N-Glycosylation And GPI-Anchor Modification Mediated Pathogenesis In Colletotrichum Graminicola

Anthracnose stalk rot(ASR)of maize caused by Colletotrichum graminicola occurs widely and seriously damages the production of maize worldwide,and results in 1 billion dollars in losses annually in the United States.C.graminicola can infect all parts of maize,causing symptoms such as leaf blight,top blight and stem rot.In recent years,because of the release of genome sequencing data,more researchers have paid attention to the study of C.graminicola.Moreover,because the advantages of hemibiotrophic characteristic,cultivable and simple genetic operation,C.graminicola can be used as a good model fungus for the study of the interaction mechanism between pathogens and maize.While,at present,there are still few related studies on the pathogenic mechanism of C.graminicola.In this paper,relevant studies were carried out on the N-glycosylation and GPI anchored modification to regulate the pathogenic mechanism of C.graminicola infection.The results are as follows:(1)After treatment with tunicamycin the inhibitor of N-glycosylation,the effects of N-glycosylation inhibition on C.graminicola mainly include reduced vegetative hyphae growth,abnormal hyphal top development,malformed conidial germination,appressorium formation and penetration defects,and loss of virulence.In addition,the conidia were more resistant to tunicamycin than the mycelium in C.graminicola.According to the results of ConA-FITC staining experiment,we preliminarily speculate that the conidia stage and the early germination stage may not be grimly dependent on N-glycosylation.(2)C.graminicola has a highly homologous N-glycosylation pathway with Saccharomyces cerevisiae through homologous comparison.Functional analysis of one key gene in the N-glycosylation modification pathway indicated that CgALG3 mediated the hyphae growth,conidial development,appressorium penetration,and pathogenicity of C.graminicola.We confirmed that CgALG3 had α-1,3-mannosyltransferase function through the subcellular localization analysis and N-glycosylation analysis of the target protein CgNIS1.Therefore,CgALG3 may be involved in the regulation of C.graminicola growth and pathogenicity by mediating N-glycosylation.In addition,we found that CgALG3 mediated N-glycosylation is essential for the stability of effector protein CgNIS1.Based on these results,we speculate that CgALG3 deficiency may affect ER quality control(ERQC)and induced the ER stress,analysis by q RT-PCR of unfolded protein response(UPR)related genes we confirm that CgALG3 is essential for ER homeostasis.(3)406 N-glycosylated modified proteins and 821 N-glycosylated modified sites were identified by quantitative N-glycosylated mass spectrometry.Analysis showed that there were significant differences in the number and abundance of glycosylated modification sites in conidia,vegetative hyphae,and invasive hyphae of C.graminicola.Cluster analysis of N-glycosylated proteins showed that Nglycosylated modification had certain modification effects on cell wall synthesis related proteins,proteases,oxidoreductases,effector proteins,glycosylated hydrolases,O-glycosylated modification pathway proteins,GPI anchored modification pathway proteins,endoplasmic reticulum quality control pathway proteins.These results indicate that N-glycosylation participate in a wide range of biological processes in C.graminicola.(4)Specific glycosylation function analysis of CgCNX1 protein which was identified in the ERQC pathway showed that N-glycosylation of CgCNX1 did no effect on the growth but significant effect on the pathogenicity of C.graminicola.Further studies showed that the alternative mutant of N-glycosylation site or deletion of CgCNX1 significantly affects the secretion of CgNIS1 and CgBAS3.Therefore,we believe that N-glycosylation can mediate the function of ER chaperone to participate in the regulation of ER quality control system,and then affect the normal processing and secretion of ER protein and affect the pathogenic process of C.graminicola.(5)GPI anchor modification is another form of glycosylation modification,which is similar as Nglycosylation.Functional analysis of CgGPI7,one of the key genes in the GPI anchoring pathway of C.graminicola,showed that GPI anchoring mediated by CgGPI7 is essential for the vegetative hyphae growth,conidial morphology and development,sporulation,cell wall integrity and pathogenicity of C.graminicola.