Post-transcriptional gene silencing triggers dispensable DNA methylation in gene body in Arabidopsis

Abstract

Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3′ regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.

DNA methylation in plants: mechanisms and tools for targeted manipulation

“…A comprehensive study dissected the genetic components needed for VIGS‐induced methylation and TGS, and showed that different sizes of virus‐derived siRNAs are able to trigger Pol V‐ and DRM2‐dependent heritable DNA methylation, while Pol IV function is dispensable (Bond & Baulcombe, 2015). The Vaucheret lab recently reported similar conclusions when characterizing PTGS‐triggered methylation of a transgene (Taochy et al , 2019). Thus, these results are consistent with non‐canonical RdDM pathways, which use different entry points independent of Pol IV activity, such as viruses and Pol II transcripts, as sources for siRNAs, and which depend on the downstream RdDM components Pol V and DRM2 (Cuerda‐Gil & Slotkin, 2016).…”

Genome-wide analyses across Viridiplantae reveal the origin and diversification of small RNA pathway-related genes

“…Their biogenesis and functionalization mainly rely on Pol IV/V and RDRs (Both RDR2 and RDR6 are reported to participate in RdDM and posttranscriptional gene silencing in Arabidopsis. ) 20 , 61 . We identified two types of RDRs (RDR1/2 and RDR6) with conserved domains (additional files) which is consistent with earlier reports 61 .…”

“… 20 , 61 . We identified two types of RDRs (RDR1/2 and RDR6) with conserved domains (additional files) which is consistent with earlier reports 61 . Earlier studies mainly focused on the functional characterization of RDRs, but there is no phylogenetic analysis for these important genes.…”

Non-coding RNA polymerases that silence transposable elements and reprogram gene expression in plants

“…In fact, siRNAs derived from Pol II transcription also trigger DNA methylation via the Pol V effector machinery. RDR6, an enzyme paralogous to RDR2, uses Pol II transcripts from TEs or transgenes to synthesize dsRNAs that are diced into 21 and 22 nt siRNAs by DCL4 and DCL2, respectively, [ 138 , 194 , 195 ]. Genetic experiments suggest that these siRNAs guide AGO1 (or other AGOs) to sites of Pol V transcription for non-canonical RdDM ( Figure 4c )[ 196 ].…”