MicroRNA528 contributes to rice resistance against herbivorous insects

Rice production is chronically threatened by the piercing‑sucking brown planthopper (BPH) and the chewing striped stem borer (SSB). Although BPH‑resistance genes have been cloned, no endogenous SSB‑resistance gene is known, and characterized pest‑responsive microRNAs mostly confer pest‑specific rather than broad‑spectrum resistance. Here we show that the monocot‑conserved miR528 acts as a negative regulator of rice resistance against both BPH and SSB: its downregulation by pest feeding, together with genetic evidence from overexpression, silencing, knockout and OsAO‑related lines, demonstrates that miR528 suppression enhances dual‑pest resistance. Among four predicted targets, only OsAO is sustainably induced during herbivory and acts downstream of miR528, as verified by epistatic analysis. The miR528-OsAO module tunes L‑ascorbic acid homeostasis to modulate reactive oxygen species (ROS) bursts, with miR528‑deficient and OsAO‑overexpressing plants accumulating higher H₂O₂ levels upon pest infestation or chitin elicitation. This study identifies miR528 as a previously uncharacterized negative regulator of dual insect resistance and establishes the conserved miR528-OsAO-AsA-ROS axis, providing genetic resources for breeding multi‑insect‑resistant rice cultivars.