Molecular Mechanism Of CfVPS35 Gene To Regulata Pathogenicity In Colletotrichum Fructicola

Camellia oleifera is a unique woody plant in China It is widely distributed in the south of Yangtze River area.Anthracnose is an important disease in oil tea production,which has caused serious losses to agriculture and economy.Colletotrichum fructicola is the main epidemic pathogen of Ca.oleifera anthracnose.Understanding the pathogenic molecular mechanism of C.fructicola is of great significance for the prevention and control of anthracnose.Retromer complex was first discovered in yeast and is widely found in eukaryotes.Vps35,the core component of the retromer complex,together with Vps26 and Vps29,constitute the cargo selection trimer.The trimer is involved in the transport of substances between the endosome and golgi and mediates the retrograde transport of various receptor proteins back to golgi.We studied the function of CfVPS35 gene to provide a theoretical basis for the pathogenic molecular mechanism.The results are as follows:(1)Functional analysis of CfVPS35 gene.The CfVPS35 gene knockout vector fragment was constructed by over-lap PCR method in C.fructicola,and transferred into the protoplasts generated by using PEG-mediated method.The transformants were selected using hygromycin.The complementation construct transferred into the mutant protoplasts.The complemented strain was obtained by by screening and verification.Biological phenotypes of mutants were determined.It was found that the growth rate of △Cfvps35 mutant was significantly slower than that of wild-type strain and complemented strain.There was no chitin deposition at the mycelial tip of mutant,and the △Cfvps35 was more sensitive to CFW and CR.Further studies showed that the sporulation and appressorium formation rate of △Cfvps35 mutant were significantly reduced.During appressorium development,glycogen transport and metabolism were obstructed,intracellular turgor pressure decreased,and autophagy was defective.In addition,after the deletion of CfVPS35 gene,the pathogenicity of C.fructicola was lost,and disease spots could not be caused on the leaves.Therefore,CfVPS35 gene is involved in regulating the growth,sporulation,appressorium formation,glycogen metabolism,autophagy and pathogenicity of C.fructicola.(2)Functional analyses of CfVPS29 and CfVPS26 genes.We found that CfVps26 and CfVps29,constituent members of the retromer cargo selection subunit,interact with CfVps35 by yeast two-hybrid technique.It was found that both CfVPS29 gene and CfVPS26 gene were involved in regulating the growth and development,cell wall stress response,conidial production,appressorium formation rate,glycogen degradation,turgor formation and pathogenicity of C.fructicola.The phenotypes of the △Cfvps29 and△Cfvps26 mutants were similar to that of the CfVPS35 gene knockout mutant.These results revealed that CfVps29,CfVps26 and CfVps35 proteins could form trimers in cells and participate in the pathogenesis in C.fructicola.In summary,CfVps35 of retromer complex subunit and its interaction proteins CfVps29 and CfVps26 play important roles in the development and pathogenicity in C.fructicola.These results provide potential target sites for the development of new fungicides against Ca.oleifera anthracnose.