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In plants, double-stranded RNA can silence genes by

triggering degradation of homologous RNA in the cytoplasmand by directing methylation of homologous nuclear DNA

sequences. Analyses ofArabidopsismutants and plant viral

suppressors of silencing are unraveling RNA-silencing

mechanisms, which require common proteins in diverse

organisms, and are assessing the role of methylation in

transcriptional and posttranscriptional gene silencing.

Addresses*Institute of Molecular Biology, Austrian Academy of Sciences,Billrothstrasse 11, A-5020 Salzburg, AustriaDepartment of Biological Sciences, University of South Carolina,Columbia, South Carolina 29208, USA

e-mail: [email protected]: MA Matzke; e-mail: [email protected]

Current Opinion in Gene tics & De velopment 2001, 11 :221227

0959-437X/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsdsRNA double-stranded RNAHC-Pro helper component proteinaseH D G S homology-dependent gene silencingIR inverted repeatN M D nonsense-mediated decayPTGS posttranscriptional gene silencingPVX potato virus XRdDM RNA-directed DNA methylationRdRP RNA-directed RNA polymeraseRNAi RNA interferencesm g suppressor with morphogenetic effect on genitaliaTG S transcriptional gene silencing

IntroductionEpigenetic silencing effects that rely on recognition of

nucleic acid sequence homology at either the DNA or RNA

level have been identified in plants, fungi and animals [14].

In plants, RNA has been implicated in two types of homology-

dependent gene silencing (HDGS). Posttranscriptional

gene silencing (PTGS), which is similar to quelling in

Neurospora crassa [5] and RNA interference (RNAi) in ani-

mals [6,7], involves targeted degradation of homologousRNAs in the cytoplasm. At the genome level, RNA can

induce the epigenetic modification of homologous DNA

sequences through the process of RNA-directed DNA

methylation (RdDM) [8]. The mechanisms of these differ-

ent modes of HDGS and the characteristics of the RNAs

involved are being actively investigated.

We focus here on evidence that double-stranded RNA

(dsRNA) plays a dual role in plant gene silencing by initi-

ating both the RNA-degradation step of PTGS and

RdDM. This conclusion derives from work during the past

year on PTGS-defective Arabidopsis thaliana mutants and

on viral suppressors of PTGS as well as experiments show-

ing that transcriptional gene silencing (TGS) and promoter

methylation can be triggered by dsRNA. The emerging

view is that many cases of HDGS at both the transcrip-tional and posttranscriptional levels in plants can be

attributed to the action of dsRNA.

Advances in posttranscriptional genesilencing: a modelAlthough the ability of dsRNA molecules to trigger degra-

dation of homologous RNAs (RNAi) was recognized first

in Caenorhabditis elegans [7], it has become clear from

shared molecular features and gene products that PTGS in

plants, which was discovered more than a decade ago [1],

also involves dsRNA. Unifying studies in plants and

Drosophila have shown that silencing is accompanied by

the accumulation of small RNAs (2125 nucleotides) of

both sense and antisense orientation that are homologous

to the silenced locus or input dsRNA [9,10,11].

Silencing can be triggered locally and then spread through

the organism in plants and C. elegans, and between nuclei

in heterokaryotic strains ofNeurospora via a mobile silenc-

ing signal, the existence of which also supports a conserved

mechanism [6,12].

Several classes of gene products required for PTGS, quelling

and RNAi have been identified to date (Table 1)

[1316,1719,20,21]. So far, only one essential gene,

SGS3 which shows no similarity with any knownprotein is unique to plants [17]. The identities of the

required proteins suggest possible features common to these

processes: the synthesis and amplification of dsRNA,

unwinding of dsRNA, and targeting of mRNAs after binding

to the ribosome (Table 1). The cellular RNA-directed RNA

polymerase (RdRP) plays a central role in PTGS and is

required even in RNAi systems where the process is induced

directly by exogenously supplied dsRNA [20]. InArabidopsis,

RdRP is required for transgene-induced PTGS where one

possible function is the synthesis of cRNA from aberrant

RNA templates, leading to the formation of dsRNA.

However, it appears to be dispensable for virus-induced local

gene silencing, where dsRNA is produced by the viral RdRP[18]. PTGS can thus be separated into two branches that

differ in their requirement for the cellular RdRP (Figure 1).

Mode of action of viral suppressors ofposttranscriptional gene silencingMany plant viruses encode proteins that suppress gene

silencing [22], reflecting the natural role of PTGS as an

antiviral defense. Different viral suppressors act at distinct

steps in PTGS [22,23] and can help to elucidate the silenc-

ing pathway. Two of these viral suppressors, the helper

component proteinase (HC-Pro) of potyviruses and the

p25 cellcell movement protein of potato virus X (PVX),have been particularly informative about the underlying

mechanisms of PTGS.

