AGO 4 is specifically required for heterochromatic si RNA accumulation at Pol V‐dependent loci in Arabidopsis thaliana

Summary In plants, 24 nucleotide long heterochromatic si RNA s (het‐si RNA s) transcriptionally regulate gene expression by RNA ‐directed DNA methylation (Rd DM ). The biogenesis of most het‐si RNA s depends on the plant‐specific RNA polymerase IV (Pol IV ), and ARGONAUTE 4 ( AGO 4) is a major het‐si RNA effector protein. Through genome‐wide analysis of sRNA ‐seq data sets, we found that AGO 4 is required for the accumulation of a small subset of het‐si RNA s. The accumulation of AGO 4 ‐dependent het‐si RNA s also requires several factors known to participate in the effector portion of the Rd DM pathway, including RNA POLYMERASE V ( POL V), DOMAINS REARRANGED METHYLTRANSFERASE 2 ( DRM 2) and SAWADEE HOMEODOMAIN HOMOLOGUE 1 ( SHH 1). Like many AGO proteins, AGO 4 is an endonuclease that can ‘slice’ RNA s. We found that a slicing‐defective AGO 4 was unable to fully recover AGO 4‐ dependent het‐si RNA accumulation from ago4 mutant plants. Collectively, our data suggest that AGO 4 ‐dependent si RNA s are secondary si RNA s dependent on the prior activity of the Rd DM pathway at certain loci.