Functional Analysis Of The Peroxisome Biosynthesis Factors MoPEX1, MoPEX4 And MoPEX10 In Development And Pathogenicity Of Magnaporthe Oryzae

Rice blast, caused by Magnaporthe oryzae, is the most serious disease of rice production. The studies on gene function of rice blast fungus are conducive to better understand the biological characteristics of the pathogenic fungus, which would promote more effective control methods.Peroxisome in eukaryotic cells as a single membrane organelle involved in biochemical reaction such as P-oxidation, glyoxylate cycle and reactive oxygen species (ROS) decomposition, which affecting growth development, sporulation, invasion in pathogenic fungi, and thus indispensable in the pathogenicity of the fungal pathogens.Peroxisomes do not contain DNA, the membrane proteins and matrix proteins enzymes were encoded by nuclear genes, synthesized in the cytoplasm and recognized via Peroxisome targeting signal (PTS) and transported into the peroxisome. The proteins associated to the Peroxisome biogenesis were called peroxins, which is regulated by PEX genes. The synthesis process of the peroxisome can be roughly divided into several parts which are recognition, docking, translocation, signal recycling. Then unloaded matrix protein binding with signal protein to transport and assemble is mainly through the complete ring of fat structure encoded by PEX2, PEX10, and PEX12. PEX1, PEX4, PEX6 genes play a role to finally let the signal protein recycling in the cytoplasm. However, the research of peroxisome formation related genes in filamentous fungi are quite limited. Therefore, the research of PEX genes of M. oryzae is meaningful and necessary.MoPEX2, MoPEX12 gene which encode the ring of fat structure in rice blast fungus has been studied, and the mechanism of another structure factors Pex10p in Magnaporthe oryzae is not clear, so this paper we first focused on MoPEX10 gene in the rice blast fungus for the functional analysis, the mainly results are as followed:The PEX 10 protein sequences of Magnaporthe oryzae, Gibberella zeae and N.crassa were the most similar. The localization of GFP fusion demonstrated that MopexlOp was distributed on peroxisome. MoPEX10 knock out causes the abnormal localization of the matrix protein. Significant effect appeared in physiological and biochemical metabolism of Amopex10, include the growth rate of the mutant slow down, flat colony on the complete culture, aerial hyphal density decreased; Conidial generation decreased significantly, lipid utilization ability was destroyed, reactive oxygen species tolerance weakened, integrity of cell wall was also affected; Loss of MoPEX10 also let Magnaporthe oryzae spores germination and appressorium formation rate decreased significantly; After culture 24h, conidia still contain lipids; The turgor of appressorium decreased significantly. Through inoculation experiments on both barley and rice, it is found that the pathogenic ability of mutants decreased significantly, even some kinds of inoculation cannot produce lesion, which confirmed MoPEX10 is required for Magnaporthe oryzae pathogenicity.PEX1, PEX4 genes are known as the important regulation factors of peroxisome signal protein recycling. But so far, the pathogenic fungi in the mechanism research are not reported. Therefore, we analysis of Magnaporthe oryzae MoPEX1, MoPEX4 genes function in this paper and found that:The protein sequences of PEX1 and PEX4 in Magnaporthe oryzae were the most similar with Gibberella zeae and N.crassa. The localization of GFP fusion showed that Mopexlp and Mopex4p were distributed on peroxisome. Both â–³mopexl and Amopex4 mutants have the abnormal localization of the matrix protein. Physiological and biochemical metabolism have Significantly affected in both mutants, include the growth rate, the colony form, aerial hyphal density, conidial generation, lipid utilization ability, reactive oxygen species tolerance, integrity of cell wall, spores germination and appressorium formation rate. All functions above have decreased obviously, and symptoms of Amopexl are severer than these of Amopex4. Cause the inhibition of lipid mobilization in mutants, the turgor of appressorium decreased significantly. Through inoculation experiments on both barley and rice, it is found that the pathogenic ability of mutants decreased significantly. After wound treatment or scald treatment, the disease can have a certain recovery, but still weaker than the wild type, which confirmed MoPEXl, MoPEX4 are required for Magnaporthe oryzae pathogenicity.All above clearly reveal the relationship between MoPEXl, MoPEX4, MoPEX10 gene and fungal growth, pathogenicity, and also have a more profound understanding on research of peroxisome function in rice blast fungus.