Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing

The generation of small interfering RNA (siRNA) involves many RNA processing components, including SUP-PRESSOR OF GENE SILENCING 3 (SGS3), RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), and DICER -LIKE proteins (DCLs). Nonetheless, how these components are coordinated to produce siRNAs is unclear. Here, we show that SGS3 forms condensates via phase separation in vivo and in vitro. SGS3 interacts with RDR6 and drives it to form siRNA bodies in cytoplasm, which is promoted by SGS3-targeted RNAs. Disrupt-ing SGS3 phase separation abrogates siRNA body assembly and siRNA biogenesis, whereas coexpression of SGS3 and RDR6 induces siRNA body formation in tobacco and yeast cells. Dysfunction in translation and mRNA decay increases the number of siRNA bodies, whereas DCL2/4 mutations enhance their size. Purifi-cation of SGS3 condensates identifies numerous RNA-binding proteins and siRNA processing components. Together, our findings reveal that SGS3 phase separation-mediated formation of siRNA bodies is essential for siRNA production and gene silencing.