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RNA–DNA Interactions and DNA Methylation in Post-Transcriptional Gene SilencingLouise Jonesa, Andrew J. Hamiltona, Olivier Voinneta, Carole L. Thomasb, Andrew J. Mauleb, and David C. Baulcombeaa Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom b Department of Virus Research, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom Correspondence to: David C. Baulcombe, baulcombe{at}bbsrc.ac.uk (E-mail), 44-1603-250024 (fax)
Post-transcriptional gene silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this association, we used plants carrying a transgene encoding the green fluorescent protein (GFP). Gene silencing was induced using potato virus X RNA vectors carrying parts of the coding sequence or the promoter of the GFP transgene. In each instance, homology-based, RNA-directed methylation was associated with silencing. When the GFP-transcribed region was targeted, PTGS affected both transgene and viral RNA levels. When methylation was targeted to a promoter region, transgene RNA levels were reduced; however, viral RNA levels were unaffected. For comparison, we induced PTGS of the gene encoding the endogenous ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit (rbcS) by inoculation with potato virus X–rbcS. In this example, no methylation of the rbcS DNA was associated with the reduction in rbcS transcript levels, and viral RNA levels were unaffected. Finally, we investigated DNA methylation by using GFP-transformed plants in which PTGS was induced by localized introduction of a T-DNA carrying GFP sequences. In these plants, there was methylation of a GFP transgene associated with systemic spread of a gene-silencing signal from the infiltrated part of the plant. This transgene methylation was not affected when systemic PTGS was blocked by suppressors of silencing encoded by potato virus Y and cucumber mosaic virus. Combined, these data support an epigenetic model of PTGS in which transgene methylation is associated with an RNA–DNA interaction that ensures that PTGS is maintained.
Post-transcriptional gene silencing (PTGS) is a genetic control mechanism that operates at the level of sequence-specific RNA degradation and acts against transgenes, endogenous genes, and viruses (reviewed in
In transgenic plants, particularly in lines that carry multiple transgene copies, PTGS may be initiated spontaneously (
Several studies have indicated an association between PTGS and coding region methylation in plants (
Transgene methylation also has been implicated in PTGS induced by viruses. In a previous study, virus-induced gene silencing (VIGS) initiated by an RNA virus, pea seed–borne mosaic virus (PSbMV), was associated with de novo methylation of homologous nuclear DNA sequences ( To gain further insight into the role of RNA–DNA interactions and de novo methylation, we have investigated PTGS of a transgene and an endogenous gene in Nicotiana benthamiana. We also have used viral suppressors of silencing to dissect the pathway of events leading to PTGS. Our findings demonstrate that DNA methylation is an indicator of an RNA–DNA interaction and that this interaction may be involved in an epigenetic transition associated with many examples of PTGS.
De Novo Methylation Associated with VIGS of a Transgene Encoding the Green Fluorescent Protein To analyze the association between VIGS and methylation of the GFP transgene, we prepared DNA samples from tissues in which the GFP transgene was silenced. This DNA was digested with the methylation-sensitive restriction enzymes AluI, Sau96I, and HaeIII and analyzed after gel blot hybridization with a GFP probe. Figure 1A shows the organization of the 35S:GFP transgene, the location of restriction sites, and the sizes of digestion products of a nonmethylated GFP transgene.
