Regulatory Mechanisms Of Adaptor Protein FgAp2 And Protease FgPrb1 Of Pathogenicity In Fusarium Graminearum

Ap2(adaptor protein)complex is a hetero-tetrameric adaptor,which plays a vital role in vesicle formation and recognition cargo protein during the clathrin mediated endocytosis in fungi.Cell wall integrity(CWI)signal pathway is important for fungal growth,development,pathogenicity and adaptation to environmental stress.To date,the relationship between the Ap2 complex and CWI signal pathway has not been studied in plant pathogenic fungi.Our group has clarified the functions of CWI signaling pathway in Fusarium graminearum.The relationship between the FgAp2 complex and CWI signal pathway was further explored in this study.We identified four subunits of the FgAp2 complex named FgAp2?,FgAp2?,FgAp2? and FgAp2? in F.graminearum by BLAST using the sequences of each subunit of Ap2 complex in Saccharomyces cerevisiae as queries,and generated deletion mutants for each gene.Cell wall hydrolase and TEM assays showed that the integrity of cell wall of four mutants were significantly impaired.Yeast two-hybrid and cellular localization assays confirmed the interaction between FgAp2? and FgWsc2 b,the upstream sensor of the CWI signaling pathway.Western blot further proved that FgAp2 could positively regulate the phosphorylation of FgMgv1,the downstream MAPK of CWI pathway,indicating that FgAp2? regulates the growth,pathogenicity by mediating the trafficking of FgWsc2 b in F.graminearum.Subtilases are a very diverse family of serine proteases,and are important for various biological processes.Although the genomes of many phytopathogenic fungi have been sequenced,the biological functions of subtilases have not been systematically characterized.In this study,32 subtilases were identified in the genome of wheat scab fungus F.graminearum.Deletion mutants for each subtilase were obtained and functionally characterized.Among them,only the deletion mutant of FgPRB1(?FgPrb1)showed defects in vegetable growth and reducted virulence on the flowering wheat heads,wheat seedling leaves,wheat coleoptiles and corn silks.DON production and the expression level of DON biosynthesis-related genes were also reduced in ?FgPrb1.This study found that FgPrb1 responded to osmotic stress via high osmolarity glycerol signaling pathway.Western blot and determination of glycerin contents revealed that the mutant showed increased phosphorylation levels of FgHog1 and contents of glycerol with the accumulation of lipid droplets whatever it was treated with or without NaCl.Besides,we also found out that FgPrb1 is involved in autophagy in the assays of western blot and protein localization of FgAtg8.Taken together,FgPrb1 regulates the virulence by mediating DON production,is response to HOG signal pathway.Results of this study indicate that FgAp2 and FgPrb1 play important roles in MAPK pathways to regulate fungal development and pathogenicity.Up to now,we have not explored whether FgAp2 complex is involved in the transportation of FgPrb1.