Pathogenic Mechanism Of Verticillium Dahliae Strain Vd991

Cotton wilt disease is the most damaging disease in most cotton-growing countries. It is caused by the phytopathogenic fungus Verticillium dahliae Kleb. V. dahliae is difficult to control, due to its persistency in soil. This survival capacity is generally believed to depend on the resistant resting structure, known as microsclerotia, which can survive in soil for several years. The pathologic mechanism of V. dahlia is complex. The hypothesis of Vd-toxin was accepted widely. The Vd-toxin is the key factor leading to the occurrence of verticillium wilt. The studies showed that the Vd-toxin caused the host leaves dry, wilt and fall, or even the whole plant death. But the pathogenic mechanism of Vd-toxin is still lack of sufficient understanding. The existing studies showed that the Vd-toxin is a protein-lipopolysaccharide complex, but the single effective ingredient has not been separated successfully.Microsclerotia play an important role in the disease cycle as they are the major inoculum source. The formation of microsclerotia affects pathogenicity of V. dahliae. In order to confirm the connection between the formation of microsclerotia and pathogenicity, a virulent, defoliating of V. dahliae (Vd991) was used as material. T-DNA randomly inserted transformants were obtained by the Agrobacterium tumefaciens-mediated transformation (ATMT) method. The transformant was selected which can't produce microsclerotia for further researching. DNA hybridization (Southern blot) identified that T-DNA was inserted the genome as the single copy into the transformant. Analysis of the region flanking T-DNA integration site in the transformant showed T-DNA insertion in CYP52A11 gene by utilizing TAIL-PCR, so that the gene is not normally expressed. The cDNA sequence was cloned by using Rapid Cloning of cDNA ends (RACE), full length of the CYP52A11 gene is 1869bp. The coding region is 1548 bp, encoding a protein consists of 516 amino acids. Comparison of cDNA and DNA sequence indicated that the gene contains five exons and four introns. T-DNA was inserted into the fifth exons in the transformant. Structure of the CYP52A11 was analysised by using SMART server, the results displayed that the first region of 1-27 amino acid is the signal peptide domain, it is believed that there is a transmembrane localization process. The second region of 73-497 amino acid is P450 domain. To determine the function of CYP52A11 gene, the complemented vector was constructed successfully. Microsclerotia generation was observed in complemented transformant, the phenotype is similar to the wild type Vd991. Southern blot analysis confirmed that the transformant contains ectopic insertions of the CYP52A11-complementing construct and the original CYP52A11 of the cyp52A11 background. The pathological effection of Vd991 and cyp52A11 mutant was evaluated using cotton leaves, the virulence decreased significantly in cyp52A11 mutant. The expression of VDH1, VdPKACl, VTP1 and Cerevisin genes which are related with the forming microsclerotia were decreased significantly in cyp52All mutant by quantitative real-time PCR (qRT-PCR) analysis. This supports that CYP52A11 gene regulated the expression of VDH1, VdPKAC1, VTP1 and Cerevisin genes. These results indicated that CYP52A11 may be a determinant of microsclerotia and pathogenicity in V. dahliae.In addition, the Vd-toxin of Vd991 is the important factor leading to the occurrence of verticillium wilt. But the single effective ingredient has not been isolated successfully. In this study, the Vd-toxin were isolated and purified by affinity chromatography and gel filtration chromatography. Wilting symotoms of cotton cotyledons were observed by injecting the purified protein. The results indicated that the protein which molecular weight is around 18.4-25 kDa were the effective factors. The 20 kDa protein was analyzed by mass spectrometry, after analysis of the sequence alignment, the genenamed cell wall proteins PhiA was selected to be cloned and expressed. PhiA protein has been expressed successfully.In summary, CYP52A11 plays an important role in microsclerotia formation and virulence of V. dahliae and regulates the expression of VDH1, VdPKACl, VTP1 and Cerevisin genes. The Cloning and expression of cell wall proteins PhiA will provide the groundwork for further researching of the protein function. These findings not only provide a new experimental evidence to reveal the pathogenic mechanism but also provide a theoretical basis to control V.dahliae.