. There is little understanding of the genes that contribute to plant resistance, the genomic architecture of resistance, and the potential role of climate in shaping resistance, in part because major crops like grapevines (Vitis vinifera) are not resistant to the bacterium. Here we study a wild grapevine species, V. arizonica, that segregates for resistance. Using genome-wide association, we identify candidate resistance genes. Resistance-associated kmers are shared with a sister species of V. arizonica but not with more distant species, suggesting that resistance evolved more than once. Finally, resistance is climate dependent, because individuals from low ( < 10 °C) temperature locations in the wettest quarter were typically susceptible to infection, likely reflecting a lack of pathogen pressure in colder climates. In fact, climate is as effective a predictor of resistance phenotypes as some genetic markers. We extend our climate observations to additional crops, predicting that increased pathogen pressure is more likely for grapevines and almonds than some other susceptible crops.

Priming grapevine with lipopolysaccharide confers systemic resistance to Pierce's disease and identifies a peroxidase linked to defense priming.

Priming is an adaptive mechanism that fortifies plant defense by enhancing activation of induced defense responses following pathogen challenge. Microorganisms have signature microbe-associated molecular patterns (MAMPs) that induce the primed state. The lipopolysaccharide (LPS) MAMP isolated from the xylem-limited pathogenic bacterium, Xylella fastidiosa, acts as a priming stimulus in Vitis vinifera grapevines. Grapevines primed with LPS developed significantly less internal tyloses and external disease symptoms than naive vines. Differential gene expression analysis indicated major transcriptomic reprogramming during the priming and postpathogen challenge phases. Furthermore, the number of differentially expressed genes increased temporally and spatially in primed vines, but not in naive vines during the postpathogen challenge phase. Using a weighted gene co-expression analysis, we determined that primed vines have more genes that are co-expressed in both local and systemic petioles than naive vines indicating an inherent synchronicity that underlies the systemic response to this vascular pathogen specific to primed plants. We identified a cationic peroxidase, VviCP1, that was upregulated during the priming and postpathogen challenge phases in an LPS-dependent manner. Transgenic expression of VviCP1 conferred significant disease resistance, thus, demonstrating that grapevine is a robust model for mining and expressing genes linked to defense priming and disease resistance.

HiFi chromosome-scale diploid assemblies of the grape rootstocks 110R, Kober 5BB, and 101-14 Mgt.

Cultivated grapevines are commonly grafted on closely related species to cope with specific biotic and abiotic stress conditions. The three North American Vitis species V. riparia, V. rupestris, and V. berlandieri, are the main species used for breeding grape rootstocks. Here, we report the diploid chromosome-scale assembly of three widely used rootstocks derived from these species: Richter 110 (110R), Kober 5BB, and 101-14 Millardet et de Grasset (Mgt). Draft genomes of the three hybrids were assembled using PacBio HiFi sequences at an average coverage of 53.1 X-fold. Using the tool suite HaploSync, we reconstructed the two sets of nineteen chromosome-scale pseudomolecules for each genome with an average haploid genome size of 494.5 Mbp. Residual haplotype switches were resolved using shared-haplotype information. These three reference genomes represent a valuable resource for studying the genetic basis of grape adaption to biotic and abiotic stresses, and designing trait-associated markers for rootstock breeding programs.

Haplotype-resolved powdery mildew resistance loci reveal the impact of heterozygous structural variation on NLR genes in Muscadinia rotundifolia.

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries 2 powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. The purpose of this study was to identify candidate resistance genes associated with each haplotype of the 2 loci. Both haplotypes of each resistance-associated locus were identified, phased, and reconstructed. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with a manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat genes. Heterozygous structural variations were also found to impact the domain composition of some nucleotide-binding leucine-rich repeat proteins. By comparing the nucleotide-binding leucine-rich repeat proteins at Run1.2 and Run2.2 loci, we discovered that the 2 loci include different numbers and classes of nucleotide-binding leucine-rich repeat genes. To identify powdery mildew resistance-associated genes, we performed a gene expression profiling of the nucleotide-binding leucine-rich repeat genes at Run1.2b and Run2.2 loci with or without powdery mildew present. Several nucleotide-binding leucine-rich repeat genes were constitutively expressed, suggesting a role in powdery mildew resistance. These first complete, haplotype-resolved resistance-associated loci and the candidate nucleotide-binding leucine-rich repeat genes identified by this study are new resources that can aid the development of powdery mildew-resistant grape cultivars.

Assembly of complete diploid-phased chromosomes from draft genome sequences.