RNA-based silencing strategies in plantsMarjori A Matzke*, Antonius JM Matzke*, Gail J Pruss and Vicki B Vance


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222 Chromosomes and expression mechanisms

A prominent feature of PTGS suppression by HC-Pro is

the absence of the small RNAs associated with silencing

([24]; A Mallory, V Vance, unpublished data). In addition,

grafting experiments have shown that HC-Pro suppression

of PTGS does not interfere with either the production or

movement of the silencing signal but prevents the plantfrom responding to that signal (A Mallory, VB Vance,

unpublished data). Thus, HC-Pro suppression of PTGS

occurs downstream of the mobile silencing signal at a step

preceding the accumulation of the small RNAs (Figure 1).

A study using the yeast two-hybrid system has identified a

plant calmodulin-related protein (termed rgs-CaM) that

interacts with HC-Pro [25]. rgs-CaM, like Hc-Pro itself,

suppresses gene silencing and might, therefore, be a cellu-

lar intermediary of HC-Pro suppression of PTGS

(Figure 1). Because calmodulin and related proteins nor-

mally act by binding calcium and subsequently activating

target proteins, HC-Pro suppression of PTGS possibly

occurs via activation of rgs-CaM and its unknown target

protein (Figure 1).

In contrast to HC-Pro, the PVX p25 protein appears to sup-

press PTGS by targeting the mobile silencing signal. Using

a virus that could replicate but not move, it was shown that

a mobile silencing signal is produced in PVX-induced

PTGS, as in transgene-induced PTGS. However, the sys-

temic silencing induced by PVX could not be detectedunless the p25 coding region was either deleted or modi-

fied [26]. The simplest interpretation of these results

suggests a model in which the PVX p25 protein blocks

PTGS by suppressing only the cellular RdRP branch of

the pathway (Figure 1). HC-Pro, however, suppresses both

branches of the silencing pathway [27], suggesting that it

affects a step downstream of the junction of the two

branches (Figure 1).

Small RNAs: mobile silencing signal?The small sense and antisense RNAs associated with silenc-

ing derive from cleavage of dsRNA [11] and both

polarities are thought to incorporate into a ribonuclease,

serving as a guide to find homologous target RNAs

[10,28,29]. Small RNAs accumulate during both virus-

and transgene-induced gene silencing, indicating conver-

gence of these two branches of silencing before the

formation of the sequence-specific ribonuclease (Figure 1).

It was proposed originally that the small RNAs associated

with PTGS might be the mobile signal that spreads silenc-

ing throughout the organism [9]. Two lines of evidence

contradict this idea. First, HC-Pro suppression of silencing

interferes with accumulation of the small RNAs but does

not eliminate either the production or movement of the

silencing signal (A Mallory, VB Vance, unpublished data).Second, the PVX p25 protein interferes with the mobile

silencing signal, but does not affect the accumulation of

small RNAs produced in the viral RdRP dependent branch

of PTGS [26]. Although the identity of the mobile signal

remains unknown, the viral suppressor studies suggest that

the signal is a precursor of the small RNAs (Figure 1).

Aberrant RNAs and substrates forRNA-dependent RNA polymeraseAlthough a requirement for RdRP in PTGS phenomena is

undisputed, the nature of the substrates for this enzyme is not

fully clear [5]. In plants, dsRNA that triggers PTGS can be

produced in the nucleus by transcription through invertedDNA repeats (IRs) [3033] (Figure 1) or through the action of

RdRP, which is postulated to use pre-existing dsRNA [34] or

aberrant sense RNAs as templates for the synthesis of anti-

sense RNAs [5]. Although not yet defined, aberrant RNAs are

presumed to be either improperly spliced or terminated, per-

haps because of epigenetic modifications that interfere with

normal processing [1,5,35]. Aberrant RNAs that are miss-

pliced and polyadenylated irregularly have been detected in

a chalcone synthase PTGS system in petunia [36]. Antisense

constructs probably also produce RNAs that feed into the

dsRNA-induced degradation pathway [37,38].

A new aspect of silencing processes is the possible link with

nonsense-mediated decay (NMD), an evolutionarily conserved

Table1

Cellular proteins involved in PTGS.

