Molecular Mechanism On Transcription Factor CgSreA Mediated Iron Assimilation Pathway To Regulate Pathogenicity Of Colletotrichum Graminicola

Corn anthracnose is a worldwide disease that occurs in whole corn growth period.Its pathogen is Colletotrichum graminicola Ces.(Wils.)causes anthracnose of corn,and is a model hemibiotrophic plant pathogenic fungus.Iron is one of the essential nutrient elements to ensure the pathogenicity of C.graminicola.C.graminicola obtains iron to ensure its own virulence from the host plant by Reductive Iron Assimilation(RIA)and Siderophore Iron Assimilation(SIA)pathways in biotrophic and necrotrophic stages,respectively.Sre(siderophore biosynthesis repressor)is a key GATA transcription factor regulating iron homeostasis,which has different effects on the virulence of pathogenic fungi.However,the biological function of CgSreA and the molecular mechanism of regulating iron homeostasis on pathogenicity of C.graminicola are still unclear.In this study,the Agrobacterium tumefacien mediated transformation(ATMT)system of C.graminicola was optimized,the GATA type transcription factor SreA was analyzed roles on the growth and development,stress resistance and pathogenicity of C.graminicola and the molecular mechanism of regulating the iron homeostasis and pathogenicity of C.graminicola via RIA and SIA pathways.The main results are as follows:1.The Agrobacterium tumefaciens mediated transformation system of C.graminicola was optimized.In order to optimize ATMT system of C.graminicola for binary vector and other conditions in laboratory.In this study,the ATMT system of C.graminicola was optimized by changing the conidia state of C.graminicola,the induction medium of Agrobacterium tumefaciens and the volume ratio of conidia suspension to Agrobacterium tumefaciens.using C.graminicola conidia as receptor and Cg Nps6 as target gene.The results showed that when fresh germinated conidia suspension of C.graminicola was prepared with IAM medium and Agrobacterium tumefaciens was induced with IM medium,and the volume ratio of sporulation suspension to Agrobacterium tumefaciens was 3:1,the transformation efficiency of C.graminicola was the highest in all the treatments.In order to verify the stability of the optimized ATMT system of C.graminicola,Cg Pks18,Cg Brn1 and Cg CDC25 of C.graminicola were used as target genes for transformation,and the mutants were successfully obtained.The results indicated that the optimized ATMT system was reliable,which provided technical support for the large-scale study of the genes function of C.graminicola.2.CgSreA of C.graminicola was analyzed by bioinformatics.SreA of Aspergillus fumigatus sequence was used as template,inqured with JGI database to obtain the complete sequence of SreA gene of C.graminicola,and its bioinformatics analysis was carried out.CgSreA has a total length of 1900 bp,contains three exons and encodes 592 amino acids.Phylogenetic analysis shows that it has the closest genetic relationship with SreA of Colletotrichum higginsanum.CgSreA contains two zinc finger domains and one cysteine domain,and the amino acid sequence of domain is very conserved in many fungi.CgSreA may interact with b ZIP transcription factors Hap X,glutathione Grx4 and so on.3.ΔCgSreA was aqcuired and the effects of CgSreA on the growth,development and pathogenicity of C.graminicola were analyzedIn this study,the knockout vector of CgSreA was constructed by In-Fusion cloning technology,and successfully obtained knockout mutant ΔCgSreA by ATMT technology.By determining the mycelial growth rate,conidia production,resistance stress and pathogenicity,it was found that ΔCgSreA strain had obvious growth defects on PDA,CM and MM medium,CgSreA negatively regulated the conidia production,ΔCgSreA strain had increased sensitivity to cell wall integrity stress,osmotic stress and oxidative stress,the pathogenicity decreased significantly.In conclution,CgSreA regulated the growth and development of mycelia,spore production,cell wall integrity,oxidative stress ability and pathogenicity of C.graminicola.4.The molecular mechanism of CgSreA on iron homeostasis and pathogenicity of C.graminicola via RIA and SIA pathways was analyzedIn order to clarify the effect of CgSreA on iron homeostasis of C.graminicola,we measured the iron sensitivity of ΔCgSreA,and analyzed the expression of iron homeostasis related genes under different iron conditions.The results indicated that the sensitivity of C.graminicola to high iron conditions increased in ΔCgsre A.the iron homeostasis of C.graminicola was affected,and CgSreA negatively regulated the genes expression related to iron assimilation pathway.However,the regulation mechanism is still unclear.Therefore,the molecular mechanism of CgSreA regulating iron assimilation pathway was studied by Yeast one hybrid(Y1H),Electrophoretic mobility shift assay(EMSA)and point mutation technology.The results showed that CgSreA directly regulated iron assimilation pathway of C.graminicola by combining motif of promoter region of iron assimilation pathway genes.Moreover,different zinc finger domains of CgSreA have different binding requirements for promoter binding motifs of different target genes.CgSreA regulated iron assimilation pathway via zinc finger domain binding to promoter of iron assimilation related genes,thus regulating iron homeostasis and affecting pathogenicity of C.graminicola.