De novo genome assembly is essential for genomic research. High-quality genomes assembled into phased pseudomolecules are challenging to produce and often contain assembly errors because of repeats, heterozygosity, or the chosen assembly strategy. Although algorithms that produce partially phased assemblies exist, haploid draft assemblies that may lack biological information remain favored because they are easier to generate and use. We developed HaploSync, a suite of tools that produces fully phased, chromosome-scale diploid genome assemblies, and performs extensive quality control to limit assembly artifacts. HaploSync scaffolds sequences from a draft diploid assembly into phased pseudomolecules guided by a genetic map and/or the genome of a closely related species. HaploSync generates a report that visualizes the relationships between current and legacy sequences, for both haplotypes, and displays their gene and marker content. This quality control helps the user identify misassemblies and guides Haplosync's correction of scaffolding errors. Finally, HaploSync fills assembly gaps with unplaced sequences and resolves collapsed homozygous regions. In a series of plant, fungal, and animal kingdom case studies, we demonstrate that HaploSync efficiently increases the assembly contiguity of phased chromosomes, improves completeness by filling gaps, corrects scaffolding, and correctly phases highly heterozygous, complex regions.

Glutathione S-transferase: a candidate gene for berry color in muscadine grapes (Vitis rotundifolia).

Muscadine grapes (Vitis rotundifolia Michx.) are a specialty crop cultivated in the southern United States. Muscadines (2n = 40) belong to the Muscadinia subgenus of Vitis, while other cultivated grape species belong to the subgenus Euvitis (2n = 38). The muscadine berry color locus was mapped to a 0.8 Mbp region syntenic with chromosome 4 of Vitis vinifera. In this study, we identified glutathione S-transferase4 as a likely candidate gene for anthocyanin transport within the berry color locus. PCR and Kompetitive allele-specific PCR genotyping identified a single intragenic SNP (C/T) marker corresponding to a proline to leucine mutation within the muscadine glutathione S-transferase4 (VrGST4) that differentiated black (CC and CT) from bronze (TT) muscadines in 126 breeding selections, 76 cultivars, and 359 progeny from 3 mapping populations. Anthocyanin profiling on a subset of the progeny indicated a dominant VrGST4 action. VrGST4 was expressed in skins of both black and bronze muscadines at similar levels. While nonsynonymous polymorphisms between black and bronze muscadines were discovered in VrGSTF12, another Type I GST-coding gene in the muscadine color locus, this gene was ruled out as a possible candidate for berry color because RNA sequencing indicated it is not expressed in berry skins at véraison from black or bronze genotypes. These results suggest that the bronze phenotype in muscadines is regulated by a mechanism distinct from the MybA gene cluster responsible for berry color variation in Vitis vinifera.

The grape powdery mildew resistance loci Ren2, Ren3, Ren4D, Ren4U, Run1, Run1.2b, Run2.1, and Run2.2 activate different transcriptional responses to Erysiphe necator.

Multiple grape powdery mildew (PM) genetic resistance (R) loci have been found in wild grape species. Little is known about the defense responses associated with each R locus. In this study, we compare the defense mechanisms associated with PM resistance in interspecific crosses segregating for a single R locus from Muscadinia rotundifolia (Run1, Run1.2b, Run2.1, Run2.2), Vitis cinerea (Ren2), V. romanetii (Ren4D and Ren4U), and the interspecific hybrid Villard blanc (Ren3). By combining optical microscopy, visual scoring, and biomass estimation, we show that the eight R loci confer resistance by limiting infection at different stages. We assessed the defense mechanisms triggered in response to PM at 1 and 5 days post-inoculation (dpi) via RNA sequencing. To account for the genetic differences between species, we developed for each accession a diploid synthetic reference transcriptome by incorporating into the PN40024 reference homozygous and heterozygous sequence variants and de novo assembled transcripts. Most of the R loci exhibited a higher number of differentially expressed genes (DEGs) associated with PM resistance at 1 dpi compared to 5 dpi, suggesting that PM resistance is mostly associated with an early transcriptional reprogramming. Comparison of the PM resistance-associated DEGs showed a limited overlap between pairs of R loci, and nearly half of the DEGs were specific to a single R locus. The largest overlap of PM resistance-associated DEGs was found between Ren3 +, Ren4D +, and Ren4U + genotypes at 1 dpi, and between Ren4U + and Run1 + accessions at 5 dpi. The Ren3 +, Ren4D +, and Ren4U + were also found to have the highest number of R locus-specific DEGs in response to PM. Both shared and R locus-specific DEGs included genes from different defense-related categories, indicating that the presence of E. necator triggered distinct transcriptional responses in the eight R loci.

Rootstock influences the effect of grapevine leafroll-associated viruses on berry development and metabolism via abscisic acid signalling.

