Screening And Identification Of Candidate Effectors Of Gaeumannomyces Tritici

Wheat is a widely cultivated food crop in the world.In recent years,the trend and scope of the occurrence of wheat take-all in the whole country are increasing every year.Wheat take-all caused by the ascomycetes Gaeumannomyces tritici is a root and soilborne disease of wheat,which resulted in dark brown spots near the base and roots of wheat.G.tritici can invade the vascular bundles of wheat and multiply in large numbers,seriously affecting the transportation of water and nutrients in wheat.The diseased wheat often appears as dwarf,withered and yellowing,and further shows the typical symptoms of "whiteheads" and "blackened roots".Once the disease occurs,it will seriously affect the wheat yield.Studies have shown that the pathogenic fungi secrete a large number of effectors to successfully infect the host.Therefore,the cloning and functional analysis of effector genes of G.tritici will be helpful to explore the molecular pathogenic mechanism of G.tritici.At present,there are few studies on the effectors of G.tritici.Based on the results of bioinformatics analysis of candidate effectors of G.tritici in our laboratory,firstly,51 candidate effectors were chosen and cloned;secondly,the candidate effectors for inducing and suppressing hypersensitive response were screened on Nicotiana benthamiana by agrobacterium infiltration;finally,the gene knock-out of the candidate effectors,which could induce and suppress hypersensitive response on N.benthamiana was performed by polyethylene glycol(PEG)-mediated protoplast genetic transformation,and the functions of these effectors were preliminarily analyzed.The results showed that: 1)fifty-one candidate effector plant expression vectors were successfully constructed;2)screening 51 candidate effectors for inducing hypersensitive response on N.benthamiana resulted in 5 candidate effectors,namely Gex1,Gex5,Gex10,Gex12,and Gex15;3)twenty-three out of 51 candidate effectors were screened for suppressing Bax-induced apoptosis,and none of them could inhibit Bax-induced apoptosis;4)the gene knock-out and fusion fragments of 4 candidate effectors,namely Gex1,Gex5,Gex10,and Gex15,were successfully constructed;5)the protoplast genetic transformation led to gene knock-out transformants of 2 candidate effectors identified,namely Gex1 and Gex5;6)Gex1 and Gex5 gene knock-out mutants were different from wild-type strains in colony morphology.This paper lays a research foundation for further functional verification of Gex1 and Gex5.