Functional Study On The Pathogenesis-genes Of Magnaporthe Oryzae

Rice(Oryza sativa L.)is one of the most important crops and its stable production plays a vital role in ensuring world-wide food security.Rice blast caused by Magnaporthe oryzae seriously damages the quality and yield of rice,so it is regarded as one of the most serious diseases that endanger rice production.M.oryzae has typical infection cycles and mature molecular genetic operations,therefore it is regarded as an ideal model for studying the plant host-fungal pathogen interaction.A deep understanding of the pathogenic mechanism of M.oryzae would provide theoretical basis for controlling rice blast,and clue in understanding other plant-pathogenic fungus interaction.This study explored the pathogenic mechanism of Magnaporthe oryzae from glycoprotein function and substrate screening,small RNA(s RNA)screening,aldehyde dehydrogenase function research,etc,and achieved the following results:(1)Glycosylation modification is ubiquitous in eukaryotes,it is an important modification after the translation of secreted and membrane proteins under the catalysis of glycosyltransferase.We identified a type-2 glycosyltransferase named Mo GT2 in M.oryzae.Through gene knockout and phenotype analysis,we found that it plays an important role in the hyphal growth,conidiation and pathogenicity of M.oryzae.Through glycoprotein staining and protein mass spectrometry analysis,we identified 7 potential substrates for Mo GT2,which provided important clues for the in-depth study of glycoproteins regulating the pathogenic mechanism of M.oryzae in the future.(2)s RNA is an important gene expression regulator in organisms,and plays an important role as a transboundary secret agent in the interaction between plants and pathogenic fungi.In this study,a rice(Y33R)resistant to M.oryzae B157 race was selected from 20 rice varieties in the field.By comparing the s RNA of the rice leaves of the resistant and susceptible varieties(Co39)at different inoculation periods,in order to clarify s RNA in rice-Roles and molecular mechanisms in the interaction of rice blast fungus.The results of the study showed that 215 known mi RNAs(one of the s RNAs)and 182 new mil RNAs were identified 48 hours after the leaves of the infected varieties were infected;purified and cultured from the diseased spots of the infected varieties 59 known mi RNAs and 15 new mil RNAs were identified from the mycelium of M.oryzae;and 49 known mi RNAs and 1new mil RNAs were identified from the pure cultured rice blast fungus hyphae.The mil RNAs identified in the pure culture mycelium samples(as background)were eliminated,and mil RNAs with high expression in rice leaves after 48 hours of infection and in the purified culture mycelia of lesions were screened as potential transboundary mi RNAs.Predict the target genes of these candidate mi RNAs and enrich the KO pathway;among them,the autophagy regulation pathway and the peroxidation balance pathway were found,suggesting that during the infection of M.oryzae,the autophagy and peroxidation balance of itself or rice may be targeted to Promote pathogenic infection.By comparing the s RNAs expressed in the leaves of infected and resistant varieties 48 hours after infection,we also found 428 significant(FDR≤0.01)differentially expressed s RNAs.The difference in the regulation of target gene expression may be caused by rice Causes of differences in susceptibility or resistance after infection by specific races of M.oryzae.This study provides a lot of materials for further study of s RNAs in regulating the interaction between M.oryzae and rice and the pathogenic mechanism.(3)Indole-3-acetic acid(IAA),also known as auxin,plays an important role in regulating plant growth and development.In addition to plants,many microbes also have the ability to synthesize IAA,and may use IAA as a virulence factor in infecting the host.By sequence homology we predict two genes,Mo SUC1 and Mo BET3,encoding aldehyde dehydrogenases(ALDHs)which may act at downstream of the IPA pathway in the IAA synthesis pathway in M.oryzae.We further conduct functional analysis by gene knockout and phenotypic analysis.The results showed that the(35)mosuc1and(35)mobet3 mutants had no obvious difference in colony morphology,mycelial growth,conidiation or pathogenicity,compared with the wild type.The(35)mosuc1and(35)mobet3 mutants also have no effect on the synthesis of IAA.These results demonstrate that Mo SUC1 and Mo BET3 genes are not involved in regulating morphological differentiation,pathogenicity or IAA synthesis in M.oryzae.In summary,this thesis identified the important gene Mo GT2 that regulates the pathogenicity of M.oryzae and screened its potential glycosylation-modified proteins;screened a large number of s RNAs that may be involved in regulating the pathogenicity of M.oryzae;excluded the ALDH encoding gene Mo SUC1 and Mo BET3 is involved in IAA synthesis of M.oryzae.These results analyze the pathogenic mechanism of M.oryzae from many aspects,and provide abundant materials for further in-depth research in the future.