Grapevine leafroll-associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high-quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition-dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling-related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks.

Multiple independent recombinations led to hermaphroditism in grapevine.

Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (Vitis vinifera L. ssp. vinifera). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine.

Diploid chromosome-scale assembly of the Muscadinia rotundifolia genome supports chromosome fusion and disease resistance gene expansion during Vitis and Muscadinia divergence.

Muscadinia rotundifolia, the muscadine grape, has been cultivated for centuries in the southeastern United States. M. rotundifolia is resistant to many of the pathogens that detrimentally affect Vitis vinifera, the grape species commonly used for winemaking. For this reason, M. rotundifolia is a valuable genetic resource for breeding. Single-molecule real-time reads were combined with optical maps to reconstruct the two haplotypes of each of the 20 M. rotundifolia cv. Trayshed chromosomes. The completeness and accuracy of the assembly were confirmed using a high-density linkage map. Protein-coding genes were annotated using an integrated and comprehensive approach. This included using full-length cDNA sequencing (Iso-Seq) to improve gene structure and hypothetical spliced variant predictions. Our data strongly support that Muscadinia chromosomes 7 and 20 are fused in Vitis and pinpoint the location of the fusion in Cabernet Sauvignon and PN40024 chromosome 7. Disease-related gene numbers in Trayshed and Cabernet Sauvignon were similar, but their clustering locations were different. A dramatic expansion of the Toll/Interleukin-1 Receptor-like Nucleotide-Binding Site Leucine-Rich Repeat (TIR-NBS-LRR) class was detected on Trayshed chromosome 12 at the Resistance to Uncinula necator 1 (RUN1)/Resistance to Plasmopara viticola 1 (RPV1) locus, which confers strong dominant resistance to powdery and downy mildews. A genome browser, annotation, and Blast tool for Trayshed are available at www.grapegenomics.com.

Xylella fastidiosa causes transcriptional shifts that precede tylose formation and starch depletion in xylem.

Pierce's disease (PD) in grapevine (Vitis vinifera) is caused by the bacterial pathogen Xylella fastidiosa. X. fastidiosa is limited to the xylem tissue and following infection induces extensive plant-derived xylem blockages, primarily in the form of tyloses. Tylose-mediated vessel occlusions are a hallmark of PD, particularly in susceptible V. vinifera. We temporally monitored tylose development over the course of the disease to link symptom severity to the level of tylose occlusion and the presence/absence of the bacterial pathogen at fine-scale resolution. The majority of vessels containing tyloses were devoid of bacterial cells, indicating that direct, localized perception of X. fastidiosa was not a primary cause of tylose formation. In addition, we used X-ray computed microtomography and machine-learning to determine that X. fastidiosa induces significant starch depletion in xylem ray parenchyma cells. This suggests that a signalling mechanism emanating from the vessels colonized by bacteria enables a systemic response to X. fastidiosa infection. To understand the transcriptional changes underlying these phenotypes, we integrated global transcriptomics into the phenotypes we tracked over the disease spectrum. Differential gene expression analysis revealed that considerable transcriptomic reprogramming occurred during early PD before symptom appearance. Specifically, we determined that many genes associated with tylose formation (ethylene signalling and cell wall biogenesis) and drought stress were up-regulated during both Phase I and Phase II of PD. On the contrary, several genes related to photosynthesis and carbon fixation were down-regulated during both phases. These responses correlate with significant starch depletion observed in ray cells and tylose synthesis in vessels.

The genetic basis of sex determination in grapes.

It remains a major challenge to identify the genes and mutations that lead to plant sexual differentiation. Here, we study the structure and evolution of the sex-determining region (SDR) in Vitis species. We report an improved, chromosome-scale Cabernet Sauvignon genome sequence and the phased assembly of nine wild and cultivated grape genomes. By resolving twenty Vitis SDR haplotypes, we compare male, female, and hermaphrodite haplotype structures and identify sex-linked regions. Coupled with gene expression data, we identify a candidate male-sterility mutation in the VviINP1 gene and potential female-sterility function associated with the transcription factor VviYABBY3. Our data suggest that dioecy has been lost during domestication through a rare recombination event between male and female haplotypes. This work significantly advances the understanding of the genetic basis of sex determination in Vitis and provides the information necessary to rapidly identify sex types in grape breeding programs.

The genetic basis of grape and wine aroma.

