Functional Analysis Of The Gene Pmk1 In Colletotrichum Fructicola

Colletotrichum fructicola is the main pathogen causing Glomerella leaf spot disease,which can not only cause the early defolitation,but also cause spot lesions on young fruits,causing serious damage to apple's quality and yield.Mitogen-activated protein kinase(MAPK)is a key factor in MAPK signaling pathway and is an important component involved in cell signal transduction,cell growth,differentiation and environmental adaptation.In filamentous fungi,the Fus3/Kss1 MAPK gene Pmk1 is a master regulator of mating,growth,sporulation and pathogenicity.The objective of this study was to determine the functions of Pmk1 gene in Colletotrichum fructicola.Pmk1 gene deletion mutant was obtained by homologous recombination mediated gene knockout technique,and the function of the gene was determined by mutant phenotypic analysis and genetic complementation..The main findings are as follows:1.Based on homologous recombination and PEG-mediated protoplast transformation,52 transformants of 1104-B were obtained,and two mutants were obtained by PCR and cellophane penetration assay,which were named 1104Pmk1-37 B and 1104Pmk1-38 B respectively.2.Deletion of Pmk1 gene did not affect the growth,sporulation and spore germination of the mutant strains,but affected appressorium formation.On PDA medium,the growth rates of the mutant and wild type strains were not significantly different.In PDB liquid shake culture,deletion of Pmk1 gene did not affect conidial yield.On onion epidermis,the mutants were abolished in appressorium formation.3.Deletion of Pmk1 gene abolished pathogenicity.Conidia suspensions were used to inoculate Gala fruits and leaves with or without wounding.In all cases,the mutants completely lost pathogenicity.4.Deletion of Pmk1 gene caused increased sensitivity toward high osmotic stresses.,On PDA medium supplemented with 200 ng /mL Congo red or osmotic stress factors(0.5M NaCl,0.5M NaCl,0.5M KCl,1M Sorbitol,or 1M Sucrose),the growth rate of the mutant was significantly slower than the wild type strain,suggesting that PMK1 is important for osmotic and cell wall stress responses.5.Appressorium formation defect of the Pmk1 mutant was restored by geneticcomplementation.6.Based on native promoter driven expression of GFP fusion protein,the expression and subcellular localization of Pmk1 gene were analyzed.The results showed that the gene was expressed in conidium,germ tube,appressorium and infectious hyphae and the protein is localized in both cytoplasm and nucleus.Taken together,our results demonstrated that Pmk1 gene was a key gene regulating developments and pathogenesis of C.fructicola.