Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana

In plants, RNA-directed DNA methylation (RdDM), a mechanism where epigenetic modifiers are guided to target loci by small RNAs, plays a major role in silencing of transposable elements (TEs) to maintain genome integrity. So far, two RdDM pathways have been identified: RNA Polymerase IV (PolIV)-RdDM and RNA-dependent RNA Polymerase 6 (RDR6)-RdDM. PolIV-RdDM involves a self-reinforcing feedback mechanism that maintains TE silencing, but cannot explain how epigenetic silencing is first initiated. A function of RDR6-RdDM is to reestablish epigenetic silencing of active TEs, but it is unknown if this pathway can induce DNA methylation at naïve, non-TE loci. To investigate de novo establishment of RdDM, we have used virus-induced gene silencing (VIGS) of an active flowering Wageningen epiallele. Using genetic mutants we show that unlike PolIV-RdDM, but like RDR6-RdDM, establishment of VIGS-mediated RdDM requires PolV and DRM2 but not Dicer like-3 and other PolIV pathway components. DNA methylation in VIGS is likely initiated by a process guided by virus-derived small (s) RNAs that are 21/22-nt in length and reinforced or maintained by 24-nt sRNAs. We demonstrate that VIGS-RdDM as a tool for gene silencing can be enhanced by use of mutant plants with increased production of 24-nt sRNAs to reinforce the level of RdDM.