Mining Of CFEM Effector Protein,a Candidate Target For The Control Of Setosphaeria Turcica

As one of the three major crops in the world,maize is also the largest grain crop in China.Setosphaeria turcica can cause northern corn leaf blight,which can lead to a significant reduction on corn yield.The common in several fungal extracellular membrane(CFEM)domain is unique to fungi and consists of about 60 aa residues containing eight conserved cysteine residues.Among them,some CFEM proteins that can be secreted into the extracellular environment have been proved to be effector of pathogenic fungi.In this study,the composition of CFEM protein family in S.turcica was explored,and the potential effector proteins were identified and the mechanism of action was analyzed.The main results are as follows:1.Through homology search of the genome database of S.turcica,we learned that there were 13 members of its CFEM family,and the IDS in the JGI database were 80947,28123,181030,165528,165307,162727,139432,128616,128580,164784,135770,1432023 and 1428406.They were named StCFEMl,StCFEM2,StCFEM3,StCFEM4,StCFEM5,StCFEM6,StCFEM7,StCFEM8,StCFEM9,StCFEM10,StCFEM11,StCFEM12,and StCFEM13.2.Bioinformatic method was used to analyze the structural characteristics of 13 genes.Sequence alignment of CFEM domain and WebLogo analysis showed that StCFEM amino acids were highly conserved and contained 8 typical cysteine on the whole except StCFEM1,2,3 and 6.Phylogenetic analysis showed that the 13 proteins(StCFEM1-13)could be divided into two clades according to the presence or absence of transmembrane domains.Among them,6 StCFEM proteins(StCFEM3,4,5,10,12,13)with signal peptides and without transmembrane domains were assumed to be candidate effector proteins.By predicting the tertiary structure of the protein,it was found that the CFEM domain of the candidate effector proteins could form a helical structure and was homologous with Csa2 from Candida albicans.3.Transcriptome analysis of the CFEM family of S.turcica showed that the 6 candidate genes were expressed at different stages during fungal infection of the host,among which SCFEM3,4,5 and 12 were actively expressed during appressgenesis.4.The secretory function of candidate effector proteins was identified by the yeast pSuc2t7M13 secretion system.The results showed that all the six candidate effector proteins had strong secretory function and belonged to secretory proteins.5.The effector proteins were identified by the tobacco transient expression system.The results showed that StCFEM3,4,and 5 had no effect on BAX/INF1-induced programmed cell death(PCD)in tobacco,while StCFEM12 could inhibit INF1-induced PCD but had no effect on BAX-induced PCD.6.The fusion expression vector of StCFEM12 and GFP was constructed,and the subcellular localization of the effector protein was observed by confocal microscopy.It was found that StCFEM12 would secrete to the host after being infected by S.turcica,and would be positioned in the host’s chloroplast to play a role.7.Using StCFEM12 as bait protein,17 candidate proteins interacting with StCFEM12 were screened from yeast two-hybrid library of cDNA from maize leaves and included Shaggy-related protein kinase,Ferredoxin 1,Methionineadenosyltransferase,Pyruvate phosphate dikinase and 1 aminocyclopropane 1-carboxylate oxidase etc.The interaction relationship between StCFEM12,Ferredoxin 1 and ACC was verified by yeast two-hybrid technique.In summary,this study found that there were 13 members of the CFEM protein family of S.turcica,identified StCFEM12 as candidate effector,and explored the interaction proteins in its maize.It lays a foundation to elucidate the role of CFEM in the process of phytogenesis.