Biological Function Analysis Of Septin Protein In Botrytis Cinerea

Botrytis cinerea,the causative agent of plant gray mold,is a worldwide important plant pathogenic fungus and is regarded as a typical necrotrophic pathogen.The pathgoen can infect up to 1,400 different plants.It can infects plants or plant products during their growth periods,fruit or vegetable storage periods.Therefore,the annual economic losses caused by gray mold are very huge.B.cinerea has received much concern as a model phytopathogenic fungal pathogen,however,its molecular pathogenesis still remains obscure.Previous studies demonstrate that B.cinerea successfully invade the host,by two specific infection structures formation,i.e.,appressorium or infection cushion,then the pathogen also secretes a variety of pathogenic factors to kill host plants to obtain nutrients.Septin,as a member of highly conserved protein families in eukaryotes,participates and plays an important role in a number of life processes,such as cytoskeleton,cell cycle,cell secretion.In fungi,the septin protein family has four core members: Sep3,Sep4,Sep5 and Sep6.Amont them,Sep4 is the smallest protein.To further clarify molecular pathogenesis of B.cinerea,this study mainly focuses on the functional analysis of the core septin in the pathogenesis,including fungal development,infection structure formation and host infection of B.cinerea.Our main results are listed as the following:(1)Four core septin components,including Bc Sep3,Bc Sep4,Bc Sep5,and Bc Sep6,were found in the genome of B.cinerea by bioinformatics analysis;(2)Four core septin gene deletion mutants were obtained by Agrobacterium tumefaciens-mediated transformation(ATMT)-mediated genetic transformation of B.cinerea: ?Bcsep3,?Bcsep4,?Bcsep5 and ?Bcsep6,and through the inoculated analysis demonstrate that core septin components are essential for the pathogenicity of B.cinerea;(3)Bc Sep4 was found to be indispensable in infection structure formation and Pathogenicity of B.cinerea by phenotype comparative analysis of wild type and the Bc SEP4 deletion mutant strains.In addition,Bc Sep4 also played an important role in asexual reproduction,germ tube development,flexible growth,stress response and intracellular transport of B.cinerea;(4)Subcellular localization of BcSep4 by GFP fusion demonstrated that Bc Sep4 might play a role in the micron-scale membrane curvature formation during the growth and development as well as infection structure formation of B.cinerea;(5)Through diphenylene iodonium(DPI)andcyclic adenosine monophosphate(c AMP)treatment,the experiment results showed that Bc Sep4 mediating infection structure formation was regulated by ROS signal transduction and related to c AMP signal transduction;(6)By comparative analysis of phenotypes of gene deletion mutants,i.e.,?Bcsep3,?Bcsep4,?Bcsep5 and ?Bcsep6,and the wild type control,it was found that the core septin components were necessary for the growth and development of B.cinerea,the infection structure formation of the pathogen;(7)The subcellular localization of core septin members showed that Bc Sep3,Bc Sep4,Bc Sep5 and Bc Sep6 process different self-assembled patterns at different positions in the cell,affected on cell polarity and co-regulated the growth and development of infection structure formation of B.cinerea.In summary,this study systematically analyzes the biological function of septins in B.cinerea for the first time and provides novel insights into our understanding of the growth and molecular pathogenesis of B.cinerea.Since septins are absence in the plants,septins analyzed in this work may be promissing potential targets for fungicide development and disease management in the future.