The Construction Of Knock-Out System Of Effecters And Preliminary Analysis Of The Biological Function In Colletotrichum Fructicola From Camellia Oleifera

Camellia oleifera is an important oil cash crop in China.Camellia oleifera anthrax is the main disease in the planting process of Camellia oleifera,causing the flower and fruit loss of Camellia oleifera,resulting in 30%～40%yield reduction.Colletotrichum fructicola is the dominant strain of national camellia anthracnose.Studies have shown that pathogenic fungal effectors play an important role in the growth and development of fungi,pathogenicity,response to external stress,and inhibition of plant immune defense response.With the development of gene sequencing technology and bioinformatics,gene knockout has become a common strategy to study functional genes of filamentous fungi.However,the knock-out transformation system of effector of C.fructicola has not been established yet.Our research group obtained several candidate effectors of fruit-derived anthrax with high expression in C.oleifera during the infection process.On this basis,the knockout fragment was constructed based on the principle of homologous recombination,and the knockout system of C.fructicola effectors was constructed through PEG-CaCl2 mediated protoplast genetic transformation.In addition,the concentration of recombinant fragment,protoplast and transformation time under transformation conditions were optimized to improve the conversion efficiency of the knockout system.The mycelium growth,sporulation and pathogenicity of the knockout strain were studied,and the biological functions of each effetor in C.fructicola were preliminarily explored,providing a theoretical basis for in-depth research on the molecular mechanism of pathogenicity of pathogenic bacteria.To promote the development of genetic improvement technology of plant disease resistance,and provide some reference for the knockout work of other fungi.The specific research results as follows:1.Construction of knockout vector for C.fructicola:Based on the principle of homologous recombination,the upper and lower arm homologous sequences of effectors Cfhep3,Cfhep4,Cfhep5 and Cfhep6 were amplified,and the sequences were validated by over-the-lap PCR for PEG-CaCl2 mediated protoplast transformation.Knockout strainsΔCfhep3,ΔCfhep4,ΔCfhep5,ΔCfhep6 were constructed.After the knockout strains were cultured for 10 successive generations,genomic DNA of the knockout strains was extracted to amplify the hygromycin resistance gene.Agar gel electrophoresis verified that hygromycin could be stably inherited in C.fructicola.2.Optimization of knockout transformation conditions for C.fructicola:The conversion conditions were optimized from the concentration of homologous fragments,transformation reaction time and protoplast concentration.The optimal transformation conditions were as follows:when effector knockout was carried out,the recombinant fragment concentration was set at 300 ng/μL～350 ng/μL,the transformation time was 2.5 h～3.5 h,and the protoplast concentration was 1×1010 cells/ml～1×1011/ml,the conversion efficiency was 10%-20%.3.Study on Biological characteristics of Knockout Strain of C.fructicola The knock out of effectors Cfhep3,Cfhep4,Cfhep5,Cfhep6 significantly affected the mycelium growth and sporulation of C.fructicola,and knocked out strains ΔCfhep3,ΔCfhep4,ΔCfhep5,ΔCfhep6 almost completely lost their virulence in oil tea leaves.Knockout strains ΔCfhep3,ΔCfhep4 significantly decreased virulence in apple,but the change was not obvious in mango and Gongpear,possibly due to the existence of biological selectivity of C.fructicola.