The GFP DNA of nonsilenced PVX-infected tissue was completely digested with these enzymes, indicating that the transgene was not methylated (Figure 1B, lanes 1, 5, and 9). In contrast, in samples prepared from tissue silenced by PVX-GFP, PVX-GF, or PVX-P, there were hybridizing fragments of higher molecular weight, indicating that the GFP DNA was methylated. In nonsilenced samples, for example, AluI fragments of 0.74 and 0.3 kb were detected (Figure 1B, lane 1), whereas in the three samples from GFP-silenced leaves, there were additional fragments of 1.26, 1.0, and 0.36 kb (Figure 1B, lanes 2 to 4). Sau96I digestion gave fragments of 0.56 and 0.28 kb for nonsilenced samples (Figure 1B, lane 5) and additional fragments of 1.3, 0.84, and 0.36 kb for silenced samples (Figure 1B, lanes 6 to 8). Similarly, HaeIII digestion of the nonsilenced sample produced hybridization products of 0.4 and 0.29 kb and shorter fragments that could not be resolved on this gel (Figure 1B, lane 9), whereas samples from the GFP-silenced tissue showed additional fragments of 1.0, 0.85, and 0.63 kb (Figure 1B, lanes 10 to 12). To rule out the possibility that the differences in hybridization pattern between silenced and nonsilenced samples were due to incomplete digestion, we removed the GFP probe and reprobed the filter with the N. benthamiana heat shock protein hsp70 gene (M. Aranda and A.J. Maule, unpublished data). Figure 1C shows that for each restriction enzyme, nonsilenced (lanes 1, 5, and 9) and silenced (lanes 2 to 4, 6 to 8, and 10 to 12) samples had identical hybridization patterns using the hsp70 probe. This result confirms that all samples were digested to the same extent. Thus, the additional GFP-hybridizing fragments in the samples from GFP-silenced leaves could be explained by the presence of cytosine methylation within recognition sites for AluI, HaeIII, and Sau96I. There were four important features of the GFP DNA methylation data (Figure 1). First, from the presence of smaller GFP DNA fragments, we conclude that the methylation of the GFP DNA was never complete in these examples of PTGS. Second, we noted that the methylation profile was similar, irrespective of whether silencing was triggered by PVX-GFP, PVX-GF, or PVX-P. Thus, sites within the P portion of the transgene were affected when silencing was triggered by PVX-GF, and likewise, sites within the GF portion were affected when the initiator of silencing was PVX-P. This result indicates that methylation extends beyond the region of identity between the viral and transgene sequences. A third feature is that the methylation may be restricted to the transcribed region of the gene: sites in the 35S promoter and 3' end of the nopaline synthase terminator were not affected (Figure 1A). However, because few restriction sites were available for analysis in the promoter and terminator regions, the extent of methylation was not determined precisely. Finally, the fourth feature of these data is that methylcytosine residues were detected at both symmetrical (CpG and CpNpG) and nonsymmetrical sites, because both types of site were analyzed and shown to be affected (Figure 1A).
VIGS of GFP by a PVX Vector Carrying the 35S Promoter Sequence
RNA and DNA samples were prepared from PVX-35S–infected tissue at 20 DPI to assess GFP and PVX RNA levels and to determine whether the transgene was methylated in the silenced tissue. Figure 2C shows that GFP mRNA levels were reduced in silenced tissue of infected 16c plants (lanes 5 to 7) compared with levels in noninfected 16c tissue (lane 1) and in nonsilenced tissue of infected plants (lanes 2 to 4). This result confirms the visual observations that PVX-35S induces silencing of the 35S:GFP transgene.
Using a probe specific for the 35S promoter region, we compared viral RNA levels in infected nontransgenic tissue (Figure 2D, lane 9) to levels in nonsilenced (Figure 2D, lanes 1 to 4) and silenced (Figure 2D, lanes 5 to 8) 16c tissue that was infected. This RNA gel blot analysis detected high levels of PVX-35S in all samples tested. Due to the between-sample variation, it was not possible to determine whether the levels were the same in the silenced and the nonsilenced samples. However, if there was a reduction in viral RNA, it was only slight and much less than the 100-fold reduction of PVX-GFP RNA detected in leaves exhibiting VIGS of GFP ( For methylation analysis, DNA samples were prepared from 16c plants in which the GFP transgene had been silenced by either PVX-35S or PVX-GFP. The DNA was digested with Sau3A or Sau96I, and the products were analyzed by DNA gel blotting using probes specific for either the 35S promoter or the 3' 359 nucleotides of GFP (P). Figure 3A shows the location of restriction sites relevant for this analysis. After digestion with Sau3A and probing with the 35S promoter, a 0.7-kb fragment was detected in all samples (Figure 3B, lanes 1 to 3). However, there was an additional fragment of 1.4 kb in the PVX-35S–silenced sample (Figure 3B, lane 2). Reprobing of this filter with the 3' 359 nucleotide P probe detected fragments of 0.28 and 0.17 kb for nonsilenced and PVX-35S–silenced samples (Figure 3C, lanes 1 and 2) and additional fragments of 1.0 and 0.9 kb in the PVX-GFP–silenced sample (Figure 3C, lane 3). These results indicate that Sau3A sites within the GFP sequence are methylated when silencing is induced by PVX-GFP and not when induced by PVX-35S. Conversely, Sau3A sites within and upstream of the 35S promoter are methylated when silencing is induced by PVX-35S but not when it is induced by PVX-GFP (Figure 3A).
