Molecular Evidence Of Mitochondrial ATP-Lon Protease(MAP1) Involved In Pathogenicity And Self-protection In Magnaporthe Oryzae

Rice blast is the most important disease that affects global riceproduction, and is caused by the filamentous ascomycete fungusMagnaporthe oryzae. In this study, we build a T-DNA insertional mutantlibrary of M.oryzae by agrobacterium-mediated transformation （ATMT）method, which contained more than6000transformants, and identified apathogenicity-defective mutant ATMTlj-22by screening this mutantlibrary. TAIL-PCR analysis revealed the integrated T-DNA in themutants disrupted promoter region corresponding to gene of MAP1（Mitochondrial ATP-dependent Lon Protease, MAP1）. qRT-PCRanalysis showed that MAP1genes expressed in each period of rice blastfungus.For further analysis, a MAP1gene deletion mutant△MAP1wasgenerated by PEG mediated protoplast transformation. Compared withwild type, the hyphae growth rate of the△MAP1mutant had no obviouschange, but the aerial hyphae showed much less. The conidialmorphology of the△MAP1mutant was much shorter and wider thanthat of the wild type. Approximate20%±8%conidia can generate twoappressoria, and the larger one was easily damaged. Further researchshowed that the conidia, which produced two appressorium, could notinvade the onion epidermal cells.The△MAP1mutant showed hypersensitivity to cell wall inhibitors（SDS, Congo red） and lysing enzyme. In addition, the△MAP1mutant showed defects on cell surface hydrophobicity. These results suggest thatMAP1gene is required for cell wall integrity. Meanwhile, transmissionelectron microscope （TEM） observation showed that MAP1geneabsence affected the thickness of the cell walls of M.oryzae, and△MAP1mutant was much thinner in cell walls thickness. Furtherresearch showed that the intracellular accumulation of ROS was affected,which was involved in oxidative cross-linking of cell wall structuralproteins.The results showed that△MAP1was hypersensitivity to NaCl,Sorbitol and H₂O₂, and the MAP1expression was increased undermultiple stress factors by qRT-PCR analysis. It showed that MAP1genesplayed an important role in the self-protection process that respond toadversity factors in M.oryzae. The△MAP1mutant showed a significantreduction in virulence on rice and barley seedlings, and displayedextension defect in rice sheath cells and onion epidermal cells. Themap1-gfp fusion proteins localized to the mitochondria of fungal cells.Meanwhile, compared with wild type（²9%）, most mitochondria cristae（⁸6%）showed collapsed and number reduced in△MAP1mutantafter be treated2hours in5mM H₂O₂.Our results suggest that the MAP1gene involved in cell wallintegrity maintaines, pathogenicity and serves as a self protection factorthat help M.oryzae to conquer multi-stress conditions. Therefore, we setup the relationship among MAP1gene-stress self-protection-mitochondria-pathogenicity in M.oryzae by MAP1gene function analysis.