Functional Characterization Of Endocytic Protein Ede1 In Fusarium Graminearum

Fusarium head blight(FHB)is mainly caused by Fusarium graminearum and is a devastating disease such as wheat and barley worldwide.FHB not only causes loss of wheat,the mycotoxins produced by it is an effective protein synthesis inhibitor in eukaryotes,and threatens human and animal health seriously.So far,endocytosis has been studied in detail in humans,mammals and yeast.Ede1 is an endocytic scaffolding protein that is localized on plasma membrane of cell and has critical roles in endocytic site initiation and maturation.However,functions of Ede1 in plant pathogenic fungi are not known.In this study,we identified an Ede1 gene in F.graminearum by comparing the amino acid sequence with Saccharomyces cerevisiae.FgEde1 contains 1270 amino acids,and prediction by SMART-Pfam online analysis domain software revealed that FgEde1 consist of three EH domains,a coiled-coil domain and a UBA domain.Using MEGA 5.0 to construct the phylogenetic tree,found that the Ede1 gene of F.graminearum is closely related to the Ede1 gene of F.oxysporum and F.oxysporum.We used the overlap PCR method to connect the knockout fragments,and knocked out the FgEde1 gene according to the principle of gene recombination.In order to further confirm the role of FgEde1 in F.graminearum,we supplemented the target gene to construct a ?Ede1-C strain.We analyzed the biological characteristics of the ?Ede1 mutants and wild-type strains and found that the colony of the ?Ede1mutant were significantly smaller than the wild-type,the growth rates in the PDA plate and the CM plate decreased by 65.6 % and 69.7 %,respectively.The mutant produced slightly fewer conidiation than the wild type,germination became delayed,but the morphology of conidiation was normal.In sexual reproduction,the mutant produced slightly fewer ascomycetes and did not produce ascospores and ascus.Compared with the wild type,the pathogenicity of the ?Ede1 mutant was greatly reduced,the disease index decreased by 94 %,and the production of DON toxin only accounted for 50 % of the wild type.Compared with the wild type strain PH-1,the expression levels of TRI5?TRI6 and TRI10 in the ?Ede1 mutant decreased by 78 %,50 % and 80 %,respectively.Furthermore,the FgEde1 deletion mutant also exhibited increased resistance to osmotic and oxidative stress as well as cell-wall perturbing agents.Importantly,deletion of FgEde1 inhibited endocytosis of hyphae and increased the severity of autophagy in hyphae.Taken together,these results reveal that FgEde1 is involved in growth,asexual and sexual reproduction,virulence,stress responses,and autophagy in F.graminearum.