Study On Pathogenic Mechanisms Of Virulence Factor VdEPG1 In Verticillium Dahliae

Cotton is an important economic and natural fiber crop worldwide.Verticillium dahliae(V.dahliae)is a soil-borne plant vascular wilt fungal pathogen.Verticillium wilt,which is caused by V.dahliae,is a devastating disease in cotton.There are no upland cotton cultivars displaying high resistance to V.dahliae.Exploring the pathogenic mechanism of V.dahliae and studying the interaction between V.dahliae and cotton can provide theory foundation and gene resources for helping molecular breeding.V.dahliae genome encodes many secreted proteins to battle with host effect during its infection.Glycoside hydrolase(GHs)act as virulence factors and regulate the plant immune responses during pathogen infection.In this study,the function of VdEPG1(a member of VdGH28)was studied,and its interaction protein in cotton was identified using Y2H.These results are listed as followings.1.In this study,we identified 11 GH28 members in V.dahliae genome.Under the cotton root inducing,the expression of all GH28s were up-regulated in the resuspension of Vd991 spore,this indicated that GH28 genes may do contribution to infection of V.dahliae.In the corresponding roots,the expression level of VDAG?04977(VdEPG1)was the highest.This result suggests VdEPG1 plays an important role during infection.Agrobacterium-mediated transient expression assay showed that all GH28 genes in V.dahliae can't induce the cell death in N.benthamiana leaves,but the VdEPG1 can suppress the cell death induced by BAX.Furthermore,the pathogenesis-related genes Nb Acre31,NbCYP71D20,Nb LOX,Nb PR4 and Nb WRKY7 activated by BAX were suppressed when VdEPG1 was co-expressed with BAX.Yeast signal sequence trap system assay suggests that VdEPG1 can be secreted outer space of V.dahliae cells during infection.These results suggest that VdEPG1 may function as virulence factor to suppress some plant immunity pathways during infection.2.Homologous recombination was used to generate the ?VdEPG1 mutants and complementary strains.The ?VdEPG1 mutant strains showed significantly decreased in mycelial growth on PDA,had hypersensitivity to osmotic stresses,such as KCl,NaCl,SDS and sorbitol.And the ability of carbon source utilization and sporulation of deletion strains were decreased.Deletion strains could not penetrate the cellophane membrane,and had the disorderly arrangement mycelium on the cellophane membrane,showed decrease pathogenicity in cotton.3.Furthermore,GhOPR9 was screened to interact with VdEPG1 by yeast two-hybrid assay.And then,the interaction was confirmed by Y2H,BiFC and LCI in vitro and vivo.Bi FC assays showed YFP fluorescence was observed at the plasma membrane,and subcellular localization displayed VdEPG1-GFP and GhOPR9-GFP could be observed at the plasma membrane in N.benthamiana leaves.These results suggested that VdEPG1 can interact with GhOPR9 at the plasma membrane.4.Through bioinformatics analysis about OPR gene family,we found that GhOPR8 and GhOPR9 were tandem duplication events.And we confirmed VdEPG1 have no interaction with GhOPR8 via Y2H assays.Under the NaCl and V.dahliae stresses,the expression of GhOPRs were induced.And under the PEG6000 stress,the expression of GhOPRs were decreased.Moreover,the expression of GhOPR9 in cotton root under V.dahliae stress was significantly increased.Through virus induced gene silenceing(VIGS)for knocking down of GhOPR9,the cotton seedlings showed more susceptible to V.dahliae.With overexpression of GhOPR9 in Arabidopsis,the transgenic lines displayed more resistance to V.dahliae.These results indicated that GhOPR9 plays a positive role in resistance to V.dahliae.5.When knock-down the GhOPR9 in plants,other genes expression of JA synthesis was analysis by q RT-PCR.The results showed the genes expression were increased to against the V.dahliae infection.And JA content was detected by ELISA.The JA content was decreased in GhOPR9 silenced plants.These results showed that GhOPR9 regulate JA synthesis in cotton.Furthermore,the GhOPR9 expression in cotton roots after inoculation by WT,?VdEPG1-8,?VdEPG1-11,C-?VdEPG1-8 and C-?VdEPG1-11 strains were analysis by q RT-PCR.The GhOPR9 expression was decreased in deletion strains treated plants,But the JA content was increased in deletion strains treated plants.These results suggested that VdEPG1 can regulate the JA content via modulating the GhOPR9 expression under the V.dahliae infection.In summary,VdEPG1 acts as a virulence factor,can interact with GhOPR9.And GhOPR9 plays a positive role in resistance to V.dahliae.The VdEPG1 may regulate the plant immune responses by activating the GhOPR9-mediated JA synthesis during pathogen infection.These results can provide new views of theory foundation to help molecular breeding of disease resistance.