Functional Analyses Of Genes Identified From The Simultaneous Transcriptome Profiling Of Both Botrytis Cinerea And Tomato During Their Interaction And Pathogenic Mechanisms Of BcCGF1

The necrotrophic phytopathogenic fungus Botrytis cinerea causes gray mold on over 1400 plant species.Gray mold is a worldwide fungal disease that is difficult to control.The pathogen infects almost all vegetables and fruit crops including numerous economically important crops such as grapevine,strawberry,raspberry,ornamental flowers and annually causes enormous economic losses worldwide.The pathogen attacks many plant organs,including stems,fruits,leaves and flowers,at both pre-and post-harvest stages.The huge economic losses,especially in the rapid development stage of modern facility agriculture that provides suitable environmental conditions for the occurrence of gray mold,are increasing year by year.Due to economic and scientific importance,B.cinerea is considered as an important pathogen for studying pathogenesis of necrotrophic fungal pathogens,which ranks second in the top ten important fungal pathogens in molecular plant pathology.At present,there is no effective method for the control of gray mold.The traditional solution is to use a large amount of pesticides,which causes not only a big concern for food safety,but also environmental pollution.Therefore,an in-depth study of the molecular pathogenesis of B.cinerea is helpful to find an effective and environmentally friendly strategy for controlling the disease.Transcriptome analyses of both host plants and pathogens,and functional validation of the identified differentially expressed genes?DEGs?allow us to better understand the mechanisms underlying their interactions.Here,we analyse the mixed transcriptome derived from B cinerea infected tomato?Solanum lycopersicum?leaves at 24 hr post inoculation,a critical time point at which the pathogen has penetrated and developed in leaf epidermis,whereas necrotic symptoms have not yet appeared.Our analyses identified a complex network of genes involved in tomato–B.cinerea interaction.The expression of fungal transcripts encoding candidate effectors,enzymes for secondary metabolite biosynthesis,hormone and reactive oxygen species?ROS?production,and autophagy-related proteins were up-regulated,suggesting that these genes may be involved in the initial infection processes.Moreover,tomato genes involved in phytoalexin production,stress responses,ATP-binding cassette?ABC?transporters,pathogenesis-related proteins,and WRKY DNA-binding transcription factors were up-regulated.We functionally investigated several B.cinerea DEGs via gene replacement and pathogenicity assays.Our results demonstrated that BcCGF1 was a novel virulence-associated factor that mediates fungal development and virulence via regulation of conidial germination,conidiation,infection structure formation,host penetration,and stress adaptation.The fungal infection-related development was controlled by BcCGF-mediated ROS production and exogenous cAMP restored the mutant infection-related development.Our findings provide new insights into the elucidation of the simultaneous tactics of pathogen attack and host defence.Our systematic elucidation of BcCGF1 in mediating fungal pathogenesis may open up new targets for fungal disease control.In addition,genes regulating autophagy,secondary metabolites,cell wall degrading enzymes,and hypoxia that may affect the pathogenicity of B.cinerea were chosen for further functional verification.These genes included two autophagy-related genes?BcATG1 and BcSNF1?,two genes that regulate secondary metabolites?BcLAE1,BcVEL1?,and six cell wall-degrading enzyme-related genes ?-glucosidases?BcBGS1 to BcBGS6?,BCIN₀3g01540,a gene encoding a glucose-methanol-choline?GMC?oxidoreductase that may be involved in B.cinerea response to hypoxic environment.We generated the gene knockedout mutants of BcSNF1,BcATG1,BcLAE1,BcVEL1,BcBGS1 to BcBGS6,and BCIN₀3g01540 and performed related functional studies.Our findings indicated that BcSNF1,BcATG1,BcLAE1,BcVEL,BcCGF1 regulate the pathogenicity of B.cinereaWe also optimized the Agrobacterium tumefaciens-mediated transformation?ATMT?in B.cinerea.Compared to the previus ATMT in B.cinerea,the efficiency of our optimized transformation improves about 8-10 folds.We generated a B.cinerea T-DNA insertion mutant library containing about 50,000 transformants and systematically dissected molecular mechanisms of pathogeneses of some novel B.cinerea factors identified from the library.We obtained the upstream and downstream flanking sequences of a T-DNA inserted gene that encodes an alcohol dehydrogenase from a pathogenicity-attenuated mutant strain identified from our library by using the approach of thermal asymmetric interlaced polymerase chain reaction?TAIL-PCR?.Interestingly,the gene was also identified from our list of DEGs and displayed a significant up-regulation during interaction between B cinerea and tomato.Taken together,this work has laid a foundation for our further systematic dissection of molecular mechanisms underlying B.cinerea-host interactions.