| Nitric oxide (NO) is a Non-polar radical and liposoluble gas molecule. As an intra-and inter-cellular signaling molecule in mammals、plants and microorganisms, it plays a dichotomous regulatory roles in numerous physiological and pathological events. Although the mechanisms of NO in plants and mammals has been documented, there are limited reports about NO in fungi. In recent years, it was reported that NO signaling pathways in fungi involved in regulate the cellular development、morphogenesis、sporulation, spore germination、appressorium formation and apoptosis. However, it was still unclear about the mechanisms of NO synthesis, degradation and its functions in the fungi.Recently, it is well known that NO is synthesized by Magnaporthe oryzae during spore germination and appressorium formation, and is required to the formation of pathogenic-infected structure and pathogenicity. In Saccharomyces cerevisia, sfal and yhbl genes involved in the NO metabolic process, and as the results of BLAST in genome of M. oryzae, MoGNO1and MoHB1are their homologous genes respectively. To understand the function of MoGNO1and MoHBl genes in NO metabolic process and morphogenesis and pathogenicity in M. oryzae, in the study, MoGNO1and MoHB1genes were cloned, and their expression characteristics were analyzed during the stage of conidia germination and appressorium differentiation, and their null mutants were constructed.The results of yeast complementation showed that the MoGNO1gene restored the formaldehyde metabolic ability of sfal-null mutant strains, and the crude protein extracts from the MoGNO1-expressed yeast strain had the higher activity of S-nitrosoglutathione reductase about1002.1nmol NADH/mg/min than sfal-null mutant strain (145.2nmolNADH/mg/min). Also, the MoHB1gene restored GSNO metabolic ability of yeast yhb1-mill mutant. The results implied that the MoGNO1and yhbl genes had the NO-metabolic function like sfal and yhbl genes in yeast, respectively.The expression levels of MoGNO1and MoHBl genes were analyzed by real-time PCR in the process of the spores germination and appressorium formation. The results showed that the expression of MoGNO1gradually increased over time. However, the expression levels of MoHBl gradually increased to reach a peak at10hpi, then gradually declined. The results also implied that MoGNO1and MoHB1maybe involved in the germination and infection structure formation of M. oryzae. This result suggests that MoGNO1and MoHB1may be involved in the growth, development and pathogenic infection process in the rice blast fungus.After Agrobacterium-mediated transformations with their respective gene-replaced plasmids, se venty-two hygromycin-resistance transformants for MoGNO1and fifty-two for MoHB1were obtained. Finally, two gene-deleted mutants for each gene were confirmed by PCR and Southern blot at least. The preliminary phenotypic analysis of these gene-deleted mutants were performed. The results showed that sporulation and pathogenicity were delayed, and appressorium collapse rate increased. The vegetative growth of their gene-deleted mutants did not differ from the wild type strain at the condition of high osmotic pressure and oxidative Stress.Subsequently, the expression of these two genes were analyzed in the gene-deleted mutants. In general, the MoHB1expression in the MoGMO1-deleted mutants declined, and the MoGNO1expression in MoHBl-deleted mutants increased. Interestingly, their expression patterns were altered against in the wild type strain, respectively. In MoGNO1-deleted mutants, the expression level of MoHBl was less at10hpi in the MoGNO1-deleted mutants than Guy11, while the levels of MoGNO1did not show gradually increasing trend. This maybe implied that there were some crosstalk between these two genes. |
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