Identification Of Rice MicroRNAs Invovled In Rice-Magnaporthe Oryzae Interaction

Rice blast is one of most serious diseases in rice production. So the study on the interaction of rice and rice blast fungus is important. In plants, miRNA can regulate plant growth and development, biotic and abiotic stress. But the miRNAs involved in the interaction of rice and rice blast fungus has not been reported. Therefore, our objective is to identify rice miRNAs involved in the interaction of rice and rice blast fungus.We compared the level of defense responses between the susceptible accession Lijiang xintuan heigu (LTH) and two resistant lines IRBLkm-Ts and IRBLz5-CA and chose LTH and IRBLkm-Ts for further investigation. Then six small RNA library were constructed by using samples from 0,12, and 24 hours post inoculation of the blast fungus. By deep sequencing analysis, the miRNAs exihibited differential responses to the infection of the blast fungus were screened. Then, we obtained transgenic rice plants overexpressing miR160a and miR398b and their roles in regulation of defense were investigated. Our main results are as follows.First, IRBLkm-Ts showed stronger defense responses than that of IRBLz5-CA. Fewer lesions were observed on IRBLkm-Ts than on IRBLz5-CA. The expression of four defense related genes was significantly higher in IRBLkm-Ts than IRBLz5-CA. The accumulation of hydrogen peroxide in IRBLkm-Ts was more than in IRBLz5-CA. These results indicate that IRBLkm-Ts is a material better than IRBLz5-CA to identify miRNAs involved in rice defense.Second,33 miRNAs were selected with diffential responses to the infection of the blast fungus. We further classified the miRNAs into three classes based on their change upon M. oryzae infection:(1) no obvious change in LTH but significantly increased in IRBLkm-Ts or reduced in LTH but no obvious change or increased in IRBLkm-Ts; (2) no significant change in LTH but decreased greatly in IRBLkm-Ts or increased in LTH but no obvious change or decreased in IRBLkm-Ts; (3) increased in both LTH and IRBLkm-Ts upon M. oryzae infection. Theoretically, the first two classes of miRNAs should play positive and negative roles in rice resistance, respectively, while the third class may be involved in the regulation of basal responses to the M. oryzae infection because of their up-regulation in both susceptible and resistant lines.Third, over-expression either miR160a or miR398b could enhance resitance to the blast fungus. The expression of miR160a was not much changed in LTH but was increased approximately 10-fold upon M. oryzae infection in IRBLkm-Ts, suggesting that miR160a represents positive regulators of resistance. The expression of miR398b was increased in both LTHand IRBLkm-Ts upon M. oryzae infection, suggesting that miR398b represents regulators acting in basal responses. Transgenic plants over-expressing miR160a or miR398b exhibited fewer lesions than the control, reduced relative mycelial growth biomass, fewer number of spores,suggesting that miR160a and miR398b can significantly enhance disease resistance.Fourth, miR160a and miR398b might differently regulate rice defense. The expression of early defense-related genes showed no difference in the transgenic lines overexpressing miR160a from that in the control line, while late-defense-related marker genes were significantly enhanced. To the contrast, the expression of both early and late defense-related marker genes was significantly enhanced in the transgenic lines overexpressing miR398b. These results suggest that miR398b may participate in PTI and ETI, while miR160a may only participate in ETI.