Verticillium wilt, caused by the notorious fungal pathogen
V. dahliae
, is one of the main limiting factors for agricultural production. Metalloproteases played an important role in the pathogenic mechanism of pathogens.
Glycoside hydrolase (GH) family members act as virulence factors and regulate plant immune responses during pathogen infection. Here, we characterized the GH28 family member endopolygalacturonase VdEPG1 in Verticillium dahliae. VdEPG1 acts as a virulence factor during V. dahliae infection. The expression level of VdEPG1 was greatly increased in V. dahliae inoculated on cotton roots. VdEPG1 suppressed VdNLP1‐mediated cell death by modulating pathogenesis‐related genes in Nicotiana benthamiana. Knocking out VdEPG1 led to a significant decrease in the pathogenicity of V. dahliae in cotton. The deletion strains were more susceptible to osmotic stress and the ability of V. dahliae to utilize carbon sources was deficient. In addition, the deletion strains lost the ability to penetrate cellophane membrane, with mycelia showing a disordered arrangement on the membrane, and spore development was affected. A jasmonic acid (JA) pathway‐related gene, GhOPR9, was identified as interacting with VdEPG1 in the yeast two‐hybrid system. The interaction was further confirmed by bimolecular fluorescence complementation and luciferase complementation imaging assays in N. benthamiana leaves. GhOPR9 plays a positive role in the resistance of cotton to V. dahliae by regulating JA biosynthesis. These results indicate that VdEPG1 may be able to regulate host immune responses as a virulence factor through modulating the GhOPR9‐mediated JA biosynthesis.
The ergosterol biosynthesis pathway plays an important role in model pathogenic bacteria Saccharomyces cerevisiae, but little is known about the biosynthesis of ergosterol in pathogenic fungus Verticillium dahliae. In this study, we identi ed the VdERG2 gene encoding sterol C-8 isomerase from V. dahliae and investigated its function in virulence by generating gene deletion mutants (ΔVdEGR2) and complemented mutants (C-ΔVdEGR2). Deletion of VdERG2 reduced ergosterol content. The conidial germination rate and conidial yield of ΔVdERG2 decreased signi cantly, and abnormal conidia were produced. In spite of VdERG2 did not affect the utilization of carbon sources by V. dahliae, but ΔVdERG2 observed a decrease in melanin production when cellulose and pectin were used as sole carbon sources, respectively. The ability of mutants ΔVdERG2 to produce microsclerotia and melanin decreased and the knockout of VdERG2 led to a signi cant decrease in the expression of microsclerotia and melanin-related genes Va M, Vayg1, VDH1, VdLAC, VdSCD and VT4HR. In addition, mutants ΔVdEGR2-1 and ΔVdEGR2-2 were very sensitive to congo red (CR), sodium dodecyl sulfate (SDS) and hydrogen peroxide (H 2 O 2 ) stresses, indicating that VdEGR2 was involved in cell wall and oxidative stress response. The absence of VdERG2 weakened the penetration ability of mycelium on cellophane and affected the growth of mycelium on cellophane. Although ΔVdERG2 could infect cotton, its pathogenicity was signi cantly impaired. These phenotypic defects in ΔVdERG2 could be complemented by reintroduction of a fulllength VdERG2 gene. In summary, as a single conservative secretory protein, VdERG2 played a crucial role in ergosterol biosynthesis, nutritional differentiation and virulence in V. dahliae.
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