Genome-wide identification of endogenous RNA-directed DNA methylation loci associated with abundant 21-nucleotide siRNAs in Arabidopsis

In Arabidopsis, the 24-nucleotide (nt) small interfering RNAs (siRNAs) mediates RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of transposable elements (TEs). In the present study, we examined genome-wide changes in DNA methylation and siRNA accumulation in Arabidopsis induced by expression of the Cucumber mosaic virus silencing suppressor protein 2b known to directly bind to both the 21/24-nt siRNAs as well as their associated Argonaute proteins. We demonstrated a genome-wide reduction of CHH and CHG methylation in the 2b-transgenic plants. We found that 2b suppressed RdDM not only at the previously annotated loci directed by 24-nt siRNAs but also a new set of loci associated with 21/22-nt siRNAs. Further analysis showed that the reduced methylation of TEs and coding genes targeted by 21/22-nt siRNAs was associated with sequestration of the duplex siRNAs by the 2b protein but not with changes in either siRNA production or transcription. Notably, we detected both the deletion and/or the transposition of multicopy TEs associated with 2b-induced hypomethylation, suggesting potential TE reactivation. We propose that the silencing of many TEs in Arabidopsis is controlled by the 24- and 21-nt endogenous siRNAs analogous to Drosophila TE silencing by PIWI-interacting RNAs and siRNAs.