Mechanism Of Gene Cluster VdDfs In Verticillium Dahliae Regulating Host Plants Defoliating Phenotype

Verticillium dahliae is a typically soil-borne vascular fungal with abundant population diversity,pertaining to asexual reproduction.According to the characteristics of infected host?cotton,okra,etc.?,Verticillium dahliae were classified into two pathogenic types:defoliation and non-defoliation.The etiological analysis showed that the defoliating strain was widely popular worldwide due to its strong pathogenicity and adaptability.Defoliating strains has become the dominant population of verticillium wilt.However,the molecular underpinnings for defoliation have yet to be elucidated.Previous research in our laboratory uncovered that the high virulence of Vd991?from cotton?contain a lineage specific region G-LSR2 compared to Vdls.17?from lettuce?and JR2?from tomato?by comparative genomics.The seven genes?VdDf1-VdDf7,VdDfs?were obtained by horizontal gene transfer from Fusarium,and regulated the dominance of Verticillium dahliae to host cotton.Simultaneously,group resequencing and genome comparative was analyzed by 75 strains?including 59defoliating strains and 16 non-defoliating strains?from the main cotton area of our country,reveling that VdDfs was only found in defoliating strains.Knockout mutant loses the ability to cause the defoliation of the host.The results showed that the seven genes had the function to enhance the defoliation to cotton.Expression analysis of VdDf1-VdDf7 revealed that the transcript levels of seven genes was significantly up-regulated after inoculation of cotton,especially VdDf5 and VdDf6,which were strongly up-regulated at 48 hours.We have obtained deletion mutants of seven genes by homologous recombination,identification of defoliating phenotype display only deletion of?Df5 or?Df6 in the Vd991 background resulted in loss of the D phenotype in cotton four weeks after inoculation.Accordingly,simultaneous complementation of VdDf5 and VdDf6 together?VdDf5₆?in the?Dfs strain and non-defoliating strain VDG78,resulted in recovery of the defoliation phenotype.Above results support the finding that VdDf5 and VdDf6 are the key genes to regulate the defoliating phenotype to cotton.The analysis of genetic evolution showed previously that seven genes was horizontally transferred from F.oxysporum f.sp.Vasinfectum.And the sequence alignment results displayed that although the homologous gene sequences in the F.oxysporum were highly consistent,there were still significant polymorphism sites.In the VdDf5 sequence comparisons with its corresponding ortholog,three non-synonymous mutations were observed,and eight SNPs resulting in four non-synonymous changes in VdDf6,as well as a 18 bp truncation resulting in a different start codon;VdDf5₆^FO expression did not confer either the D phenotype to the cotton ND isolate?VDG78?.These results indicated that VdDf5and VdDf6 had undergone natural selection following the horizontal transfer from F.oxysporum and finally resulted in higher virulence/defoliation to cotton.Global gene expression patterns were examined in?Dfs deletion strains and compared with the wild-type strain 48 hours after inoculation of cotton plants by expression profile,showing that the redox gene VEDA₀5052 was significantly regulated by VdDfs gene cluster.Transcripts of the gene encoding short-chain dehydrogenase?VEDA₀5052,hereafter name VdDH1?were especially abundant during infection of cotton,and showed lower expression in?Dfs,?Df5₆ mutant and non-defoliating strain VDG78.Moreover,re-introduction of VdDH1 into the?Dfs and?Df5₆ led to recovery of the defoliation phenotype,suggesting VdDfs can cooperate with VdDH1 to endow defoliation phenotype.The growth phenotypic identification of?Dfs,?Df5₆ and?DH1 mutant under oxygen tension showed that the germination of mycelia and the ability of producing spores decreased significantly compared to normal conditions.Electron microscopy showed that?Dfs,?Df5₆ and?DH1 mutants loss penetrating ability and were restricted to the epidermal surface.These experiments manifested that key functional genes VdDf5,VdDf6 and affected genes VdDH1 play an important role during V.dahliae responsing to environmental changes in oxygen tension,promote adaptation to vascular oligotrophic environment,eventually led to defoliation phenotype to cotton.The annotation of 22 genes encoded by G-LSR2 predicted a variety of functions.VdDf5 and VdDf6share homology with polyketide synthases,and VdDf4 and VdDf7 share homology with a major facilitator superfamily?MFS?gene and a N-acyl-phosphatidylethanolamine-hydrolysing phospholipase D?NAPE-PLD?,respectively.Therefore,G-LSR2 appears to be a typical gene cluster involved in secondary metabolisms.Cotton seedlings were treated with the extracts from culture supernatants from the?Dfs strain and the?Dfs strain simultaneously complemented with VdDf5 and VdDf6.As expected,cotton leaves displayed no defoliating phenotype after treatment with?Dfs strain culture supernatant.Quantification of NAEs showed that the concentration of NAE12:0 was significantly lower in culture suspensions of the?Dfs mutant relative to the wild-type,and NAE content was recovered after re-introduction of VdDf5 and VdDf6 together into the?Dfs mutant and non-defoliating strain VDG78.Analysis of population revealed the concentrations of NAE 12:0 were significantly lower in cultures of ND strains than in D strains,Correspondingly,cotton plants inoculated with the ND isolate VDG78 also displayed D phenotype seven days when combined with treatment with the NAE 12:0.Excessive NAE12:0 results in FAAHs expression in the host cotton,causing the host to become more sensitive to the abscess acid and the hormone imbalance and eventually leading to defoliation.The above results showed that the gene cluster VdDfs was involved in the synthesis of the secondary metabolite NAE12:0,which affected the regulation of defoliation.Taken together:VdDfs gene cluster was horizontally transferred from F.oxysporum to V.dahliae by natural selection,and is found exclusively in the genomes of defoliating?D?strains;The gene cluster was involved in the stress response to environmental changes in oxygen tension of the host vascular tissue by cooperating with other redox genes.the gene clusters by participating in secondary compounds NAE12:0 metabolism interfere with the cotton FAAHs gene expression,leading to the host of ABA sensitivity enhancement and imbalance in hormone levels and resulting in phenotype.