Screening And Functional Analysis Of Pathogenic-related Genes In Botrytis Cinerea

Grey mould,caused by Botrytis cinerea,is one of the most important disease in the world,which causes serious economic losses every year.B.cinerea is a typical necrotrophic plant-pathogenic fungus,which has the characteristics of wide host,strong invasion,rapid propagation and fungicide resistance.Control of B.cinerea still faces great challenges.Therefore,the study of molecular mechanisms of the interactions between B.cinerea and its host will provide new strategy and potential target for the control of B.cinerea.Previous studies in our laboratory found that disruption of the G?-like gene Bcgbl1 resulted in the failure to form infection cushions and loss of pathogenicity in B.cinerea.Based on the transcriptome data of wild-type strain B05.10 and Bcgbl1 knockout mutants inoculated with hydrophobic surface(simulating the formation of infection cushions)for 16 h and 24 h,we screened the genes related to infection cushions formation regulated by Bcgbl1.In this study,three infection cushions formation-related genes,catalase gene Bccat6,GPI anchored protein gene Bcgpi and metallopeptidase gene Bcmp,were screened from transcriptome data.The expression levels of the three candidate genes were significantly up-regulated during the infection cushions formation stage.In order to verify the function these three genes,we generated the knockout mutants of these three genes using the Split-Marker technology.Results showed that the extracellular protease activity of the metallopeptidase gene Bcmp knockout mutant decreased significantly.However,the knockout of the three genes had no significant effect on infection cushions formation,mycelial growth,sclerotia formation and pathogenicity of B.cinerea.In order to further screen pathogenic-related genes in B.cinerea,five mutants with reduced virulence were identified from the T-DNA insertion mutant library,namely AT2,AT11,AT22,AT617 and AT919.Southern blot results showed that all the five mutants were single copy T-DNA insertion,which resulted in the reduction of the mycelial growth rate.The mutant AT919 showed defects in infection cushions formation,abnormal colony morphology,lack of sclerotia formation,increased sensitivity to high salt stress,decreased acid production ability,and enhanced resistance to succinate dehydrogenase inhibitor fungicide boscalid.hi-TAIL PCR result showed that the T-DNA insertion disrupted the expression of the succinate dehydrogenase flavoprotein subunit gene Bcsdh A.It is suggested that Bcsdh A is involved in the growth,development,pathogenicity and SDHI fungicides resistance in B.cinerea.In summary,three genes(Bccat6,Bcgpi,and Bcmp)which were up-regulated during the infection cushions formation stage,were knocked out in this study.The knockout mutants of these three genes displayed no defects in infection cushions formation and pathogenicity.We analyzed the biological characteristics of the mutant AT919 and identified the T-DNA insertion sites.Our results suggested that the succinate dehydrogenase flavoprotein subunit gene Bcsdh A is very important for the growth,development and pathogenicity of B.cinerea.