| Magnaporthe oryzae, which causes rice blast disease, is the most destructivepathogen of cultivated rice worldwide. All the steps of Magnaporthe life cycle,including spore germination, germ tube elongation, appressorium formation,differentiation of penetration peg, and hyphae infection were controlled by specificgenes directly or indirectly. Therefore, to understand the molecular mechanism of M.oryzae pathogenicity, nurture long-lasting anti-plague rice varieties, develop lastingcontrol strategy of rice blast has theoretical value and is an imminent researchproposition.Research showed that, pre-mRNA splicing is prevalent in eukaryotes from yeaststo mammalian and regulates various life activities. Through previous studies in ourlaboratory found that splicing factors were related with M. oryzae toxicity, even as thedirect toxic factors.Splicesome protein U1A plays an important role in pre-mRNAsplicing, transcription, and polyadenylation inhibition acidification as a specialcomponent protein of small nuclear ribonucleoproteins U1SnRNP, but its role in the M.oryzae, or the other pathogenic fungi is not clear.Like most eukaryotic gene expression pattern, M. oryzae is also need to thealternative splicing with splicesome’s participation to transform the pre-mRNA tomature mRNA. Deletions or mutations of the MU1A gene of M. oryzae (MU1A) maycause abnormal mRNA splicing, abnormal expression of proteins, abnormal growth ofM. oryzae, eventually affect the virulence of M. oryzae.In this study, we constructed theMU1A knockout vectors, MU1A gene knockout linear fragments, RNA interferencevector, over expression vector taking splicesome protein MU1A of wild type M. oryzae as the main object. Using two methods: Agrobacterium tumefaciens mediatedtransformation and PEG-mediated protoplast transformation to knock out the MU1A, wegot14single exchange transformants, a dual exchange but not MU1A gene deletiontransformant. Unfortunately, we had not got the MU1A deletion mutants. Thus, wepresume that the MU1A gene may be the functional gene of M. oryzae to survival.Deletion of it can cause death，or another possibility is that there is unknownmechanism in M. oryzae can prevent the occurrence of double exchange MU1A gene.Using the RNA interference and gene over-express technology to M. oryzaepathogenic strains70-15(Wt70-15), this study got series MU1A Knock-Down Strainsand MU1A over-expression Strains. We selected two strains, the99th transformant,which had48%expression of wild-type strain70-15in MU1A (Kd-99); the10thtransformant, which had four times expression of wild-type strain70-15in MU1A(Op-10) to analysis the biological function of MU1A. Kd-99and Op-10had no obviousdifference in morphology and growth rate compared with Wt70-15under normal cultureconditions. The conidias of Kd-99were abnormal in shapes and the malformation ratewas73.3%. Interestingly, under the hydrogen peroxide, congo red, SDS stressconditions, Kd-99strain showed obvious resistance to these stress compared withWt70-15，however, the Op-10strain showed weak resistance. Preliminary judgment that,MU1A may be related with the growth Controling to M. oryzae under stress or abnormalconditions, because to most fungis, the temporary stop growing (or sleep) is a commonway to keep individual survival.The research also anlysised the three strains about the spore germination,appressorium formation, and infection of plant cells.The results show that although theabnormal conidias of Kd-99can germ and even formed appressorium, but appressoriumwere smaller, had less pigmentation, and can not invade onion epidermal cells. TheOp-10and Wt70-15remained the same basically. Thus, the MU1A may be necessaryfor life activities of M. oryzae, reducing gene expression of MU1A made mycelium losesensitivity to adversity stress, then caused the abnormal growth uncontrollably; Meanwhile, MU1A gene may play a role in conidial normal growth, thus affect theinfection process to plant cells, and involved in the pathogenic process. |
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