Functional Analysis Of CgMk1 MAPK And The Upstream Receptors In Colletotrichum Gloeosporioides

Poplars are widely cultivated in China and of great value in both economic and ecologic value.The phytopathogenic fungus Colletotrichum gloeosporioides is the causal agent of poplar anthracnose,which causes considerable economic losses in China.C.gloeosporioides is also one of the most ubiquitous plant pathogens and causes destructive anthracnose on numerous crops and woody plants worldwide.Appressorium is the infection structure for rupturing the host cuticle in C.gloeosporioides,causing necrotic lesions that are the typical symptoms of anthracnose.Therefore,the study on theformation of appressorium plays a vital role in the illustrating of the molecular mechanisms of pathogenicity.In the initial stage of plant-pathogen interaction,phytopathogens recognize various host surface signals using receptors located in cell membrane,and activate the downstream pathogenicity MAP kinase 1(Pmk1)mitogen-activated protein kinase(MAPK)to play various functions.In plant pathogenic fungi,Pmk1 MAPK was reported as the key regulator of pathogenicity related functions.In addition,Pmk1 MAPK play conserved role in appressorium formation in appressorium-forming fungi.Therefore,this study utilized the gene knockout method to illustrate the functions of putative upstream receptors mucin CgMsb2,membrane spanning proteins Cg Sho1 and CgMk1 MAPK in appressorium formation,penetration,pathogenicity,response to stressors and their activation relationships.The main findings in this study are described below:1.Identification and functional analysis of the putative upstream receptors of CgMk1 MAPK including CgMsb2 and Cg Sho1.In this study,gene knockout mutants ?CgMsb2,?Cg Sho1 and?CgMsb2Sho1 were obtained.Results showed that CgMsb2 played a major role in the recognition of various host surface signals and the deletion of CgMsb2 resulted in significant defects in appressorium formation,appressorium penetration,cellophane membrane penetration and pathogenicity.Cg Sho1 played a minor role and together with CgMsb2 in regulating host signal recognition,cellophane membrane penetration and pathogenicity,and the deletion of Cg Sho1 resulted in an expansion defect of infection hyphae.In addition,both CgMsb2 and Cg Sho1 were shown to be required for oxidant adaptation and vegetative growth under nitrogen limitation.2.Identification and functional analysis of the component in CgMk1 MAPK.In this study,we identified and knockout the putative adaptor proteins Cg Ste50,MAPKKK Cg Ste11,and MAPKK Cg Ste7.Deletion of Cg Ste50,Cg Ste11,and Cg Ste7 resulted in the loss of appressorium formation,penetration of the cellophane membrane,invasive growth and the vegetative growth under nitrogen-limited environment.Besides,pathogenicity of the deletion mutants of Cg Ste50,Cg Ste11,and Cg Ste7 was lost on intact or wounded poplar leaves,similar to the defects observed in the CgMk1 mutant.3.Determined the acitation relationships between CgMsb2,Cg Sho1 and CgMk1 MAPK.In this study,we found that there are complex upstream and downstream regulatory relationships in the intracellular signaling network.The western blot and dominant active assays revealed that CgMsb2 contributed to the phosphorylation of the mitogen-activated protein(MAP)kinase CgMk1,which was essential for infection-associated functions,while Cg Sho1 was unable to activate CgMk1 alone but rather cooperated with CgMsb2 to activate CgMk1.In addition,Cg Ste50,Cg Ste11,and Cg Ste7 positively regulate the phosphorylation of CgMk1.