Sequestering microRNA396 expression enhanced alfalfa resistance to Spodoptera herbivory

Abstract Alfalfa (Medicago sativa) is the most important leguminous forage crop. Its yield and forage quality will be severely affected by the outbreak of defoliator insects, especially Spodoptera litura. Here, we found that the expression of alfalfa precursor miR396 genes (pre-MsmiR396a/b) was significantly down-regulated firstly and then up-regulated in wound treatment that simulating feeding injury of defoliator insects. To verify the function of microRNA396 (miR396) in insect resistance of alfalfa, we generated MIM396transgenic alfalfa plants with significantly down-regulated expression of miR396 by Agrobacterium-mediated genetic transformation. The MIM396 transgenic alfalfa plants exhibited improved resistance to S. litura larvae with increased lignin content but decreased JA content. Further research revealed that the expression of miR396 putative target genes MsGRF1a/1c/4b/5b was increased in five MIM396 transgenic lines, and the response of MsGRF1c/5b to wound treatment was just opposite to the expression pattern of precursor miR396. By RNA sequencing analysis, we found that the differently expressed genes related to insect resistance between WT and MIM396 transgenic plants mainly clustered in lignin, flavonoids and glucosinolates biosynthesis pathways. In addition to the phenotype of increased insect resistance, MIM396transgenic plants also showed reduced biomass yield and quality. Our results broaden the function of miR396 in alfalfa and provide genetic resources for studying alfalfa insect resistance.