Functional Analysis Of Effector CgCFEM14 Of Colletotrichum Graminicola

Maize（Zea mays L.）is one of the three major food crops in the world,which is widely planted in the world.The yield and quality of maize are seriously damaged due to diseases every year.Colletotrichum graminicola,a semi-living vegetative pathogen,is a representative pathogenic fungus on maize,and also an important limiting factor affecting the high and stable yield of maize.It is known that the effective factors of C.graminicola mainly promote host infection by affecting host immune response,which is an important pathogenic factor of C.graminicola.Iron is not only an important nutrient element and coenzyme factor in organisms,but also involved in regulating the pathogenicity of plant pathogenic fungi.Fungi-specific extracellular membrane structural protein CFEM（Common in Fungal Extracellular Membrane）often regulates iron absorption and participates in pathogenicity as an effector.In this study,the effector CgCFEM14 of C.graminicola was used as the target gene to study the regulation and pathogenicity of CgCFEM14 on iron uptake,and analyze the main functions of CgCFEM14.The main results are as follows:1.The phylogenetic tree of CFEM protein in C.graminicola was constructed.The screened and identified CgCFEM14 gene of C.graminicola and its physicochemical properties were analyzed by bioinformatics.There are 24 proteins with CFEM domain in C.graminicola,of which 22 have signal peptides and 10 have signal peptides without transmembrane structure.The full length of CgCFEM14 was 519 bp,encoding 172 amino acids,molecular weight of16737.02 Da,isoelectric point of 5.03,and signal peptide.The CFEM domain is located between the 21st and 91st amino acids.Phylogenetic tree analysis showed that CgCFEM14 had the closest relationship with CgCFEM20.2.The role of CgCFEM14 in the growth,development,stress resistance and pathogenicity of C.graminicola was studied.The mutant strain ofΔCgCFEM14 was constructed.The biological studies showed that the deletion of CgCFEM14 did not affect the formation of hyphae and spores of C.graminicola,but the sporulation,spore germination rate and appressorium formation of the mutant were significantly decreased.The mutant strain was more sensitive to exogenous H₂O₂stress.The growth rate of the mutant was significantly decreased under iron deficiency of 50μM BPS and iron excess of 250μM.The pathogenicity test showed that the pathogenicity of the mutant strain was significantly decreased,and the expression of CgCFEM14 in the host was significantly increased at 48 h after infection with wild-type Cg M2.3.The regulatory effects of CgCFEM14 on iron homeostasis-related gene expression in response to iron ion concentration were analyzed.The iron deficiency condition could induce the expression of CgCFEM14.The expression levels of Cg NRPS2 and Cg NRPS6,a key enzyme in the siderophore synthesis pathway,were significantly down-regulated inΔCgCFEM14 strain compared with wild-type Cg M2 under Fe deficiency and Fe excess conditions.The expression levels of cytochrome synthesis gene CgCYC and heme synthesis gene Cg EMHA in iron consumption pathway,vesicular iron transporter CgCCCA in iron storage pathway were significantly down-regulated inΔCgCFEM14 strain compared with wild-type Cg M2 under iron excess condition.These results indicated that CgCFEM14 was involved in the maintenance of iron homeostasis in the condition of iron deficiency and iron excess.