A similar conclusion was obtained by analyzing Sau96I digestion products. Probing with the 35S promoter sequence detected a hybridization product of 0.56 kb for the nonsilenced sample (Figure 3B, lane 4). In the PVX-35S–silenced sample, there were additional fragments of 1.31, 1.26, and 0.96 kb (Figure 3B, lane 5). The 0.96-kb fragment was most predominant, indicating that the Sau96I site within the 35S promoter was more often methylated than the upstream sites. The 1.3-kb fragment in the PVX-GFP–silenced sample (Figure 3B, lane 6) probably is a consequence of methylation of the Sau96I sites in the transcribed region of the transgene. Reprobing with the P probe detected a fragment of 0.28 kb for nonsilenced and PVX-35S–silenced samples (Figure 3C, lanes 4 and 5) and additional fragments of 1.3 and 0.84 kb for PVX-GFP (Figure 3C, lane 6). From these data, combined with the data in Figure 1, we conclude that viral RNA can mediate methylation of both the transcribed and promoter regions of the GFP transgene. Methylation can spread within the transcribed region when silencing is initiated by PVX-GFP. When silencing is initiated by PVX-35S, methylation is directed to the promoter but does not extend downstream into the transcribed region.
De Novo Methylation Is Not Associated with VIGS of an Endogenous Gene
Figure 4B shows the results of an RNase protection experiment using a probe to detect endogenous rbcS transcripts. Levels of rbcS mRNA in leaves exhibiting the chlorotic phenotype were at least 10-fold lower than in mock-inoculated leaves or in control leaves infected with PVX or PVX-ABP. RNA gel blot analysis with a PVX-specific probe showed that viral RNA levels were high in tissue infected with PVX-rbcS, wild-type PVX, or PVX-ABP (Figure 4C). These results suggest that the endogenous rbcS transcript, but not PVX-rbcS, is a target of VIGS. The possible methylation of rbcS DNA was investigated in DNA samples prepared at 25 DPI from N. benthamiana infected with either PVX–ß-glucuronidase (PVX-GUS) or PVX-rbcS. The DNA was digested with AluI, HaeIII (data shown), Sau3A, or Sau96I (data not shown) and probed with an rbcS- specific probe. Figure 4D shows that for each enzyme, there was an identical hybridization profile in the two samples, indicating that de novo methylation is not associated with VIGS of rbcS. Once again, equal digestion of samples was confirmed by reprobing of the filter with hsp70 (data not shown).
De Novo Methylation Is Associated with Systemic Silencing The oldest mature leaf on N. benthamiana line 16c plants that had three fully expanded leaves was infiltrated with Agrobacterium carrying the 35S:GF T-DNA, in which the 3' part of the GFP coding sequence was deleted. Using 35S:GF allowed us to distinguish between the GFP transgene and any introduced GFP sequences by using the P probe, which comprises the 3' 359 nucleotides of GFP, in DNA gel blot analysis. Figure 5A shows that 6 days postinfiltration there was silencing of GFP in the infiltrated patch. Control infiltrations by using MgCl2 (Figure 5A) or Agrobacterium that did not carry the GFP sequence did not cause silencing. After 20 DPI, silencing of GFP was visible in all newly emerging tissue, and eventually (~35 DPI) even mature, noninfiltrated leaves became silenced.
RNA and DNA samples were prepared from tissue infiltrated 7 days earlier with either MgCl2 (sample 1) or Agrobacterium 35S:GF (sample 2) and from systemically silenced tissue at 35 DPI that either was (sample 3) or was not (sample 4) mature at the time of infiltration. Figure 5B shows the results of gel blot analysis of these RNA samples. As expected, GFP RNA levels were lower in tissues in which GFP fluorescence was visibly reduced (Figure 5B, lanes 2 to 4) compared with unsilenced tissue (Figure 5B, lane 1). The corresponding DNA samples were digested with the methylation-sensitive enzyme Sau96I, and the products were analyzed by DNA gel blotting with the P probe (Figure 5C). Similar to the results described for VIGS of GFP, the predicted (unmethylated) hybridization pattern was observed only for unsilenced tissue, and all silenced tissue showed additional hybridizing fragments. Thus, a fragment of 0.28 kb was detected in the sample from unsilenced tissue (Figure 5C, lane 1). Mature tissue that had been infiltrated with 35S:GF showed additional fragments of 2.0 and 0.84 kb (Figure 5C, lane 2), and the tissues that were systemically silenced showed additional fragments of 1.3, 0.84, and 0.36 kb (Figure 5C, lanes 3 and 4). The sizes of these fragments indicate that the integrated transgene was methylated both in the 5' region corresponding to the infiltrated GF sequence and in the nonoverlapping 3' regions (cf. Figure 1). To confirm equal digestion of samples, we stripped and reprobed the blot with the N. benthamiana hsp70 gene. Figure 5D shows that all four samples had identical hybridization patterns, indicating that all samples were digested to the same extent and that the differences in hybridization pattern with the 3' 359-bp P probe are likely due to methylation. These results demonstrate that GFP DNA methylation occurs in the developed tissue in which silencing is triggered initially and also is associated with systemic silencing in both developed and developing tissue. Methylation of GFP DNA never was observed in unsilenced leaves (data not shown), indicating that de novo methylation is not simply a consequence of normal development.
