A phased small interfering RNA acts in an OsAGO2-dependent manner to mediate OsARF7 cleavage and promote male fertility in rice

In monocots, a class of small RNAs, phased small interfering RNAs (phasiRNAs) are specifically expressed in anthers and are closely associated with male fertility. 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 STTM-knockdown of osphasiR1_1 lead 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 hyper-activates target genes such as the key fertility-related gene Ubiquitin-fusion ribosomal protein L40 4 (UbL404) and Heat-shock protein 70-4 (cHSP70-4). Collectively, the identified osphasiR1_1-OsAGO2-OsARF7 regulatory module not only fills a critical gap in the phasiRNA pathway but also expands our understanding of phasiRNA-mediated gene regulation, thereby advancing insights into the molecular mechanisms underlying male fertility control in monocots.