Characterization Of COS1-regulated Genes RGS1and CMR1in Magnaporhe Oryzae

Rice is feeding more than50%of the world’s population, which is one of the most important food crops. However, rice blast, caused by fungal pathogen Magnaporhe oryzae, has reemerged as an important factor affectting rice production and quality. Rice blast occurs at almost every rice growing region, causing up to30%loss of rice yield each year. How to control the spread of rice blast is a worldwide problem, so we need to understand clearly invasion mechanism of Magnaporhe oryzae.A previous study found that COS1may function as a transcription factor controlling genes responsible for conidiation, and COS1is dispensable in pathogenicity of rice blast fungus. COSl may have a role in mycelial infection. Since the COS1protein is a transcription factor, which genes are regulated?To identify genes influenced by COS1in M. oryzae, RNA-Seq technique was used to screen genes that differentially expressed in COS] mutant strain (M2942). These genes, showed a log2Ratio larger than1or below-1and a FDR (False Discovery Rate) of0.001or less were screened. By this criterions,442genes were identified. Of these,88genes were up-regulated and354were down-regulate in M2942. Further ananlysis indicated that there was a conserved A4GA3motif in the promoters of these genes. This motif was validated to be COSl-binding by electrophoresis motivation shift assay (EMSA). Among them, expression of gene RGS1was up-regulated, while expression of gene CMR1was down-regulated. RGS1(Regulators of G-protein signaling1) is a regulatory factor in G-protein signaling pathway which regulate conidiation. CMR1is a regulator, regulating ofmelanin biosynthesis.To further investigate function of RGS1and CMR1and their relationship with COSl. RGS1and CMR1knockout mutants were generated by PEG-mediated transformation. Colony morphology observation indicated that, in comparison with the wild strain Y34, RGS1Δ grew slightly slowly, and produced much less conidia and melanin. Inoculation of mycelia of RGS1A caused visible symptoms on unwounded leaf blades and RGS1Δ completely prevented the growth of tice roots, suggesting that RGS1plays an important role in the regulation of conidiation, vegetative growth, melanin production and pathogenicity. Similar experiments demonstrated that mutation of CMR1had no obvious effect on vegetative growth, production of conidia and shape of conidium, comparing with Y34. However, CMR1Δ losed its pathogenicity on rice, suggesting that CMR1is pathogencity-related gene. RGSl has been reported to involve in G-protein signaling and regulating conidiation. We then analysed expression of the conidiation-related genes in RGSIA, CMRl Δ, COS1Δ by qRT-PCR. The results showed that when mutation of RGSl down-regulated flbA, Mackbl, CMR1, Mockb2, MagC, PLAl, MPSl and MAC1, while up-regulated C0Nl, COSl and CMRl. Mutation of COSl down-regulated CMRl, while mutaion of CMRl did not affect exppresion of COSl sugesting that COSl may regulate expression of CMRl indrectly.