Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

Verticillium dahliae is a widespread soilborne fungus that causes Verticillium wilt on numerous economically important plant species. In tomato, until now, three races have been characterized based on the response of differential cultivars to V. dahliae, but the genetic basis of race divergence in V. dahliae remains undetermined. To investigate the genetic basis of race divergence, we sequenced the genomes of two race 2 strains and four race 3 strains for comparative analyses with two known race 1 genomes. The genetic basis of race divergence was described by the pathogenicity-related genes among the three races, orthologue analyses, and genomic structural variations. Global comparative genomics showed that chromosomal rearrangements are not the only source of race divergence and that race 3 should be split into two genotypes based on orthologue clustering. Lineage-specific regions (LSRs), frequently observed between genomes of the three races, encode several predicted secreted proteins that potentially function as suppressors of immunity triggered by known effectors. These likely contribute to the virulence of the three races. Two genes in particular that can act as markers for race 2 and race 3 (VdR2e and VdR3e, respectively) contribute to virulence on tomato, and the latter acts as an avirulence factor of race 3. We elucidated the genetic basis of race divergence through global comparative genomics and identified secreted proteins in LSRs that could potentially play critical roles in the differential virulence among the races in V. dahliae. IMPORTANCE Deciphering the gene-for-gene relationships during host-pathogen interactions is the basis of modern plant resistance breeding. In the Verticillium dahliae-tomato pathosystem, two races (races 1 and 2) and their corresponding avirulence (Avr) genes have been identified, but strains that lack these two Avr genes exist in nature. In this system, race 3 has been described, but the corresponding Avr gene has not been identified. We de novo-sequenced genomes of six strains and identified secreted proteins within the lineage-specific regions (LSRs) distributed among the genomes of the three races that could potentially function as manipulators of host immunity. One of the LSR genes, VdR3e, was confirmed as the Avr gene for race 3. The results indicate that differences in transcriptional regulation may contribute to race differentiation. This is the first study to describe these differences and elucidate roles of secreted proteins in LSRs that play roles in race differentiation.

Background

Plant pathogens and their hosts undergo adaptive changes in managed agricultural ecosystems, by overcoming host resistance, but the underlying genetic adaptations are difficult to determine in natural settings. Verticillium dahliae is a fungal pathogen that causes Verticillium wilt on many economically important crops including lettuce. We assessed the dynamics of changes in the V. dahliae genome under selection in a long-term field experiment.

Results

In this study, a field was fumigated before the Verticillium dahliae race 1 strain (VdLs.16) was introduced. A derivative 145-strain population was collected over a 6-year period from this field in which a seggregating population of lettuce derived from Vr1/vr1 parents were evaluated. We de novo sequenced the parental genome of VdLs.16 strain and resequenced the derivative strains to analyze the genetic variations that accumulate over time in the field cropped with lettuce. Population genomics analyses identified 2769 single-nucleotide polymorphisms (SNPs) and 750 insertion/deletions (In-Dels) in the 145 isolates compared with the parental genome. Sequence divergence was identified in the coding sequence regions of 378 genes and in the putative promoter regions of 604 genes. Five-hundred and nine SNPs/In-Dels were identified as fixed. The SNPs and In-Dels were significantly enriched in the transposon-rich, gene-sparse regions, and in those genes with functional roles in signaling and transcriptional regulation.

Conclusions

Under the managed ecosystem continuously cropped to lettuce, the local adaptation of V. dahliae evolves at a whole genome scale to accumulate SNPs/In-Dels nonrandomly in hypervariable regions that encode components of signal transduction and transcriptional regulation.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.