Verticillium Dahliae Kleb (Vd991) T-DNA Mutant Library Construction And Screening Of Pathogenic-Related Genes

Verticillium dahliae Kleb is an important cause agent of vascular disease——Verticillium wilt in cotton. In recent years, Verticillium wilt has spread rapidly in the northern and southern cotton fields in China, causing big losses of cotton yields. Traditional methods demonstrated helplessly in the management of Verticillium wilt. By now, we knew less about pathogenic mechanism of V. dahliae and it is difficult to find resistant cotton cultivars. To investigate the pathogenesis of V. dahliae, in this study, we used the methods of T-DNA insertional mutagenesis and Agrobacterium tumefaciens-mediaed transformation to construct a T-DNA mutant library of fungus V. dahliae (Vd991). Furthermore, we screened and identified some of mutants to provide theories basis on the study of pathogenic mechanism of V. dahliae and Verticillium wilt management.A binary vector pCTHyg contained a Hyg gene which promoted by TrpC from Aspergillus nidulans has been constructed, and then the T-DNA in pCTHyg was transported to V. dahliae (Vd991) by Agrobacterium tumefaciens-mediated transformation. We totally obtained 15131 Vd991 T-DNA mutants. 30 mutants have been analyzed by phenotype screening, including growth rate in different medium which contains different carbon source, conidia-product ability, and pathogenicity. Finally, we analyzed flanking sequences of mutants by high-efficiency TAIL-PCR. The results revealed that:1. T-DNA insertion will change growth rate of mutants in different medium which contains different carbon source. Compared with wild-type Vd991, we found that 76E01 and 92G09 showing reduced growth rate in the cellulose and pectin medium.2. T-DNA insertion will change conidia-product ability of mutants in Czapek liquid medium. Compared with wild-type Vd991, we found that 76B11, 76E01, 78A12, 78C02, 78C07-2 and 92B12 showing decreased spores production.3. T-DNA insertion will change pathogenicity of mutants. Compared with wild-type Vd991, we found some of identified mutants showing decreased virulence.1) 424, 68E02, 76E01, 77D01, 77F07, 78C05, 78C07-1, 78C07-2, 91D08, 91H07 and 92A03 showed virulence decreased in the cotton cultivar Ji 11.2) 424, 68E02, 76E01, 77C09, 77D01, 78C02, 78C07-2, 91F03, 92A03, 92B12, 92D02 and 92E07 showed virulence decreased in the cotton cultivar Yu 21.3) 76A12, 76E01, 77B06, 77F07, 77H02, 78A12, 78C07-2, 85H04, 91F03, 91H07 and 92A12 showed virulence decreased in the cotton cultivar Lu 28.4) 424, 68E02, 76E01, 77D01, 78C07-2 and 91D08 showed virulence decreased significantly in all the three cotton. 78C03 showed virulence decreased in all the three cotton.4. T-DNA flanking sequences of 13 mutants were further identified by hiTAIL-PCR. BLAST search and alignment, found T-DNA insertion site of 11 mutants.