An extensive forward genetic screen identifies the HWS-AGO1 axis as pivotal for target mimicry-mediated miRNA degradation in Arabidopsis.

Target RNAs have emerged as key regulators of miRNA stability, influencing development and physiology in both plants and animals. We previously identified the F-box protein HAWAIIAN SKIRT (HWS) as a key player in target mimicry directed miRNA degradation in plants (pTDMD) using a STTM160-triggered miR160 degradation reporter system. However, the precise mechanism by which HWS functions remains unclear. Here, we conducted an extensive forward genetic screen and identified P14 additional hws alleles that near-completely restored STTM160-induced developmental defects, underscoring the central role of HWS in pTDMD. Intriguingly, we discovered two adjacent amino acid substitutions (R421K and G422D) in the PAZ domain of AGO1 that strongly suppressed the STTM160 phenotype. Although the R/G substitutions only impacted a small set of endogenous miRNA levels, they were sufficient to rescue the developmental defects caused by HWS overexpression (HWS-OE). Similar to HWS dysfunction, the AGO1 R421K substitution significantly retained target mimicry RNAs in AGO1 immunoprecipitates, suggesting that the R421 residue of AGO1 may coordinate with HWS for the clearance of AGO1-miRNA-target mimicry complexes. Our findings thus provide solid genetic evidence for the coordination of HWS and AGO1 in pTDMD.