Suppression of Gene Silencing Does Not Reverse DNA Methylation
PTGS of the GFP transgene in line 16c seedlings was initiated by infiltration by using Agrobacterium 35S:GFP. Once silencing was established at 20 DPI, plants were inoculated with either the potyvirus potato virus Y (PVY) or CMV. As described in Figure 6A shows the results of RNA gel blot analysis confirming earlier findings that GFP fluorescence correlates with levels of GFP mRNA. Thus, GFP mRNA levels in nonsilenced plants (lane 1) were similar to levels in the new leaves infected by CMV (lane 4) and in PVY-infected tissue (lane 5). Levels of GFP mRNA were low in silenced plants (lane 2) and in the old leaves of a CMV infection (lane 3).
To investigate whether suppression of silencing was associated with a change in DNA methylation status, we digested the DNA samples with AluI and HaeIII, and the products were analyzed using a GFP-specific probe. In the sample from nonsilenced tissue, in which silencing had not been triggered, there were fragments of 0.74 and 0.3 kb for AluI (Figure 6B, lane 1) and of 0.4 and 0.28 kb for HaeIII digestion (Figure 6B, lane 6). These are the sizes that would be expected if there were no methylation of the GFP DNA (cf. Figure 1A). The samples from CMV- and PVY-infected tissue showed identical additional hybridizing fragments of 1.26, 1.0, and 0.36 kb for AluI (Figure 6B, lanes 3 to 5) and of 1.0, 0.85, and 0.63 for HaeIII digestion (lanes 8 to 10). This pattern of additional hybridizing fragments is the same as that observed for systemic silenced samples (Figure 6B, lanes 2 and 7). As for the previous experiments, the filter was reprobed with the N. benthamiana hsp70 gene to confirm equal digestion of samples (Figure 6C). Thus, the methylation associated with PTGS is maintained after infection by CMV or PVY, even though silencing was suppressed.
From the data reported here, it is shown that viral RNA has the potential to interact with homologous transgenes and that sequence-specific methylation of DNA is associated with this interaction. This type of interaction had been implicated by previous reports. However, it remained possible that there could be either DNA–DNA interactions between homologous transgenes ( Using VIGS, we found that both transcribed and promoter regions participate in the RNA–DNA interaction and were targeted for methylation. However, the consequences of the interaction varied, depending on the region involved. Viral RNA interactions with the transcribed region led to PTGS of the transgene and elimination of the virus from infected cells, whereas interactions with the promoter led to transcriptional silencing, and virus accumulation remained high (Figure 2).
Previous analyses showed that VIGS of GFP induced by PVX-GFP involves separate initiation and virus-independent maintenance stages. Initiation is characterized by simultaneous accumulation of the virus and suppression of the GFP transgene RNA, whereas, in the maintenance stage, PTGS of the transgene persists even though the viral RNA has been eliminated. The previous analyses also showed that PVX induces PTGS in the absence of homologous DNA. This DNA-independent PTGS ( Combining the findings from these previous analyses with the results presented here, we now propose that the initiation stage of VIGS is due to RNA-mediated defense and does not require any sequence similarity between the virus and the genome of the host plant. We further propose that the maintenance stage requires the presence of a transgene that is similar to the viral RNA and is dependent on an RNA–DNA interaction leading to methylation of the transcribed part of the transgene. Thus, with PVX-GFP and derivatives, progression into the maintenance stage would follow an interaction between GFP RNA derived from the virus and the homologous transgene. In this scenario, the RNA–DNA interaction forms the basis for an epigenetic change in gene expression that ensures maintenance of PTGS in the absence of the continued presence of the virus. It remains to be determined how an RNA interaction could lead to DNA methylation and how the interaction would influence PTGS. In principle, transcription could be affected by the formation of an RNA–DNA duplex or triplex or by DNA methylation. From the pattern of GFP DNA methylation (Figure 1), it is likely that transcription of the GFP transgene is involved. As demonstrated using PVX-GF and PVX-P to initiate VIGS, the methylated DNA extends beyond the region of identity between viral RNA and the GFP transgene but appears to be restricted to the transcribed region. In terms of RNA-directed methylation, this pattern could be explained if the initial methylation corresponds to this region of identity. Aberrant transcripts of this DNA then would mediate more extensive secondary methylation that corresponds to the transcribed region of the GFP transgene. This cycle of RNA–DNA interactions and production of aberrant RNA may account for maintenance of methylation at nonsymmetrical cytosines that is otherwise difficult to explain in terms of a conventional maintenance methylase. Such a cyclical process also can explain the failure of VIGS initiated by PVX-35S or PVX-rbcS to progress into the virus-free maintenance stage (Figure 2 and Figure 4). For VIGS of GFP by using PVX-GFP, silencing is maintained in the absence of virus accumulation, whereas for PVX-35S and PVX-rbcS, silencing of the corresponding nuclear sequence always correlated with the presence of high levels of virus. With PVX-35S, the maintenance stage of VIGS would be absent because the targeted DNA was not transcribed (Figure 2 and Figure 3). With PVX-rbcS, there was no detectable methylation of the rbcS DNA (Figure 4), indicating that this endogenous gene did not participate in the RNA–DNA interaction; therefore, the ability to perform PTGS was not imprinted.
