Arabidopsis small RNAs associated with RDR6-dependent RNA-directed DNA Methylation

Background: The majority of plant transposable elements (TEs) are found in a silenced state that is epigenetically propagated by the maintenance of symmetrical DNA methylation. TE methylation is established and reinforced by a mechanism of RNA-dependent DNA methylation, which is dependent on transcription of the PolIV RNA polymerase. Recently, a pathway has been described that initiates de novo DNA methylation dependent on components of the RNAi post-transcriptional silencing pathway, independent of PolIV. To define the function of this new pathway, we have focused on the RDR6 protein, which plays a central role in the pathway we refer to as RDR6-dependent RNA-directed DNA Methylation (RDR6-RdDM). Methods: We have sequenced total small RNAs from wild-type and three mutant genotypes: ddm1, ddm1/rdr6, and rdr6. Conclusions: We demonstrate that RDR6-RdDM utilizes 21 and 22 nucleotide siRNAs to de novo methylate actively transcribing TEs. In addition, we find that the RDR6-RdDM pathway functions in the efficient initiation and re-establishment of TE transcriptional silencing, while it is not necessary to maintain trans-generational epigenetic silencing. Examination of flower bud small RNAs from wild-type and 3 mutant genotypes