Indentification Of The Verticillium Dahliae Kleb. Insertional Mutant Phenotypes And Analysis Of The T-DNA Flanking Sequences Of Mutants

The cotton Verticillium Wilt caused by Verticillium dahliae is one of the most devastating diseases of cotton worldwide.At present,there aren't any high resistant or immunity material sources,high resistanct cotton varieties on V.dahliae,and due to lack of good effective fungicides used for cotton Verticillium wilt,so it is difficulty to manage it.Till now,the pathogenic molecular mechanism of V.dahliae is still unknown.Screening of pathogenicity defective mutants obtained by Agrobacterium tumefaciens-mediaed transformation?ATMT?,then isolating and identifying pathogenicity-associated genes on the whole genome level of V.dahliae,is helpful to reveal the molecular pathogenesis V.dahliae and provide theoretical basis for the control of cotton Verticillium wilt.Previously,a mutant library has been constructed by Agrobacterium tumefaciens-mediated transformation?ATMT?from a virulent defoliating V.dahliae Kleb.FGH2 in our lab?In this study we identified the mutans with increased microsclerotia formation,colony growth rate and pathogenicity defects,and then isolate the flanking sequence of T-DNA insertion mutants of V.dahliae.The results are as follows:1.The V.dahliae FGH2 isolate produced a large number of microsclerotia on PDA medium.Several mutants were abtained.while eight T-DNA insertion mutants lost the ability for forming microsclerotia.Six T-DNA insertion mutants growed slow significantly,while eight T-DNA insertion mutants had an increase in growth significantly.The mutant V181 can't grow on WA medium.By dipping roots of Yinshan 1 cotton seeding with FGH2 spore suspension of 186 transformants,the virulence of the 10 mutants to cotton plants decreased significantly,and 3 mutants displayed dramatic decrease in virulence2.The flanking sequences of T-DNA insertional sites from 12 mutants were obtained by hi TAIL-PCR,and sequences of amplified hi PCR products were used for BLAST to the database of V.dahliae VDLs.17,and the 9 T-DNA insertional locus were identified,located in 6 chromosomes.and the flanking sequences showed 97-100% identity with the corresponding site genome of VDLs.17.There were 3 mutants with T-DNA tag in coding region,2 mutants T-DNA tag located in 500 bp upstream from start codon.3.In mutant V181,T-DNA inserted in coding region of VDAG₀8393.1,which encodes aminodeoxychorismate synthase?ADCS?.The main phenotype effects of this insertion were significantly reduced in pathogenicity on Yinshan 1.The conidiospores of the mutant can abnormally germinate,and can not formate colonies,determined to be a auxotroph mutant.And it recoved the growth cultivated in water agar?WA?adding PABA or folic acid.4.In mutant V546,T-DNA inserted in coding region of VDAG₀7282.1,which encodes hypothetical protein gene.The mutant colony were velum type.The main phenotype effects of this insertion defectived microsclerotia development capability and significantly reduced in pathogenicity.5.In mutant V1009,T-DNA was inserted in coding region of VDAG₀1673.1,which encodes eroxisomal membrane protein PEX16.The mutant colony were velum type.The main phenotype effects of this insertion defectived microsclerotia development capability and virulence.6.In mutant V113-1 had a significant decrease in microsclerotia production,the T-DNA insertion site locate in the gene VDAG₀9526.1,coding FADbindingdomain-containing protein 857 bp upstream,and hypothetical protein gene VDAG₀9525.1 3509 bp downstream.Mutant V162-1 also had a significant decrease in microsclerotia production,whose T-DNA insertion site locate in the gene VDAG₀1672.1 164 bp upstream,and hypothetical protein gene VDAG₀1671.1 1471 bp downstream.Mutant V971-1 completely lost the ability of development microsclerotia,the T-DNA insertion site locate in the gene hypothetical protein gene VDAG₀5438.11371 bp downstream and hypothetical protein gene VDAG₀5439.1 2291 bp downstream.Mutant V319 has a decrease of the ability of microsclerotia production and the T-DNA insertion site locate in the gene hypothetical protein gene VDAG₀5673.1271 bp upstream and hypothetical protein gene VDAG₀5672.11081 bp downstream.Mutant V11-2 also completely lost the ability of microsclerotia,whose T-DNA insertion site locate in the gene coding cytidine diphosphate-2 acylglycerol-inositol 3-phosphoric acid acyl transferase VDAG₀7014.1 2968 bp upstream and in the gene coding glycosides 5-phosphate decarboxylase VDAG₀7014.1 969 bp downstream.The mutant V26-2 with microsclerotia development defect,the T-DNA insertion site locate in the gene RO10 protein VDAG₀2793.1 716 bp downstream and the gene coding infiltration induced protein VDAG₀2794.1 2906 bp upstream.