| Used the ATMT (Agrobacterium tumefaciens-mediated transformation) technology, we obtained the T-DNA insertion mutant library of Botrytis cinerea. One appressoria-deficiency mutant, BCt41, was found by screening the ATMT mutant library of B. cinerea. The mutant gene of BCt41was identified as BcADRl, encoding an unknown function protein, by TAIL-PCR, PCR, Southern blotting and RT-PCR techniques. In orded to identify the function of BcADR1, the expression pattern of BcADR1in the wild-type BC22was examined by semi-quantitative RT-PCR and Real-Time PCR. We constructed the complementing vector and obtained the BcADR1-complementing mutant BCt41/BcADR1. Phenotype and pathogenicity analysis of BC22, BCt41and BCt41/BcADR1were performed to identify that the function of BcADR1in growth, development and pathogenicity in B. cinerea. Cell wall degradation enzyme activity, toxin activity, acid production, appressoria formation and penetrating power, expression levels of pathogenicity-related genes, and sensitivity to stress of BCt41and BCt41/BcADRl were detected to analyze the regulatory mechanisms of BcADR1. These works could help us to understand the regulatory mechanisms of BcADR1in growth, development and pathogenicity of B. cinerea.1. The tissue-specific expression pattern of BcADR1in the wild-type BC22was examined by semi-quantitative RT-PCR and Real-Time PCR. The results revealed that BcADR1was highly expressed in mycelia, but not expressed in sclerotia and conidia.2. The BcADR1gene complementing vector was constructed and transformed into the T-DNA inserted mutant strain BCt41by PEG mediated protoplast transformation. The BcADR1-complementing mutant(BCt41/BcADRl) was obtained by screening using glufosinate ammonium, and identified by PCR, Southern blotting and Real-Time PCR techniques.3. Phenotype and pathogenicity of BC22, BC41and BCt41/BcADR1were analyzed in this study. The BCt41mutant growed slowly, hyphae slender, light color, did not produce sclerotia, conidial yield and pathogenicity significantly enhanced. These results showed that the BcADRl gene is positive regulation growth and sclerotia development, negative regulation in pathogenicity and conidia formation of B. cinerea.4. The activity of pectin methyl transelimination enzyme (PMTE), polygalacturonic acid transeliminase (PGTE), cellulase (CX), polymethylgalacturonase (PMG), and endopolygalacturonase (PG) in the T-DNA insert mutant BCt41was significantly lower than wild-type strain BC22(WT) and mutant BCt41/BcADR1. The toxin activity and acid production of BC41were significantly decreased while compared with WT and BCt41/BcADR1. It was determined that the BcADRl gene is involved in regulating cell wall degradation enzyme activity, toxin activity and acid production.5. Penetration ability and appressorium formation were performed on cellophane and onion epidermis. It was found that WT, BCt41and BCt411/BcADR1can penetrate the cellophane and form colonies on the PDA medium respectively. But the appressoria of the BCt41appeared significant difference compared to WT and BCt41/BcADR1. These results indicated that the BcADR1might regulate appressorium development and does not affect penetration.6. Real-Time PCR technology was used to detect the expression levels of pathogenicity-related genes in BC22, BCt41and BCt41/BcADR1. It was found that the expression level of pathogenicity-related genes, i.e., Bcg2, BcReg1, PkaR, Bac, Bcp1, Bmp1, Bcg3, Sod1, and Bos1was obviously up-regulated in mutant BCt41. Therefore, the BcADR1gene is involved in regulating the expression of pathogenicity-related genes in B. cinerea.7. The sensitivity of BC22, BCt41and BCt41/BcADR1to NaCl, KCl, fluconazole and CFW were detected by measuring the growth rate of three thrains. It was found that the growth rate of BCt41in NaCl, KCl, fluconazole and CFW medium was significantly decreased while compared with WT and BCt41/BcADR1. These findings suggested that BcADR1might regulate osmotic stress, cell wall and membrane integrity in B. cinerea. |
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