Protein Mutant name/organism Possible roles

RdRp qde1/Neurospora[13] 1. Synthesis of cRNAsde1/Arabidopsis[18] 2. Amplification of dsRNA

sgs2/Arabidopsis [17] 3. Signaling of methylation

ego1/C. elegans[20] 4. Synthesis of mobilesignal5. Development6. Viral defense

eIF2C-like qde2/Neurospora[21] 1. Target PTGS toribosomes

rde1/C. elegans[16] 2. Signaling of methylation

ago1/Arabidopsis [19] 3. Development

RecQ DNA qde3/Neurospora[14] 1. Unwinding dsRNA

helicaseRNase D-like mut-7/C. elegans[15] 2. Transposon defenseRNA helicase mut-6/Chlamydomonas 3. Ribonucleaseactivity [40]4. Nuclear step?

Coiled-coil Sgs3/Arabidopsis[17] 1. Signaling of protein methylation

2. Viral defense

NMD Proteins smg2/C. elegans[39] 1. ATPase/ helicase/RNA

smg5/C. elegans[39] binding (smg2)smg6/C. elegans[39] 2. Dephosphorylation

of SMG2 (smg5/smg6)

rgs-CaM Nicotiana tabacum[25] 1. Suppression of PTGS2. Development

ago1, argonaute1; ego1 , enhancer of glp-1; qde, quelling defective;rde, RNA interference deficient; rgr-CaM, regulator of gene silencing-calmodulin-like protein; sde, silencing defective; sgs, suppressor of


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RNA-based silencing strategies in plants Matzke et al. 223

pathway in which mRNAs that contain a premature stop codon

are selectively degraded. Three out of six differentsmg(sup-

pressor with morphogenetic effect on genitalia) genes required forNMD in C. elegans were found to be required for persistence of

RNAi [39]. One of these genes,smg-2, is homologous to yeast

Upf1, which encodes an adenosine triphosphatase with RNA-

binding and helicase properties. A DEAH-box RNA helicase

(Mut6) that is involved in degrading misspliced and non-

polyadenylated transcripts was also shown to be required for

transgene and transposon silencing in the unicellular green alga

Chlamydomonas reinhardtii[40]. These results suggest a partial

overlap between NMD and RNAi/PTGS pathways and

provide new insights for unraveling the PTGS pathway.

RNA-directed DNA methylationRdDM was first discovered with viroids, which are plant

pathogens consisting solely of noncoding, highly base-paired,

rod-shaped RNAs several hundred nucleotides in length.

During viroid infection of tobacco, viroid cDNA copies

integrated into nuclear DNA became methylated de novo,implicating replicating viroid RNA in DNA modification

[41]. RdDM of nuclear transgenes has been observed

recently in plants infected with cytoplasmic RNA viruses

carrying transgene sequences [42,43] and in a nonpatho-

genic transgenic system [44]. RdDM results in dense

methylation at most symmetrical and nonsymmetrical

cytosines within the region of homology between the

inducing RNA and the target DNA [45]. DNA targets as

short as 30 bp can be modified [46]. Whereas previous

models for methylation associated with HDGS invoked

DNADNA pairing, which triggers methylation of

sequence duplications in some fungi [3], RdDM provides

an alternate means to induce the sequence-specific methy-

lation observed in both PTGS and TGS.

Figure 1

A model for RNA-based transcriptional andposttranscriptional silencing. Steps involvingdsRNA and steps that are affected by viralsuppressors of PTGS and in various PTGS

mutants are shown. TGS may be triggereddirectly by transcription of inverted repeatsequences in the nucleus and methylation ofhomologous promoter regions in the genome.In addition, dsRNA and other aberrant RNAsformed in the nucleus may be transported to thecytoplasm and enter the PTGS pathway. Twomodes of dsRNA production lead to PTGS inthe cytoplasm: first, virus-induced genesilencing mediated by the viral RdRP, andsecond, transgene-induced gene silencingmediated by the cellular RdRP. The dsRNA fromeither of these sources can be targeted by aputative dsRNA specific ribonuclease whichgenerates 2125 nucleotide RNAs of bothpolarities (small RNAs). These small RNAs are

incorporated into a ribonuclease and act asguides for sequence-specific degradation ofhomologous RNAs. dsRNA from the cytoplasmmay trigger methylation of homologous genomicsequences presumably by transfer of a signalmolecule into the nucleus. Similarly, PTGS canbe induced locally and then spread throughoutthe organism via production and transport of amobile silencing signal. The identity of thesignaling molecule(s) that induces methylationand systemic PTGS is unknown but is likely toincorporate an RNA component, possiblydsRNA as shown here or processed forms ofdsRNA, such as the small RNAs. HC-Prosuppresses gene silencing at a step upstream

of the accumulation of the small RNAs butdownstream of the mobile silencing signal,probably via activation of an endogenouscellular suppressor of PTGS, rgs-CaM. ThePVX p25 suppressor of PTGS prevents theaccumulation and/or transport of the mobilesilencing signal, probably by interfering with thecellular RdRP branch of the pathway.

