Identification And Functional Analysis Of The CFEM Effectors Of Gaeumannomyces Tritici

Wheat take-all is an important widespread and destructive soil-borne disease all over the world.The causal agent of the wheat take-all is a hemibiotrophic fungus,Gaeumannomyces tritici,mainly infects the root and rhizome of the wheat,casuing enournmous yield loss and even crop failure.Plant pathogenic fungi secrete effectors into host plant tissues to interfere with their normal physiological functions and defense responses,thus promoting their infection.Common in Several Fungal Extracellular Membrane(CFEM)protein is a unique protein domain of fungi.CFEM effectors are found to be associated with the pathogenicity of the pathogenic fungi.However,until now,there are no reports on the screening,cloning,identification,and functional analysis of CFEM effectors of G.graminis.First,based on the annotated information of the whole genome of the wheat take-all fungus,a total of 16 CFEM proteins were obtained and named GgtCFEM1-16.Further analysis of signal peptide,subcellular localization,and transmembrane domain of GgtCFEM1-16 was performed using SignalP,TargetP,and TMHMM bioinformatics softwares,respectively.Seven out of 16 GgtCFEMs were found to be predicted extracelluar secreted proteins and defined as candidate CFEM effectors of G.graminis.Second,6 out of 7 candidate CFEM effectors were cloned and expressed in Nicotiana benthamiana through Agrobacterium-medidted transient expression method.The results showed that GgtCFEM1,GgtCFEM9,GgtCFEM10,and GgtCFEM11 could induce Hypersensitive Response(HR)in N.benthamiana.Using both Bax,a cell apoptosis inducing factor,and Infl,a pathogen associated molecular pattern of Phytophthora infestans,which can induce HR as elicitors,all of 6 cloned candidate CFEM effectors were expressed in N.benthamiana.None of them were found to be able to suppress HR induced by Bax or Infl in N.benthamiana.Finally,GgtCFEM9 gene was knocked out using Polyethylene Glycol(PEG)-mediated protoplast transformation method.Compared to the wild type,the knockout mutant of GgtCFEM9 showed difference in colony color and aerial mycelium but not in pathogenicity.These results lay a foundation for further study the function of GgtCFEM9 gene.