Systemic Silencing In the infiltrated leaf, in which the systemic silencing was initiated, there was methylation of the integrated GFP transgene due to an interaction either with the infiltrated T-DNA or with transcripts of this DNA (Figure 5). We interpret this methylation as an indicator that the GFP transgene would participate in the cycle involving transcription and RNA–DNA interactions, as proposed above for the maintenance stage of VIGS. From the methylation of the GFP transgene in the noninfiltrated leaves (Figure 5), we infer that this cycle subsequently was repeated in cells receiving the signal of systemic silencing. Further support for the involvement of the RNA–DNA interaction cycle in systemic silencing is based on the distribution of methylated cytosine residues in the integrated GFP transgene (Figure 5): when systemic silencing was initiated by the 5' part of the GFP sequence, there were methylated residues in the nonoverlapping 3' part of the integrated GFP transgene. Correspondingly, the target of systemic silencing extended beyond the sequence used to initiate systemic silencing. Thus, the 3' part of the GFP sequence was a target when systemic silencing was initiated by the nonoverlapping 5' region and vice versa.
Viral Suppressors According to these suggestions, if HC-Pro was able to block maintenance of silencing, it should have interfered with the cycle of RNA–DNA interactions, and the level of GFP DNA methylation would have been reduced in the leaves of PVY-infected plants. Similarly, if the 2b protein was able to block movement of the silencing signal or to prevent initiation of silencing in the differentiating leaves, the GFP transgene in the newly emerging leaves of CMV infected plants would have been unmethylated. However, both of these predictions were wrong. The GFP transgene was as highly methylated when systemic silencing of GFP was suppressed by PVY or CMV as it was in noninfected plants, thereby showing that PTGS and methylation can be uncoupled. Because methylation is maintained in the absence of PTGS, we conclude that methylation is not a consequence of PTGS. However, we cannot distinguish between the possibilities that methylation is a cause of silencing or that methylation and PTGS represent separate pathways that share a common trigger. To account for our observations, we now consider that both HC-Pro and 2b act at a stage in the PTGS mechanism that is downstream of the cycle of RNA–DNA interactions involved in maintenance and signal production. To account for the ability of HC-Pro to suppress silencing in all parts of the plant, we conclude that this protein targets a labile component of the PTGS pathway. In contrast, the 2b protein must act against a stable component of the PTGS pathway. Presumably, because the 2b protein cannot reverse systemic silencing in the old leaves of the plant, it prevents formation of this component, at the level of either synthesis or assembly.
Epigenetic and Genetic PTGS
The difference between genetic and epigenetic PTGS is likely to involve the various ways that the gene-silencing mechanism is activated and maintained, although the components that target and degrade RNA may be identical. Similarly, in fungi and animals, there may be certain components, including double-stranded RNA and a host-encoded RNA-dependent RNA polymerase (
Plant Material
Wild-Type and Recombinant Viruses PVX-rbcS carries a 500-bp fragment of an N. benthamiana ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit (rbcS) cDNA (nucleotides 2 to 502) that was polymerase chain reaction–amplified using primers based on the N. tabacum rbcS sequence (GenBank accession number X02353).
Agrobacterium Induction of Post-Transcriptional Gene Silencing
GFP Imaging
DNA Extraction and Gel Blot Analysis
RNA Extraction, Gel Blot Analysis, and RNase Protection Assay
We are grateful to the Gatsby Charitable Foundation for support of this work, to our colleagues in the Sainsbury Laboratory and Virus Research department for valuable discussions, and to Lu Rui for plasmid pGR107. Received July 9, 1999; accepted October 15, 1999.
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