Functional Analysis Of OsNPSN11in Resistance To Rice Blast

Rice (Oryza sativa L.) is one of the important monocotyledonous crops in the world, and provides the staple food for more than half of the world’s population. Rice blast, caused by Magnaporthe oryzae, is one of the most widespread and destructive diseases of rice, which causes the reduction of rice yield and decline in quality of rice. It will be useful for blast-resistance breeding by identifying more resistance-related genes and elucidating the mechanism of resistance. The membrane fusion in vesicle trafficking in the cells of eukaryotic organisms is mediated by SNARE (Soluble-N-ethyl-maleimide-sensitive fusion protein attachment protein receptor) proteins, which are highly conserved from various species. SNAREs are involved in the formation of the cell plate, interacting with ion channel proteins, and play important roles in plant growth, development and response to abiotic and biotic stresses. OsNPSN11protein is Novel Plant-specific SNARE, which could be induced by blast. Overexpression of OsNPSN11increased the resistance to rice blast. In order to study the role of OsNPSNll in disease resistance, OsNPSN11overexpression transgenic rice, OsNPSN11suppressing transgenic rice and non-transgenic control rice were used to compare the differences before and after inoculation from the pathological, physiological, biochemical and molecular levels, and the main results as follows:1. The germination and infection process of the spores were observed in transgenic and non-transgenic control lines after hyphae fluorescence staining and DAB staining, the result showed that most of the spores stayed at appressorium stage and unable to infect the cells on the OsNPSN11overexpression transgenic lines and disease-resistant rice (24hpi), the spores accounted for65.5-76.5%at this stage, significantly higher than the OsNPSN11suppressing transgenic rice, empty vector transgenic rice and non-transgenic control lines (33.5-35%); at48h after inoculation, the leaves of OsNPSN11overexpression transgenic lines showed large area of brown patches for hypersensitive response, but little patches on the leaves of OsNPSN11suppressing transgenic rice and non-transgenic control rice. 2. Comparative analysis of the content of H2O2and activity of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) between OsNPSN11transgenic rice and non-transgenic control rice at8、24、48、72h post-inoculation, the results showed that the content of H2O2in OsNPSN11overexpression transgenic rice was quickly accumulated within24-48h after inoculated with Magnaporthe oryzae, following which the activity of the three antioxidant enzymes significantly increased; however the accumulation of H2O2in the non-transgenic control rice was obviously less than that in OsNPSN11overexpression transgenic rice, and the increasing of enzymes activity was lower than that in the OsNPSNll overexpression transgenic rice, which indicated the abundant accumulation of H2O2in short time is the key link to resistance to pathogens.3. Gene chips were used to analyze the differences of transcriptom between OsNPSN11overexpression transgenic rice and non-transgenic control rice, the results indicate that at least259differentially expressed genes were related to DNA/RNA metabolism and regulation, fatty acid metabolism, hormone metabolism, protein metabolism, primary metabolism, secondary metabolism, energy metabolism, oxidation/reduction, signal transduction, cell wall synthesis and development, defense response and other biological processes. There are three up-regulated genes respectively encode peroxidase, L-ascorbic acid oxidase and CMPG1, the expression of which in OsNPSN11overexpression transgenic rice were obviously higher than the expression in non-transgenic control rice after being inoculated with Magnaporthe oryzae. This suggested that these three genes were related to the role played by OsNPSNl1in resistance to rice blast.