AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V dependent loci in Arabidopsis thaliana
Genome 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. Summary: 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 Summary In 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.