Functional Analysis Of IMPDH And The Secreted Proteins Regulated By Snare Proteins Movam7 And Mosec22 In Magnaporthe Oryzae

Rice Blast caused by Magnaporthe oryzae resulting in a serious losses in rice, threatening global food security. Prevention and control of this disease is mainly used for resistant cultivars, supplemented by chemicals. Since the pathogen of physiological racesis variability, so prevention the rice blast has always been a problem on rice production. Understanding the molecular mechanisms of rice blast in growth, development and pathogenesis promote to develop effective and long-term control strategies. With the completion of the whole genome sequence of Magnaporthe oryzae, it is more convenient that molecular biology techniques have been used in the study of the pathogenic mechanism of Magnaporthe oryzae. In recent years the proteomics technology is widely used in various research areas to detect a difference in protein expression. Membrane fusion need energy which provide by GTP. In this paper, we investgated the biological function of the inosine-5’-phosphate dehydrogenase in GTP biosynthesis and the secreted proteins regulated by SNARE proteins MoVam7 and MoSec22.There are two purine metabolism ways in vivo, one is de novo synthesis pathway, the other is salvage pathway, and the main way is de novo synthesis pathway. Inosine-5’-monophosphate dehydrogenase is a key enzyme in GTP de novo synthesis pathway and it is a rate-limiting reaction in relation to the later steps. In this paper, we investgated biological function of the inosine -5’- phosphate dehydrogenase enconding MoIMD4 of Magnaporthe oryzae in GTP biosynthesis.We found that the growth rate, the adhesion of appressorium, the colonization and expansion in the host cells and the virulence of the AMoimd4 mutant decreased. When the exogenously inosine monophosphate （IMP） was added, the growth rate, the adhesion of appressorium, the colonization and expansion in the host cells and the virulence of the AMoimd4 mutant can not be restored; but when the exogenously xanthine nucleotides （XMP） was added, the phenotype of the AMoimd4 mutant in the growth rate, the adhesion of appressorium, the colonization and expansion in the host cells and the virulence can be recovered. Further observation, we found that MoImd4-GFP fusion protein located in the entire cytoplasm. Through prediction functional domains, we found that MoIMD4 has two conserved CBS domains which did not affect the localization, but two conserved CBS domains are involved in the growth and pathogenicity of Magnaporthe oryzae and are necessary for exercising the biological function. In conclution, MoImd4 is involved in energy metabolism, the vegetative growth and pathogenicity of Magnaporthe oryzae, providing a new direction for drug development targeting to the enzyme of energy metabolism.SNARE proteins （Soluble NSF attachment protein receptor proteins） are a key factor of vesicular transport in the process of membrane fusion in endocytosis and play an important role in endocytosis and exocytosis. To further investigating the secreted proteins regulated by SNARE proteins MoVam7 and MoSec22, we used a proteomic approach to obtain the secreted proteins of the wild-type Guy 11 and the mutants AMovam7 and AMosec22. Twenty-six, four and fifteen repeated protein spots were detected, which mainly focused on the molecular weight of 15-120 KD and the isoelectric point of 4-7. Using the MALDI-TOF-TOF MS analysis and protein database searches, we indentified eight and fifteen significant differences in the expression of the proteins （greater than 1.5 or less than 0.66）. The fifteen identified proteins of the mutans AMosec22 contain the eight identified proteins of the mutant ΔMovam7. We construct the knockout vectors of these twelve genes that have been knockouted successfully. We measured the phenotype in vegetative growth, sporulation and pathogenicity of these twelve mutants. The results suggest that these twelve genes are not involved in the pathogenicity. SNARE protein MoSec22 affect the vegative growth through regulating the expression of these three genes which Gene ID is MGG₀8041、MGG₁3252 and MGG₁4917. These three genes which Gene ID is MGG₀3817、MGG₀8041 and MGG₀7877 involved in sporulation, SNARE proteins MoVam7 and MoSec22 influence the sporulation may through regulating the expression of these three genes.