The Polycistronic miR166k-166h Positively Regulates Rice Immunity via Post-transcriptional Control of EIN2

Raquel Salvador-Guirao, Yue-ie Hsing, Blanca San Segundo

MicroRNAs (miRNAs) are small RNAs acting as regulators of gene expression at the post-transcriptional level. In plants, most miRNAs are generated from independent transcriptional units, and only a few polycistronic miRNAs have been described. miR166 is a conserved miRNA in plants targeting theHD-ZIP IIItranscription factor genes. Here, we show that a polycistronic miRNA comprising two miR166 family members, miR166k and miR166h, functions as a positive regulator of rice immunity. Rice plants with activatedMIR166k-166hexpression showed enhanced resistance to infection by the fungal pathogensMagnaporthe oryzaeandFusarium fujikuroi, the causal agents of the rice blast and bakanae disease, respectively. Disease resistance in rice plants with activatedMIR166k-166hexpression was associated with a stronger expression of defense responses during pathogen infection. Stronger induction ofMIR166k-166hexpression occurred in resistant but not susceptible rice cultivars. Notably, theethylene-insensitive 2(EIN2) gene was identified as a novel target gene for miR166k. The regulatory role of the miR166h-166k polycistron on the newly identified target gene results from the activity of the miR166k-5p specie generated from the miR166k-166h precursor. Collectively, our findings support a role for miR166k-5p in rice immunity by controllingEIN2expression. Because rice blast is one of the most destructive diseases of cultivated rice worldwide, unraveling miR166k-166h-mediated mechanisms underlying blast resistance could ultimately help in designing appropriate strategies for rice protection.