Screening Of Verticillium Dahliae Pathogenicity-related Mutants And Functional Analysis Of The Pathogenic Gene VdCYP1

Verticillium wilt, caused by the fungus Verticillium dahliae, is a soil borne vascular disease. Verticillium wilt has become the main impediments to cotton productions in China. Verticillium wilt is particularly difficult to control because of the complex pathogenic mechanism of V. dahliae. To elucidate the pathogenic mechanism, the identification of pathogenicity-related genes of V. dahliae has become a current research hotspot.Agrobacterium tumefaciens-mediated transformation（ATMT）, played an important role in functional genomics, is a powerful tool for isolating and identifying novel genes. Previously, using Agrobacterium tumefaciens-mediated transformation, we generated a T-DNA mutant library of fungus V. dahliae（Vd991） contained 15131 randomly inserted T-DNA mutants. Traditional identifications of Verticillium wilt in green house have been applied to rapid screening the pathogenicity of V. dahlia, but its efficiency is low and unavailable to the study of screening and identification of the pathogenicity-related mutants. Therefore, the method, using for rapid, high-throughput identification of the pathogenicity-related mutants, are important for the study of the function of pathogenicity-related mutants of V. dahliae.In this study, the results showed that the conidial concentration up to 5.0×105 CFU/mL could obvious infect in cotton and cause the Verticillium wilt symptom. The standard culture method of V. dahliae was constructed, and the preparation of spore suspension was simplified. Subsequently, the inoculation steps were optimized for construction the rapid process of V. dahliae pathogenicity identification, which included single spore isolation, expanding culture, conidial suspension preparing, inoculation, and results investigation. Moreover, the rapid screening system of pathogenicity-related mutants in V. dahliae was constructed after coordinate design. By identifying the pathogenic phenotype of 1344 inserted mutants, results suggest:（1） 69 mutants showed decreased pathogenicity in the cotton cultivar Jun-1, 16 mutants significantly decreased or loss of pathogenicity;（2） 6 mutants showed significantly decreased pathogenicity in the cotton cultivar Jun-1 by traditional root-dipping group verification;（3） the mutant M01C06 and M01E010 mostly loss of pathogenicity by the analysis of the disease index. The T-DNA insertion site’s right flank sequence of mutant M01A06, M01C06, M03B07 and M4H05 was determined by hiTAIL-PCR.Through sequencing and BLAST analysis, it’s indicated that mutant M01C06 T-DNA insertion point is located in the fourth chromosome and between gene VDAG₀5889 and gene VDAG₀5890. The VDAG₀5889 encoding protein was predicted as the translation initiation factor 5, and the VDAG₀5890 encoding protein was predicted to cytochrome 3A13 P450, named VdIF5 and VdCYP1, respectively. Analysis of gene expression levels of VdIF5 and VdCYP1 by quantitative PCR, the result indicates the expression of VdCYP1 gene was significantly decreased, and speculates that the gene VdCYP1 affects the pathogenicity of M01C06. The deletion mutant of ?VdCYP1 was obtained by Agrobacterium tumefaciens-mediated transformation method. By measuring Spores production, phenotypic identification and pathogenicity, the results suggest:（1） the growth rate and phenotypic of the deletion mutant of ?VdCYP1 and the inserted mutant M01C06 are unchanged;（2） the spores production and pathogenicity of ?VdCYP1 and M01C06 are significantly decreased. The conlusions evidence that the pathogenicity of M01C06 is significantly decreased because of T-DNA insertion affected the expression of VdCYP1, and VdCYP1 is the pathogenicity-related gene of V. dahliae.The full length genome sequence of VdCYP1 is 1767 bp, the full length cDNA of VdCYP1 is 1458 bp, which encoding 485 amino acids, and containing 5 exons and 4 inxons. Though the SMART and TMHMM 2.0 software to analyze the sequence of the gene VdCYP1, the gene Vd CYP1 contains a transmembrane domain and a cytochrome P450 domain, the predictable function of VdCYP1 is related to the synthesis of secondary metabolites and the pathogenicity of the fungus. Using quantitative PCR to analyze the expression of VdCYP1 in the V. dahliae infected cotton tissue, found the expression up-regulated significantly in the 1 to 3 days after inoculation. These results suggested that the function of VdCYP1 is closely related to the V. dahliae early infection progress. The over expression vector of VdCYP1 were constructed and transferred into the mutant M01C06 and deletion mutants ?VdCYP1 to construct the overexpression strains of mutant M01C06 and complementary transformants of deleted mutants ?VdCYP1. The pathogenicity test indicated that the pathogenicity of overexpression strains and complementary transformants were significantly enhanced on cotton, the disease index values are 60.11±1.94 and 62.49±3.13 respectively which are similar to the wild type strain（the disease index value is 68.05±3.62）. These results demonstrate that the pathogenic function of the cytochrome P450 gene is involved in the infection of V. dahliae.