Bioinformatic analysis and functional characterization of the cfem proteins in maize anthracnose fungus Colletotrichum graminicola

Fungal secreted proteins that contain the Common in Fungal Extracellular Membrane(CFEM)domain are important for pathogenicity.The hemibiotrophic fungus Colletotrichum graminicola causes the serious anthracnose disease of maize.In this study,we identified 24 CgCFEM proteins in the genome of C.graminicola.Phylogenic analysis revealed that these 24 proteins(CgCFEM1–24)can be divided into 2 clades based on the presence of the trans-membrane domain.Sequence alignment analysis indicated that the amino acids of the CFEM domain are highly conserved and contain 8 spaced cysteines,with the exception that CgCFEM1 and CgCFEM24 lack 1 and 2 cysteines,respectively.Ten CgCFEM proteins with a signal peptide and without the trans-membrane domain were considered as candidate effectors and,thus were selected for structural prediction and functional analyses.The CFEM domain in the candidate effectors can form a helical-basket structure homologous to the Csa2 protein in Candida albicans,which is responsible for haem acquisition and pathogenicity.Subcellular localization analysis revealed that these effectors accumulate in the cell membrane,nucleus,and cytosolic bodies.Additionally,5 effectors,CgCFEM6,7,8,9 and 15,can suppress the BAX-induced programmed cell death in Nicotiana benthamiana with or without the signal peptide.These results demonstrate that these 10 CgCFEM candidate effectors with different structures and subcellular localizations in host cells may play important roles during the pathogenic processes on maize plants.

Bioinformatic analysis and functional characterization of CFEM proteins in Setosphaeria turcica

Common in Fungal Extracellular Membrane(CFEM)domains are uniquely found in fungal extracellular membrane proteins which are important for pathogens.This study identified 13 StCFEM proteins in the genome of Setosphaeria turcica,the hemibiotrophic fungus that causes northern corn leaf blight.Sequence alignment and WebLogo analysis of their CFEM domains indicated that the amino acids were highly conserved and that,with the exception of StCFEM1,2,3,and 6,they contained eight cysteines.Phylogenic analysis suggested that these 13 proteins(StCFEM1–13)could be divided into 2 clades based on the presence of the trans-membrane domain.Six StCFEM proteins with a signal peptide and without a trans-membrane domain were considered as candidate effector proteins.The CFEM domain in the candidate effector proteins could form a helical-basket structure homologous to Csa2 in Candida albicans.Transcriptome analysis suggested that the 13 genes were expressed during fungal infection and a yeast secretion assay revealed that these candidate effectors were secreted proteins.It was also found that StCFEM3,4,and 5 couldn't affect BAX/INF1-induced programmed cell death(PCD)in Nicotiana benthamiana and while StCFEM12 could suppress INF1-induced PCD,it showed no effect on BAXinduced PCD.This study found that there were 13 members of the S.turcica CFEM protein family and that StCFEM12 was a candidate effector.This study laid the foundation for illustrating the roles of CFEM proteins during the pathogenic processes of phytopathogens.

A candidate effector protein PstCFEM1 contributes to virulence of stripe rust fungus and impairs wheat immunity

Common in Fungal Extracellular Membrane(CFEM)domain proteins are considered to be unique to fungi and closely related to pathogenicity.However,the Puccinia striiformis f.sp.tritici(Pst)effector containing the CFEM domain has not been reported.Here,we obtained an effector,PstCFEM1,containing a functional N-terminal signal peptide sequence and the CFEM domain from Pst race CYR31.qRT-PCR assay indicated that the transcript levels of PstCFEM1 were highly induced during the early stages of infection.Overexpression of PstCFEM1 suppressed Pst322(an elicitor-like protein of Pst)-trigged cell death,reactive oxygen species(ROS)accumulation and callose deposition.Host-induced gene silencing(HIGS)experiments showed that knockdown of PstCFEM1 decreased the virulence of Pst,while ROS accumulation in silenced plants increased near the infection site.In addition,wheat containing the PstCFEM1-silenced construct increased resistance to multiple races of Pst.Our data suggest that PstCFEM1 suppresses wheat defense by inhibiting ROS accumulation and contributes to increased virulence of Pst.