期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：3
页码：917-922
DOI：10.1073/pnas.1413053112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceUsing 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. 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 naive, 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.
