Bioinformatics Analysis Of Snare Proteins And Functional Characterization Of Mgsec22 In Magnaporthe Grisea

The filamentous fungus Magnaporthe grisea causes rice blast, the most serious disease of cultivated rice. Functional genomics approaches are providing new insight into the biological characteristic and molecular pathological mechanism of the fungi and leading to the prospects for sustainable control of rice blast.The membrane fusion process is highly conserved in all eukaryotes. The central components driving membrane fusion events involved in vesicle delivery to target membranes are a set of integral membrane proteins called SNAREs. Four SNAREs from Qa-, Qb-, Qc- and R-subfamilies formed a 4-helical bundle to mediate membrane fusion process. According to the SNAREs reported in S.cerevisiae and A.oryzae, a set of 21 putative SNAREs in M. grisea was identified and analyzed. SNARE proteins were classified as four subfamilies by structural and phylogenetic analysis. Sequence blast of SNAREs proteins suggested ER, Golgi, and endosomal SNAREs are largely conserved in filamentous fungi. The differences of SNAREs in secretion pathway are the presence of the apparently non-redundant plasma membrane Qa-SNAREs. Such features might raise partial explanation of the key differences between yeasts and filamentous fungi, in terms of polarized growth, morphogenesis, and secretion.A putative gene encoding a SNARE protein was cloned and named Mgsec22. Southern blotting showed it was unique in M. grisea genome. The phylogenetic analysis of protein sequence revealed that although Mgsec22 was much divergent to yeast Sec22p, it was somewhat similar functionally to Sec22p. For further analysis, method of target gene knockout was used to obtain the Mgsec22 mutant. Mgsec22 mutant seemed lacking aerial hyphae and growing slowly comparing with that of wild-type strain Guyll. Deletion of Mgsec22 also resulted in severely decreasing of conidial production and loss of pathogenicity. The Mgsec22 mutant displayed a more tolerance to the cell wall stressors. CFW straining revealed a change of cell wall structure, and the chitin content was increased. ROS was related to cell differentiation and pathogenicity. NBT staining assay showed that the ROS level was altered, and quantitative real-time PGR analysis suggested the expression of two NADPH oxidases Nox1, Nox2 was down-regulated respectively. Peroxidases play a crucial role for the interaction of the plant-pathogen. The colorimetric determination of enzyme activity revealed the activity of peroxidases and another extracellular enzymes, laccase, were both abolished. Various staining assays and transmission electron microscopy studies were used to test the morphology of vacuole and the endocytsis. It was suggested that the morphology of vacuole did not change, but the endocytsis of the mutant was dramatically inhibited.