| Gray mold is a common fungal disease in agricultural production which mainly affects dicotyledon. In protected cultivation of fruits and vegetables, gray mold has become a major disease. It can affect as many as two hundred kinds of plants. According to some reports, this single disease has already led to an economical loss of ten billion to one hundred billion dollars globally. The pathogen of this disease is Botrytis cinerea, which is a fungus belonging to Ascomycota. Botrytis cinerea is a typical necrotrophic pathogenic fungus. Research shows that Botrytis cinerea could generate a variety of pathogenic factors including multiple cell wall-degrading enzymes(CWDEs), cutinase, toxins, phytohormones, resistance enzymes, small RNAs and other small molecules, thus participate in the pathogenic process. However, little is known about the pathogenic mechanism of Botrytis cinerea. Related studies in this field, such as identification of pathogenic genes of Botrytis cinerea, can not only help to reveal the molecular mechanism of this necrotrophic pathogenic fungus, but also allow us to discover protein factors that probably work as a fungicide target. This will establish a solid theoretical and technological basis for the development of highly efficient reagent dealing with gray mold as well as other similar diseases.In this study, we performed an Agrobacterium tumefaciens-mediated transformation(ATMT) using strategy of forward genetics. By improving the co-culture conditions, we finally achieved a transformation efficiency of 80-100/105 conidia. Then we constructed a T-DNA random insertional transformant library which contains 12000 transformants and inoculated them on detached leaves to test their pathogenicity. A total number of 463 pathogenicity-defecient mutants were screened out, including mutants with less pathogenicity and with significantly enhanced pathogenicity. By performing southern-blotting, we discovered that the single copy rate of T-DNA random insertion into Botrytis cinerea’s genome reached 53.8% in this mutant library, which provided a scientific research foundation for future experiments on large scale screening looking for pathogenicity-related genes in Botrytis cinerea. Also, by inoculating on different host plants, we found that some of the mutants have host-specificpathogenicity.Based on the analysis of T-DNA insertion site of the mutants mentioned above, we identified a pathogenicity-defecient mutant which has a T-DNA insertion at the position of 169 bp downstream of the stop codon of ATM1. Atm1 s are ABC transporters located on inner mitochondrial membrane and evolutionary consered in eukaryotes. It plays an important role in maintaining normal biological activities of the cell. But until now, the role of Atm1 in the pathogenic process of Botrytis cinerea still remains unknown. In order to verify its function, we applied reverse genetics strategy and knocked out the gene using homologous replacement. Phenotype analysis shows that the germination of the mutant ΔBcatm1’s conidia is slow and asynchronous while the growing speed of its hypha is also significantly slower than wild type. Detached leaf inoculation experiments show that the mutant ΔBcatm1 hasalmost lost pathogenicity. After penetrating into host cells, it can only cause tiny lesions near the inoculation site without expansion. Complementation of the mutant ΔBcatm1 using a complete Bc ATM1 gene could partially or fully rescue the abnormal phenotype mentioned above. This indicates that Bc ATM1 is a key pathogenic gene which participate in the process of conidial germination, hypha growth and host infection in Botrytis cinerea. |
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