Studies On The Biological Functions Of The Autophagy And Endocytosis Regulatory Factors In The Rice Blast Fungus

Mganaporthe oryzae,a filamentous pathogenic fungus,has typical characteristics in growth,development,and plant infection.Therefore,many plant pathologists have used it as one of a model to study the interaction between pathogens and host plants,and it is significant to study the pathogenesis of M.oryzae to control rice blast.Autophagy and endocytosis are the two important pathways in eukaryotes,which are highly conserved in transportation,degradation,absorption,and intracellular materials recycle.The two pathways are necessary to maintain the growth and development of organisms.In M.oryzae,the autophagy and endocytosis affect the growth and development,the production of conidia,and the pathogenicity.In this study,three Vps9 domaincontaining proteins MoVps9,MoMuk1,MoVrl1,and two VASt domain-containing proteins MoVast1,MoVast2 were identified and analyzed systematically.We clarified their functions on autophagy and endocytosis pathways,and regulation mechanism of the pathogenicity.We identified three VPS9 domain-containing proteins,MoVps9,MoMuk1 and MoVrl1,which are redundant in functions.Deletion of MoVps9 resulted in severe defects in growth,conidiation and pathogenicity.Loss of MoMuk1 or MoVrl1 has no significant effect on the growth and development.However,the phenotype of ΔMovps9ΔMomuk1(the double-gene-deletion mutant)are more severe than that of ΔMovps9.The ΔMovps9ΔMomuk1 mutant lost of pathogenicity completely.These results indicated that MoVps9 and MoMuk1 are functionally redundant in part.MoVps9 and MoMuk1 are localized to vesicles,and co-localized with MoVps21,a recognized marker of early endosomes.In addition,deletion of MoVPS9 resulted in severe defects in autophagosome degradation.Deletion of the MoMUK1 gene in the ΔMovps9 mutant background(ΔMovps9ΔMomuk1 mutant)exhibited more severe defects in development,autophagy,and endocytosis compared with the ΔMovps9 mutant.These indicated that MoMuk1 also play roles in autophagy and endocytosis.Furthermore,yeast two-hybrid and co-immunoprecipitation assays confirmed that MoVps9 directly interacts with the GDP form of MoVps21.Thus,our study demonstrated that MoVps9 is a key protein involved in autophagy and endocytosis.Two VASt(VAD1 Analog of StAR-related lipid transfer)domain-containing proteins,MoVast1 and MoVast2,were identified via co-immunoprecipitation by GFPMoAtg8 in M.oryzae.Deletion of MoVAST1 showed conidial defects,impaired appressorium formation,and reduced pathogenicity.Through Filipin and FliptR staining,autophagic flux detection,MoAtg8 lipidation,and drug sensitivity assays,we uncovered that MoVast1 is a novel autophagy regulating factor,and monitors MoTOR activity by regulating PM tension and sterol homeostasis to control autophagy and pathogenicity.