The Function Of Chitin Synthases In Colletotrichum Fructicola Of Camellia Oleifera

Camellia oleifera is a woody edible oil tree species native to China,which plays an important role in regional agricultural economy.Anthracnose is one of the main diseases of Ca.oleifera,causing huge economic losses.Colletotrichum fructicola is the major pathogen of anthracnose on Ca.oleifera.So,studying the gene function and elucidating the pathogenic mechanism of C.fructicola can provide potential targets for the development of safe and efficient fungicides against anthracnose on Ca.oleifera.Cell wall plays an important role in growth,development and pathogenesis in fungi.Chitin is one of the main components of fungal cell wall.Chitin synthase is involved in the synthesis and transport of chitin.Chitin synthase is one of the ideal targets for designing fungicides because chitin and chitin synthase do not exist in humans and other vertebrates.In this study,seven chitin synthase genes(CfCHSl-CfCHS7)were identified in the whole-genome of C.fructicola by bioinformatics analysis.Based on encoded amino acid sequences,CfChs were divided into 7 categories by domain prediction and phylogenetic analysis.The chitin synthase gene knockout mutants ΔCfchs1-ΔCfchs7 were obtained by gene knockout according to the principle of homologous recombination.Each gene produced at least two independent empty mutants with the same phenotype.First,we found that the mycelial growth rates were significantly slowed down.Then,results showed that cell wall stress response of ΔCfchsl,ΔCfchs3,ΔCfchs5 and ΔCfchs6 was defective.However,there are no significant changes in these two aspects of ΔCfchs2,ΔCfchs4 and ΔCfchs7.Next,compared to wild-type CFLH16,the pathogenicity ofΔCfchsl,ΔCfchs5 and ΔCfchs6 had significantly reduced,whereas that of ΔCfchs2,ΔCfchs3,ΔCfchs4 and ΔCfchs7 showed not significantly altered.Subsequent studies on gene knockout mutants with altered pathogenicity were carried out.We constructed the vectors PYF11::CfCHS1,PYF11::CfCHS5 and P YF11::CfCHS6,and obtained complementation strains ΔCfchs1/CfCHS1,ΔCfchs5/CfCHS5 and ΔCfchs6/CfCHS6 by protoplast transformation.Then,we performed biological phenotypic assays.The mutants ΔCfchs1,ΔCfchs5 and ΔCfchs6 were found to be defective in hyphae growth,conidiation,appressorium formation,stress responses,cell wall integrity and pathogenicity compared to the wild type.And the corresponding complementation strains restored these defects.Among them,the mycelial growth of ΔCfchs1,ΔCfchs5 and ΔCfchs6 were significantly slowed down,the pathogenicity of them were significantly weakened,and the cell wall integrity of them were affected to a certain extent.For ΔCfchs1,the conidiation defects were serious and the response to osmotic pressure,oxygen pressure and endoplasmic reticulum stress were inhibited.For ΔCfchs5 and ΔCfchs6,they did not produce aerial hyphae and their colony color was abnormal.Besides,their conidiation decreased significantly,most of their conidia were abnormal,and the appressorium formation rate decreased significantly.The difference of them is that ΔCfchs5 have defects in resistance to osmotic pressure,oxygen pressure and endoplasmic reticulum stress,while Cfchs6 only have reduced tolerance to oxygen pressure stress.In summary,this study found that CfCHS2,CfCHS4 and CfCHS7 genes did not affect the growth,cell wall stress response and pathogenesis of C.fructicola,while CfCHS1,CfCHS3,CfCHS5 and CfCHS6 were involved in the regulation of mycelial growth and cell wall stress response of C.fructicola.CfCHS1,CfCHS5 and CfCHS6 are involved in the regulation of conidiation,appressorium formation,external stress,cell wall integrity and pathogenesis of C.fructicola.This study can provide reference for elucidating the molecular mechanism of chitin synthase involved in regulating the pathogenesis of C.fructicola,and provide theoretical basis for developing new antifungal agents using chitin as the target site.