| Rice blast caused by Magnaporthe oryzae is one of the devastating diseases that threaten global rice production.In order to effectively control rice blast,we need to understand the growth and pathogenic mechanisms of Magnaporthe oryzae.Studies have shown that Magnaporthe oryzae secreted proteins play an important role in the pathogenic process of Magnaporthe oryzae.In previous studies,we analyzed the secreted proteins differential expression during the appressorium development of Magnaporthe oryzae.A secretory protein Mo RMD1（Sporulation protein RMD1）with significant differential expression was selected,which is speculated to be related to the pathogenicity of Magnaporthe oryzae.According to related literature,there is no research on the function of Mo RMD1 protein corresponding gene in Magnaporthe oryzae.In this study,the effect of Mo RMD1 gene on the pathogenicity of Magnaporthe oryzae was studied by gene knockout.The results are as follows:（1）Bioinformatics analysis of Mo RMD1 protein.The results showed that the Mo RMD1 protein was composed of 633 amino acids with a molecular weight of 69910.35,and a theoretical isoelectric point（p I）of 5.39.Mo RMD1 protein contained a DUF155domain（DUF:a domain of unknown function）.Signal P 4.1 Server performs N-terminal signal peptide prediction analysis on the amino acid sequence of Mo RMD1 protein,and the results show that it doesn’t have an N-terminal signal peptide;The Secretome P 1.0 Server was used to analyze the protein amino acid sequence again,the NN value was 0.398759.Therefore,Mo RMD1 protein does not conform to the characteristics of non-classical secretory protein.（2）To refer the homologous recombination strategy and PEG-mediated protoplasts transformation,Five?Mo RMD1 mutants were obtained by PCR,Southern blot and GFP fluorescence microscopy.Two of?Mo RMD1 mutants were selected for subsequent experiments.Two?Mo RMD1-com complement strains were obtained by PCR and GFP fluorescence microscopy,and one of them was selected for subsequent experiments.（3）Phenotypic test of the?Mo RMD1 mutants.The results showed that the colony morphology,growth rate,mycelial morphology and conidia morphology of?Mo RMD1were not significantly different from the wild-type of Magnaporthe oryzae.However,the and appressorium formation of decreased significantly.The sporulation and appressorium formation of?Mo RMD1-com complement strain recovered to the wild-type level.（4）Oxidative stress and osmotic stress test of the?Mo RMD1 mutants.The results showed that under different concentrations of H2O2 stress,compared with wild-type of Magnaporthe oryze,there was no significant difference in the inhibition of?Mo RMD1 and?Mo RMD1-com by H2O2.Under the stress of 0.5 mol/L Na Cl,0.02%SDS and sorbitol,compared to the wild-type of Magnaporthe oryze,there was no significant difference in the sensitivity of?Mo RMD1 and?Mo RMD1-com to 0.5 mol/L Na Cl,0.02%SDS and sorbitol;However,?Mo RMD1 is not tolerant to 0.05%SDS.（5）Assay of cell wall integrity of?Mo RMD1 mutants.The results showed that under congo red（200μg/m L）stress.Compared with wild type and complement strains of Magnaporthe oryze,the sensitivity difference of?Mo RMD1 is not significant.（6）The q RT-PCR analysis was performed on the four conidiation-related genes of?Mo RMD1.The results showed that compared with the wild-type and complement strains of Magnaporthe oryze,the gene expression of Mo Sec22,Mo Hox2 and Mo Con8 in?Mo RMD1 was significantly increased,but there was no significant difference in the expression of Mo Cos1.（7）The pathogenicity of the?Mo RMD1 mutants were analyzed.In vivo and in vitro inoculation tests showed that compared with wild-type and?Mo RMD1-com of Magnaporthe oryze,the area and number of disease spots caused by?Mo RMD1 were significantly reduced and decreased.The disease index statistics showed that the pathogenicity was significantly lower than the wild-type of Magnaporthe oryze and?Mo RMD1-com;The results of tissue transparency showed that the lethality of?Mo RMD1on rice cells is significantly reduced. |
