Constructing Of The Muntant Library Of Verticillium Dahliae V07Df2via T-DNA Insertion And Screening The Mutants With Phenotypic Variability

Verticillium dahliae Kleb., a soil-borne pathogenetic fungus, causes Verticillium wilt on many crops such as the cotton and eggplant around the world. However, there is little understanding about the pathogenesis of the pathogen and the host-pathogen interaction. No method has been proved to manage the disease effectively. Our study provides the basis of analysis the pathogenesis.Two strains isolated from cotton in Jiangsu, highly virulent defoliating strain V07DF2and medial virulent nondefoliating strain Bp2, were labeled with green fluorescent protein (GFP) by Agrobacterium tumefaciens-mediated transformation (ATMT). The transfer region of the GFP-expressing plasmid1300-HYG-sGFP was integrated into the genome of the strains. After optimizating the process of ATMT, we can obtain1,000～2,000mutants among106spores and the transformation efficiency is up to0.1%～0.2%. In order to observe the process of infection and colonization, the transgenic strains with wild phenotypes and high GFP expressing level were selected and inoculated to the Arabidopsis seedlings. The results indicate that the progresses of the infection and colonization of the GFP-tagged strains can be observed after inoculation. Additionally, the results laid a good foundation for Agrobacterium tumefaciens-mediated transformation of V. dahliae, and prepared research materials for the further analysis of the mechanisms of the infection and the pathogenicity.Promoter trapping is inserting a reporter gene without promoter such as GFP gene near the border of the T-DNA region, which compose the promoter capture vecter with other selectable marker genes. If the reporter gene insert into the promoter region or express correctly with other gene, is will be activated, which is helpful for decreasing the rate of meaningless insertion.A new vector named bidirectional promoter-capture vector, which was transformed into the genome of strain V07DF2by ATMT, was successfully constructed and used to constructe the T-DNA insertional mutant library containing6,000mutants. The T-DNA region of this vector contains the GFP sequence with ORF and terminator, which was used to capture the promoter of expressing gen, Hygromycin resistance box screening the transformants and plasmid rescue component amplifying flanking sequence. Analysising molecular genetics of mutants, the function of the bidirectional promoter-capture vector was estimated. And the single spores islation is a necessary process, which can provent cross-contamination and ensure the resource stability of mutants.Changes in morphology, such as growing rate, hyphal density, pigmentation traits and sGFP expression level, were observed in the1,000randomly selected transformants to validate the effectiveness of the mutant library. As a result, a total of108mutants were obtained, including four slowly grow mutants, two mutants with mycelial morphology variation, two thin-hyphal density mutant, two mutant produting atropurpureus pigment, thirty-eight mutants with different capability of sporulation, and sixty-five mutants with green fluorescence. Some mutants had overlapping morphology. Pathogenicity identification analysis of the significant variation62candidates indicated that four mutants lose the virulent and ten mutants have weak virulent. All the mutants will be useful for the analysis of the pathogenicity-related molecular mechanism of Verticillium dahliae.