Characterization Of Vesical Trafficking Associated Protein FgMon1 And Its Binding Proteins FgRab5/7 In Fusarium Graminearum

Fusarium graminearum(teleomorph:Gibberella zeae)is an economically important plant pathogen that causes Fusarium head blight(FHB)or head scab disease on wheat and barley worldwide.In addition to the high economic impact of FHB,the infected cereals are often contaminated with deoxynivalenol(DON)and zearalenones(ZEA)which poses an extremely threat to human and animal health.However,efficient strategies to control the FHB have not been well established to date,and the current means is primarily dependent on fungicides that often exhibit many negative traits.Therefore,it is of high urgency to identify the molecular mechanism of F.graminearum on growth and disease,in order to develop novel and effective control strategies for FHB.Within the endomembrane system of eukaryotic cells,protein and lipid are packaged into vesicles at donor organelles and transported to acceptor membranes which depend on multiple fusion and fission events.Mon1 and Ccz1 were the first identified genes which are essential for the cytoplasm to vacuole targeting(Cvt)pathway and autophagy in yeast.Further evidences showed that Ccz1 and Mon1 are also essential for yeast homotypic vacuole fusion and regulating vesicle traffic at the tethering/docking stage,Recent studies reported that the Mon1-Ccz1 complex serve as the Rab7 GEF.We characterized FgMonl in F.graminearum,the homolog of Monl in S.cerevisiae.Phylogenetic analysis showed Monl was well conserved in filamentous fungi.In addition,we generated the FgMON1 gene deletion mutants of F.graminearum.The multiple defects of the ?Fgmon1 mutant implicate that FgMonl is a key protein for the development,infection?sexual development and DON production of F.graminearum.and we found conidial morphology of the ?Fgmon1 mutant was also changed when compared with the wide type PH-1.Besides,the ?Fgmon1 mutant was hypersensitive to cell wall perturbing agents,indicating FgMon1 might have a role in cell wall integrity maintenance.Cytological examination revealed that the ?Fgrmon1 mutant was also defective in vacuole fusion and autophagy,and delayed in endocytosis.Our data show that GFP-Monl localized in the cytosol and vacuoles in F.graminearum.This would be compatible with the recruitment of Monl to some membranes,as expected for a function in vacuolar fusion of Cvt vesicles and autophagosomes.Yeast two hybrid and in vitro GST-pull down assays approved that FgMonl physically interacts with a Rab GTPase FgRab7 which is also important for the development,infection,membrane fusion and autophagy in F.graminearum.Further research showed that FgMonl physically interacts with FgRab7 Q67L and FgRab7 T22N.FgMon1 likely acts as a GEF of FgRab7 and constitutively activated FgRab7 was able to rescue the defects of the AFgmonl mutant.In summary,our study provides evidences that FgMonl and FgRab7 are critical components that modulate vesicle trafficking,endocytosis and autophagy,and thereby affect the development,plant infection and DON production of F.graminearum.In yeast and animal cells,maturation of late endosomes from early endosomes require the conversion of Rab5-to-Rab7.Mon1-Ccz1 complex,the effectors of Rab5,could be recruited to the membrane by activated Rab5 protein and then bind Rab7.To investigated the fuctions of FgRab5-FgMonl-FgRab7 in F.graminearum,Yeast two hybrid assay approved that FgMon1 physically interacts with FgRab5A and FgRab5B.We obtain the corresponding mutants by gene knockout methods.The results showed that the ?Fgrab5A?Fgrab5B mutants were defective in vegetative growth,asexual/sexual development,conidial germination,plant infection and deoxynivalenol production.Cytological examination revealed that the mutants were also defective in vacuole fusion and delayed in endocytosis.In summary,we speculated that FgMonl and FgRab5A?FgRab5B?FgRab7 may function as a critical component that modulate vesicle trafficking,endocytosis and autophagy,and thereby affect the development,plant infection and DON production of F.graminearum.Taken together,GEF?Rab GTPase and tethering complexes are of key importance for the endolysosomal system.We conclude that a dynamic balance likely exist between FgMon1,FgRab5 and FgRab7,which might regulate the fusion between endosome and vacuole.As they are involved in vacuole fusion,thus influence endocytosis and autophagy,and eventually affects the development and infection of the F.graminearum.