AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V-dependent loci in Arabidopsis thaliana

Significance statementGenome-wide characterization of AGO4-dependent siRNAs revealed that AGO4 is required for the accumulation of a small subset of heterochromatic siRNAs in Arabidopsis thaliana. These AGO4-depdenent siRNAs are likely secondary het-siRNAs produced by a self-reinforcing loop of RdDM. Slicing-defective AGO4 is unable to fully complement het-siRNA accumulation from an ago4 mutant, demonstrating the critical role of AGO4 catalytic ability in het-siRNA accumulation.\n\nSummary: 152; Introduction: 618; Results: 1291; Discussion: 1013; Experimental procedures: 881; Acknowledgements: 24; Figure legends: 568; Author contribution: 25; Conflict of interest: 13; Funding: 34; References: 1289\n\nSummaryIn plants, 24 nucleotide long heterochromatic siRNAs (het-siRNAs) transcriptionally regulate gene expression by RNA-directed DNA methylation (RdDM). The biogenesis of most het-siRNAs depends on the plant-specific RNA polymerase IV (Pol IV), and ARGONAUTE4 (AGO4) is a major het-siRNA effector protein. Through genome-wide analysis of sRNA-seq data sets, we found that AGO4 is required for the accumulation of a small subset of het-siRNAs. The accumulation of AGO4-dependent het-siRNAs also requires several factors known to participate in the effector portion of the RdDM pathway, including RNA POLYMERASE V (POL V), DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1). Like many AGO proteins, AGO4 is an endonuclease that can slice RNAs. We found that a slicing-defective AGO4 was unable to fully recover AGO4-dependent het-siRNA accumulation from ago4 mutant plants. Collectively, our data suggest that AGO4-dependent siRNAs are secondary siRNAs dependent on the prior activity of the RdDM pathway at certain loci.