| Verticillium dahliae is a soil-borne fungus causing destructive and worldwide vascular wilt diseases on cotton, which is one of the most serious diseases in cotton-planting countries and regions.According to its stable dormancy microsclerotium, strong variability and host co-evolution, the pathogenic molecular mechanism of V. dahliae still remain unclear. In recent years, the public availability of the genome sequence of V. dahliae(Vd Ls.17) to speed up the research progress. To help elucidate the complex pathogenic molecular mechanism of V. dahliae and to promote the development of effective targeted control strategies, several technological methods, including both forward and reverse genetics measures, have been used for functional analysis of pathogenicity-related genes. Which has the vital significance for targeted control of verticillium wilt on cotton.In our previous study, we obtained 25 low virulent mutants with single T-DNA inserted from an ATMT mutant library of the highly virulent isolate Vd080. In this study, We focused on two low pathogenicity mutants, cloning pathogenicity related gene Vd PR1 and Vd PR3 to investigate their function. The main results were as follows:1. Vd PR1 located in the first chromosome, the full length is 2239 bp, including 4 exons and 3introns, coding 464 deduced amino acid residues. The prediction of encoded protein showed that Vd PR1 belongs to typical secreted proteins. The full-length of Vd PR3 was 762 bp, encoding 106 amino acids,with ORF of 321 bp, which also located in the first chromosome.2. Based on the principle of homologous recombination, knockout vectors were constructed with the combination of fusion PCR, nested PCR and Gateway technology; complementary vector were structured by using enzyme digestion method. The knockout mutants and complementary mutants were obtained by Agrobacterium tumefaciens-mediated transformation method, respectively.3. We assessed the role of pathogenicity related genes Vd PR1 and Vd PR3 in growth development and pathogenicity of V. dahliae. Compared with wild type strain Vd080, knockout mutant ΔVd PR1 andΔVd PR3 colony morphology has changed, the ability of microsclerotium formation decreased significantly. In the medium with cellulose as the sole carbon source, the growth rate of knockout mutants ΔVd PR1 and ΔVd PR3 were reduced by 21% and 53%, respectively. Pathogenicity assay showed that knockout mutants ΔVd PR1 and ΔVd PR3 postponed infectious events in cotton with less stem discoloration was observed in cotton plants. At 24 dpi, Cotton plants were highly infected with the wild type strain Vd080, the DI value reached 52.11±3.7. However, the DI values were remarkably reduced in the three ΔVd PR1 mutants, ranging from 20.42±2.0~22.28±2.7, with decline by 59%.Destroied the Vd PR3 knockout mutant ΔVd PR3, the DI values range from 20.67±3.2~26.71±0.3, with Significantly lower than the wild type strain Vd080. The infectivity of complementary transformants was recovered approximately similar to the wild type strain Vd080 level. To assess adhesion capacity and extension ability. Fungal DNA of wild-type Vd080 and mutants in cotton were quantified in the plant tissues by q PCR. The results demostrated that, Vd PR1 and Vd PR3 are both important not only forroot penetration but also for subsequent colonization of vascular tissues, contribute to the virulence of V.dahliae.In conclusion, the two pathogenicity related gene Vd PR1 and Vd PR3 are closely related to the virulence of V. dahliae, and participate in microsclerotium formation, growth rate, spore production capacity, and extracellular enzyme activity. Which play an important role in the infect process of cotton,have a significant contribution to the pathogenicity of V. dahliae. |
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