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Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana
Saturday, 2015/01/24 | 07:44:40

Donna M. Bond and David C. Baulcombe

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.

 

See: http://www.pnas.org/content/112/3/917.abstract.html?etoc

PNAS January 20, 2015 vol. 112 no. 3 917-922

 

Fig. 1.

Fig. 1.

Establishing VIGS of FWA. (A, Upper) Northern blot with TRV RNA species (Fig. S1B) and (Lower) sRNA Northern blot with FWAtr sRNAs in TRV:FWAtr-, TRV (T)-, or mock (M)-infected Col-0(FWAC) plants, 14 dpi. FDH and U6 were probed as loading controls, respectively. TRV:FWAtr is marked with a red asterisk. The black asterisks represent the 20-nt and 30-nt sRNA markers. For the sRNA Northern blot analysis, TRV:FWAtr-infected samples 7–12 were run on a separate gel as indicated by the white separating line between samples 6 and 7. (B, Upper) RT-PCR of TRV and TRV:FWAcds in TRV:FWAcds-, TRV (T)-, or mock (M)-infected Col-0(FWAC) plants, 14 dpi. FDH was assayed as an internal control. (Lower) sRNA Northern blot with FWAcds sRNAs in the same plants. U6 was probed as a loading control. The black asterisks represent the 20-nt and 30-nt sRNA markers. (C) FWA expression in TRV:FWAtr- (tr), TRV:FWAcds- (cds), TRV- (T), or mock (M)-infected Col-0(FWAC) plants, 14 dpi and 45 dpi. Each sample is represented by a black diamond and the average of all samples per treatment is represented by the red horizontal line. A two-tailed Student t test suggested FWA expression was repressed by TRV:FWAtr (*P < 0.05) and TRV:FWAcds (**P < 0.01), 14dpi. (D) Proportion of early- (black), intermediate- (dark gray), and late- (light gray) flowering V1 progeny from TRV:FWAtr-, TRV (T)-, or mock (M)-infected Col-0(FWAC) plants (A), compared with Col-0(FWACme) and Col-0(FWAC). Lines with a ratio of <1 (early):3 (late) are marked with a black asterisk.

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