Functional Analysis Of Autophagy-related Protein9(Atg9) In Botrytis Cinerea

Plant grey mold, caused by Botrytis cinerea, is a common fungal plant disease inthe world. The disease can cause huge economic losses yearly. B. cinerea is a typicalnecrotrophic pathogen that employs its conidia to infect the hosts under naturalconditions. The conidia of B. cinerea can spread through the air, and the pathogen caninfect more than200host plant species. Autophagy-related protein9(Atg9) is anessential protein in autophagosomal membrane, and protein ATG9contains117aminoacid residues. ATG9is responsible for the cellular molecule cycles which is dependenton the organelles such as autophagosom and/or lysosome. In this study, we describethe function of Autophagy-related protein9(Atg9) gene in the growth, developmentand pathogenicity of B. cinerea, which were shown as the following:1. Using B. cinerea virulent strain Bc3-4as a material, I have generated a B.cinerea T-DNA insertion mutant library containing more than3000transformants.After performing a screen for the pathogenicity deficit mutants, from the transformantpopulation, I obtained a total of148T-DNA insertion mutants with decreasinginfection phenotypes. In the pathogenicity screen, detached tomato, strawberry leaveswere separately applied to screen the pathogenicity deficit mutants. For each host, thescreen was independently performed at least three times.2. Thermal Asymmetric Interlaced PCR (Tail-PCR) was used to perform geneticanalysis of T-DNA insertion of the genes in the B. cinerea T-DNA transformants. Apathogenicity-deficit mutant with the T-DNA insertion of the promoter region of Atg9gene in Botrytis cinerea was found. Southern blot analysis showed that the mutationwas caused by the insertion of a single copy of the T-DNA. Since the reports about theregulation of autophagy genes on B. cinerea growth and pathogenicity were little. Inthis study, the roles of B. cinerea Atg9gene in the regulation of the pathogen growthand development were discussed.3. The Agrobacterium tumefaciens mediated transformation (ATMT) wasemployed to successfully knock out Atg9gene in B. cinerea. The Atg9-knockoutmutant△BcAtg9was identified by PCR approach with specific primers. 4. The comparison of the fungal growth and development between the wild-typeBc05.10and△BcAtg9mutant strains showed that the vegetative growth rate of△BcAtg9mutant was similar to that of the wild-type strain Bc05.10in PDA plates,indicating that Atg9gene does not affect mycelial growth of the pathogen. Sporulationanalysis showed that sporulation of the wild-type strain was about (3.1±0.4)×107conidia/plate, and sporulation of△BcAtg9mutants was about (1.5±0.3)×107conidia/plate, indicating that the sporulation production decline to50%of the control,which was significantly different. Pathogenicity test showed that after7days postinfection, the lesion area caused by the wild-type strains was (1.24±0.14) cm2, whilethe lesion area caused by△BcAtg9mutant were (0.34±0.06) cm2, indicating that thepathogenicity of△BcAtg9mutant was significantly reduced. Above results wereconsistent with the attenuated pathogenicity caused by T-DNA insertion into thepromoter region of B. cinerea Atg9gene.The results from this study show that Atg9gene of B. cinerea does not affect thenormal growth of mycelia and the formation of spores, however, deletion of the genehas a significant impact on the sporulation and pathogenicity of the pathogen,suggesting that Atg9genes is involved in the sporulation and pathogenesis in B.cinerea.