Functional Analysis Of Molecular Marker And Itsnearby Genes In Verticillium Dahliae Defoliatingstrain

The pathogenic mechanism of Verticillium dahliae has always been the focus of the cotton Verticillium wilt research.In this study,we located the defoliating molecular marker(MM15)on V.dahliae genome by bioinformatics analysis and knocked out MM15 and its nearby genes in wild-type defoliating strain XJ592 by method of Agrobacterium tumefaciens-mediated transformation(ATMT).In order to explore their functions on vegetative growth and pathogenicity,we compared the differences between knock-out mutants and wild type strain on several phenotypes.The functions of MM15,Vdcut14 and VDAG＿00446 are all reported for the first time.The results are as follows:1.Bioinformatics analysis indicated that MM15 is located between gene VDAG＿00445(Vdcut14)and gene VDAG＿00446 and the length is 15 bp by BLAST.2.Vdcut14,VDAG＿00446 and MM15 in the wild type defoliating strain XJ592 were knocked out by the way of ATMT and obtained 10 knock-out mutants.The mutants are VdΔcut14-3,13,16,VdΔ00446-4,24,VdΔMM15-7,13,14,15,and 17.Southern blot showed that VdΔcut14-3 and 13 were single insertion mutants.We genetically complemented VdΔcut14-3 by ATMT and obtained three complementary mutants VdΔcut14/Vd CUT14-27,29,44.3.The results of pathogenicity test showed that the wild type defoliating strain XJ592 and knock-out mutants of VdΔMM15 had higher disease incidence than mutants of VdΔcut14-3 and VdΔ00446 and wild type non-defoliating strain XJ511 at 1-3 weeks post inoculated.But disease index of all treatments were above 80% besides XJ511 and VdΔ00446-24 at 5 weeks post inoculated.The result suggest that the 15 bp molecular marker has nothing to do with the pathogenicity of Verticillium dahliae and Vdcut14 enhances the ability of pathogenicity.4.Hydrogen peroxide stress experiment indicated that the inhibition zone of the knock-out mutant strain VdΔcut14-3 was significantly reduced compared with the wild-type strain XJ592 at 3 dpi.The yield of microsclerotium in VdΔcut14-3 was less than XJ592 and VdΔcut14/Vd CUT14-27,29,44.These results showed that Vdcut14 could inhibit the sensitivity of V.dahliae to hydrogen peroxide.5.The results of spore yield experiment showed that the spore yield of VdΔcut14-3 was significantly increased compared with wild type strain XJ592 and VdΔcut14/CUT14-27,29,44,indicated that Vdcut14 gene inhibits spore production of Verticillium dahliae.There was no significant difference between VdΔcut14 and wild type strain XJ592 in pressure stress,penetration ability and growth rate experiments indicated that Vdcut14 gene does not participate in pressure stress response,vegetative hyphae growth and penetration.