Functional Analysis Of Guanine Nucleotide Exchange Factor FgCdc25 In Fusarium Graminearum

Wheat is the staple food for almost half of the population in China.Ensuring production and quality of wheat is critical for national food security.Fusarium head blight(FHB),caused mainly by Fusarium graminearum,is one of the most devastating wheat diseases in China.More seriously,F.graminearum produces harmful mycotoxins in infested grains,such as deoxynivalenol(DON),causing a grave threat to food security.The deciphering of the molecular mechanism of pathogenicity and DON biosynthesis will provide an important theoretical basis for developing new fungicides to FHB and mycotoxin management.The cAMP(Cyclic Adenosine Monphosphate)signaling pathway plays an important role in fungal pathogenicity.Previous studies have reported that adenylate cyclase(AC)and GTPase Ras2 in the cAMP pathway play important roles on pathogenicity and DON biosynthesis in F.graminearum.However,the guanine nucleotide exchange factors(GEFs)regulating Ras GTPase activity in this pathway has not been identified.In this study,two genes encoding putative RasGEFs,FgCDC25 and FgSDC25,were identified in F.graminearum.Combing with genetic and biochemical methods,the biological functions of FgCdc25 in pathogenicity,toxin production and abiotic stress response were analyzed.The results showed that:(1)FgCdc25 directly interacted with FgRas2,but not with FgRas1.FgSdc25 didn't interact with both Ras GTPase proteins;(2)FgCdc25 was involved in mycelial growth,asexual and sexual development;(3)The mutant ?FgCdc25 significantly reduced the cAMP biosynthesis under DON inducing medium,resulting in the decreased expression of TRI genes,and affected DON toxisome formation and DON production.The addition of exogenous cAMP was able to rescue the defect in DON production caused by the loss of FgCdc25;(4)FgCdc25 interacted with FgStell,a key kinase in the pathway associated with the penetration structure formation to regulate the phosphorylation level of downstream kinase FgGpmkl and influence the penetration structure formation;(5)FgCdc25 interacted with the upstream kinase FgBck1 in the cell wall integrity pathway,negatively regulating the cell wall integrity of pathogens.Phosphorylation level of FgMgvl in the mutant AFgCdc25 was significantly increased,resulting in increased resistance to cell wall damaging agent Congo Red;(6)Virulence assays on flowering wheat head indicated that ?FgCdc25 totally abolished the virulence,even on the inoculated spikelets.In addition,we further analyzed the roles of different domains in FgCdc25,and found that RasGEF_N and RasGEF domain were important for the proper localization of FgCdc25 protein and the interaction with FgRas2.In this study,it was the first time to identify the GEF protein FgCdc25 of FgRas2 in the cAMP pathway and analyze its function in F.graminearum.Results indicated that FgCdc25 was critical for cAMP biosynthesis,mycotoxin biogenesis and pathogenicity.More importantly,we found that FgCdc25 regulated cell wall integrity and penetration structure formation by interacting with FgMgvl and FgGpmkl in two MAPK(Mitogen-activated Protein Kinase)pathways.Our results provided more valuable information to better investigate the role of cAMP pathway in abiotic adaption,mycotoxin biosynthesis and pathogenicity in F.graminearum.