The grape is one of the oldest and most important horticultural crops. Grape and wine aroma has long been of cultural and scientific interest. The diverse compound classes comprising aroma result from multiple biosynthetic pathways. Only fairly recently have researchers begun to elucidate the genetic mechanisms behind the biosynthesis and metabolism of grape volatile compounds. This review summarizes current findings regarding the genetic bases of grape and wine aroma with an aim towards highlighting areas in need of further study. From the literature, we compiled a list of functionally characterized genes involved in berry aroma biosynthesis and present them with their corresponding annotation in the grape reference genome.

The population genetics of structural variants in grapevine domestication.

Structural variants (SVs) are a largely unexplored feature of plant genomes. Little is known about the type and size of SVs, their distribution among individuals and, especially, their population dynamics. Understanding these dynamics is critical for understanding both the contributions of SVs to phenotypes and the likelihood of identifying them as causal genetic variants in genome-wide associations. Here, we identify SVs and study their evolutionary genomics in clonally propagated grapevine cultivars and their outcrossing wild progenitors. To catalogue SVs, we assembled the highly heterozygous Chardonnay genome, for which one in seven genes is hemizygous based on SVs. Using an integrative comparison between Chardonnay and Cabernet Sauvignon genomes by whole-genome, long-read and short-read alignment, we extended SV detection to population samples. We found that strong purifying selection acts against SVs but particularly against inversion and translocation events. SVs nonetheless accrue as recessive heterozygotes in clonally propagated lineages. They also define outlier regions of genomic divergence between wild and cultivated grapevines, suggesting roles in domestication. Outlier regions include the sex-determination region and the berry colour locus, where independent large, complex inversions have driven convergent phenotypic evolution.

Diploid Genome Assembly of the Wine Grape Carménère.

In this genome report, we describe the sequencing and annotation of the genome of the wine grape Carménère (clone 02, VCR-702). Long considered extinct, this old French wine grape variety is now cultivated mostly in Chile where it was imported in the 1850s just before the European phylloxera epidemic. Genomic DNA was sequenced using Single Molecule Real Time technology and assembled with FALCON-Unzip, a diploid-aware assembly pipeline. To optimize the contiguity and completeness of the assembly, we tested about a thousand combinations of assembly parameters, sequencing coverage, error correction and repeat masking methods. The final scaffolds provide a complete and phased representation of the diploid genome of this wine grape. Comparison of the two haplotypes revealed numerous heterozygous variants, including loss-of-function ones, some of which in genes associated with polyphenol biosynthesis. Comparisons with other publicly available grape genomes and transcriptomes showed the impact of structural variation on gene content differences between Carménère and other wine grape cultivars. Among the putative cultivar-specific genes, we identified genes potentially involved in aroma production and stress responses. The genome assembly of Carménère expands the representation of the genomic variability in grapes and will enable studies that aim to understand its distinctive organoleptic and agronomical features and assess its still elusive extant genetic variability. A genome browser for Carménère, its annotation, and an associated blast tool are available at http://cantulab.github.io/data.

Iso-Seq Allows Genome-Independent Transcriptome Profiling of Grape Berry Development.

Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species' entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or "private" gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.

Whole-Genome Resequencing and Pan-Transcriptome Reconstruction Highlight the Impact of Genomic Structural Variation on Secondary Metabolite Gene Clusters in the Grapevine Esca Pathogen Phaeoacremonium minimum.

The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.

Condition-dependent co-regulation of genomic clusters of virulence factors in the grapevine trunk pathogen Neofusicoccum parvum.

The ascomycete Neofusicoccum parvum, one of the causal agents of Botryosphaeria dieback, is a destructive wood-infecting fungus and a serious threat to grape production worldwide. The capability to colonize woody tissue, combined with the secretion of phytotoxic compounds, is thought to underlie its pathogenicity and virulence. Here, we describe the repertoire of virulence factors and their transcriptional dynamics as the fungus feeds on different substrates and colonizes the woody stem. We assembled and annotated a highly contiguous genome using single-molecule real-time DNA sequencing. Transcriptome profiling by RNA sequencing determined the genome-wide patterns of expression of virulence factors both in vitro (potato dextrose agar or medium amended with grape wood as substrate) and in planta. Pairwise statistical testing of differential expression, followed by co-expression network analysis, revealed that physically clustered genes coding for putative virulence functions were induced depending on the substrate or stage of plant infection. Co-expressed gene clusters were significantly enriched not only in genes associated with secondary metabolism, but also in those associated with cell wall degradation, suggesting that dynamic co-regulation of transcriptional networks contributes to multiple aspects of N. parvum virulence. In most of the co-expressed clusters, all genes shared at least a common motif in their promoter region, indicative of co-regulation by the same transcription factor. Co-expression analysis also identified chromatin regulators with correlated expression with inducible clusters of virulence factors, suggesting a complex, multi-layered regulation of the virulence repertoire of N. parvum.