Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana
-
Edited by Steven E. Jacobsen, University of California, Los Angeles, CA, and approved November 18, 2014 (received for review July 10, 2014)
Significance
Using virus-induced gene silencing (VIGS) in wild-type and mutant Arabidopsis, we characterize a novel mechanism associated with the de novo establishment of heritable epigenetic marks in plants. Once established by this novel mechanism, the epigenetic mark is then reinforced by the previously characterized PolIV pathway of RNA-directed DNA methylation. A similar transition from the novel mechanism to the PolIV pathway is likely to explain many epigenetic phenomena in which RNA-directed DNA methylation is established de novo, including transposon silencing and paramutation. A practical benefit of our work is the identification of a mutant plant genotype in which the maintenance mechanism of epigenetic VIGS is reinforced. This genotype would aid the use of epigenetic VIGS for dissection of gene structure and function.
Abstract
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.
Footnotes
- ↵1To whom correspondence should be addressed. Email: dcb40{at}cam.ac.uk.
-
Author contributions: D.M.B. and D.C.B. designed research; D.M.B. performed research; D.M.B. and D.C.B. analyzed data; and D.M.B. and D.C.B. wrote the paper.
-
The authors declare no conflict of interest.
-
This article is a PNAS Direct Submission.
-
Data deposition: The sequence reported in this paper has been deposited in the Array Express database, www.ebi.ac.uk/arrayexpress (accession no. E-MTAB-3009).
-
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1413053112/-/DCSupplemental.
Freely available online through the PNAS open access option.
