Screening And Identification Of Potential Virulence Genes In Cotton Verticillium Dahliae Kleb. Strain

The Ascomycete fungus Verticillium dahliae Klebahn causes vascular wilt disease across over600plant species, including many high-value crops, such as cotton, olive, tomato, strawberry, etc. Due to less understanding of pathogenesis mechanism, Verticillium wilt is still a chronic economic problem in crop production, causing great losses in annual crop yields. To address this issue, screening and identification of pathogenicity-related factors is a key step to further decipher the mechanism of pathogenicity in V. dahliae. Accompanied by rapid development of omics techniques, including genomics and proteomics, which enable us to obtain a big data and screen the critical and useful information fastly and systematically.In this study, based on V. dahliae genome data, the gene family of Necrosis and ethylene-inducing protein （NEP） that may contribute to virulence was selected for in-depth functional characterization. More specifically, we identified eight VdNEP genes in the genome of VdGl, a V. dahliae strain isolated from cotton, designated as VdNEP1to VdNEP8. Of which, VdNEP6gene, encoding a secreted protein of256amino acids, contains a conserved heptapeptide motif GHRHD WE that is important in plasma membrane permeability and accordingly assisting triggering of plant defense response through damaging plant cells causing leaf necrosis. Especially, VdNEP6showed a high ratio of non-synonymous SNPs among twenty V. dahliae isolates which were collected from different cotton production fields, revealing their potential roles in pathogenesis in cotton V. dahliae. Functional validation of the signal peptide indicated that VdNEP6exactly located in extracellular. However, gene disruption of VdNEP6using homologous recombination and Agrobacterium tumefaciens mediated transformation （ATMT） techniques can not affect the pathogenicity, growth and conidiation of the wild type strain VdGl. Transient expression assay carried by Potato virus X （PVX） vector pGR107revealed that VdNEP6was neither involved in cell death induction nor suppression activity. Thus, we suggested that VdNEP6has no direct effect in cotton V. dahliae strain, since it failed to affect reducing the virulence of the wild type and no clear host cell death pattern was accomplished.Based on the quantitative analysis between the V. dahliae secretomes that were induced or non-induced by cotton tissue extract, numerous CAZy proteins showed a high sensitivity to cotton tissue, revealing their potential roles contributing to virulence in V. dahliae. Ten of CAZy genes that showed high expression level in the cotton tissue induced secretome were selected for functional characterization on virulence. To test cell death induction or suppression activities, ten of CAZy genes were cloned and transiently expressed in tobacco leaves via PVX （Potato virus X）-mediated expression system. The results indicated that none of genes can induce cell death in tobacco, whereas two genes including a putative cutinase and a Carboxypeptidase Y genes （VDG1₀.3897and VDG1₀.3197） can effectively suppress cell death triggered both by apoptosis regulator BAX and VdNEP1elicitor that known to trigger necrosis on cotton leaves. The suppression ability of the candidates suggested that the two genes may play potential roles in V. dahliae pathogenesis and possibly enhance the virulence through the regulation of host immunity. In addition, gene expression analysis indicated that the two candidate genes showed a strong induction in V. dahliae when cultivated in the medium with cotton tissue extract. Through homologous recombination, the gene knock-out mutants without the cutinase VDG1₀.3897candidate gene was successfully obtained. The disease assay of the mutant on cotton is in progress.In addition, to disclose why the V. dahliae specifically colonize in plant vascular niches, we conducted a comparative genomics among14sequenced plant pathogenic fungi including vascular and non-vascular pathogens. The tested fungi are ascomycetes and belong to three classes, including Sordariomycetes （Fusarium graminearum, F. oxysporum, Verticilium dahliae, V. albo-atrum, Neurospora crassa, Magnaporthe grisea and Chaetomium globosum）, Eurotiomycetes （Aspergillus nidulans and A. terreus） and Leotiomycetes （Botrytis cinerea）. The data revealed57genes that are specific to vascular wilt fungus Verticilium dahliae （VDGhl.991）. In-depth comparative analysis of these57genes among the reported135fungal genomes further revealed five genes, namely, VdVS1to VdVS5that showed high presence ratio in vascular related fungal genomes such as V. dahliae, Fusarium oxysporum and V. albo-atrum but not in non-vascular related pathogens. Further gene expression analysis indicated that five vascular-related genes of V. dahliae can be strongly induced by cotton tissue, an in vitro interaction system between V. dahliae and cotton. Gene disruption of VdVS3and VdVS5in V. dahliae wild-type strain VDGhl.991did not affect the growth and sporulation but caused virulence reduction on cotton compared with the wild type strain, indicating that VdVS3and VdVS5may play important roles in V. dahliae pathogenicity. Further functional characterization of these vascular-related candidate genes may facilitate our understanding on the pathogenicity mechanism of vascular infected fungal pathogens.In summary, this study provided the target genes related to pathogenicity in V. dahliae, further functional investigation may greatly enhance the exploration of pathogenesis mechanism of this vascular-parasitic fungus.