Abstract

A class of small RNAs, the phased small interfering RNAs (phasiRNAs), are specifically expressed in anthers and closely associated with male fertility in monocots. However, the specific 21-nt phasiRNAs involved in regulating male fertility remain unclear. Here, we identified a functional 5′-A phasiRNA (referred to as osphasiR1_1) that mediates the cleavage of OsARF7 transcripts to promote male fertility. CRISPR–Cas9 knockout and short tandem target-mimic knockdown of osphasiR1_1 led to significantly reduced male fertility and seed setting. Further mechanistic experiments revealed that osphasiR1_1 mediates OsARF7 cleavage and promotes male fertility in an OsAGO2-dependent but MEL1/OsAGO5c-independent manner. OsARF7 exhibits transcriptional activation activity, and its overexpression hyperactivates target genes such as the key fertility-related gene ubiquitin-fusion ribosomal protein L40 4 and heat shock protein 70-4 . Identification of the osphasiR1_1–OsAGO2– OsARF7 regulatory module fills a critical gap in the phasiRNA pathway and expands our understanding of phasiRNA-mediated gene regulation, providing new insight into the molecular mechanisms that control male fertility in monocots.