Mobile signal,dsRNA

QDE-2, RDE-1, AGO1?SMG-2?Ribosome

Aberrant RNA

CellularRdRP PVX

p25

Transgene

Promoter Coding

TGS PTGS

Methylation

dsRNA

Invertedrepeat

Nucleus

Virus RNA

dsRNA

Viral

RdRP

Small RNAs

Sequence-specificnuclease

PTGS

mRNAtarget

Current Opinion in Genetics & Development

HC-Pro( rgs-CaM ?) Cytoplasm


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RNA-directed DNA methylation, double-stranded RNA and transcriptional gene silencingThe ability of viroids and RNA viruses, which produce

dsRNA replication intermediates, to trigger RdDM suggest-

ed a general requirement for dsRNA in this process. This has

been confirmed in a transgenic TGS system involving pro-moter RNAs. A double-stranded RNA transcribed from an

IR containing promoter sequences was able to trigger de novo

methylation and silencing of homologous promoters in trans

[47]. The promoter dsRNA was degraded to small RNAs

~23 nucleotides in length, indicating that it entered the same

degradation pathway as dsRNAs involved in PTGS. In a sep-

arate study, a cytoplasmic RNA virus vector carrying 35S

promoter sequences was able to induce methylation and

TGS of nuclear transgenes under the control of the 35S pro-

moter inNicotiana benthamiana [43]. dsRNA thus provides a

common molecular link between RdDM, which can lead to

TGS if promoter sequences are involved, and the RNA

degradation step of PTGS. Although additional studies are

needed to assess the generality of dsRNA-mediated promot-

er methylation, preliminary work suggests that at least some

endogenous plant promoters can be silenced by this method

(W Aufsatz, AJM Matzke, unpublished data).

Methylation and posttranscriptional genesilencingMethylation in either coding or transcribed regions of trans-

genes has been detected in many cases of PTGS in plants

[1]. The triggers for de novo methylation and whether

methylation is the cause, consequence, or unrelated to

PTGS are uncertain in most cases [30,42,43,48]. The rele-vance of methylation and whether it is induced by RdDM

or other signals probably differ for various PTGS systems.

Methylation is not required for RNAi in invertebrates that

do not methylate their DNA or for quelling in Neurospora,

which has a sparsely methylated genome [49]. In contrast,

PTGS in plants can be released when transgene methyla-

tion is reduced by drug treatment [50] or in methylation and

chromatin structure mutants ofArabidopsis (H Vaucheret,

personal communication). Moreover, transgene methylation

is reduced in plants mutant for RdRP [51], AGO-1 [19],

and SGS-3 [17] (Table 1). These findings suggest that the

establishment and/or maintenance of PTGS might require

DNA epigenetic modifications. Interestingly, despite thedispensability of methylation for quelling in Neurospora, a

nuclear step is suggested by the requirement for a RecQ

DNA helicase, QDE3 [5,14] (Table 1).

Mechanism of RNA-directed DNA methylationThe mechanism of RdDM is unknown but is assumed to

involve RNADNA interactions based on sequence homol-

ogy [8]. The minimal DNA target size for RdDM of 30 bp

opens the possibility that the 2125 nucleotide RNA degra-

dation products of dsRNA could be responsible for

directing de novo methylation. The small RNAs could con-

ceivably guide the DNA methyl transferase to unmodified

homologous DNA sequences, similar to what has been pro-

posed for the RNase involved in the mRNA-degradation

step of PTGS [10,28,29]. Conflicting results have been

obtained with HC-Pro, which suppresses PTGS by

preventing the accumulation of small RNAs. HC-Pro-

expressing plants either did ([43]; A Mallory, VB Vance,

unpublished data) or did not [24] maintain methylation of a

PTG-silenced transgene. Additional studies with HC-Prousing other PTGS systems and cases of RNA-mediated

TGS, in which the silenced phenotype is not reversed by

HC-Pro ([52]; MA Matzke, MF Mette, unpublished data),

will establish whether small RNAs are indeed essential for

the initiation and maintenance of RdDM or whether intact

dsRNA is involved.

Natural roles of RNA-based silencing: hostdefense and developmentRNA-based silencing mechanisms, which are effective at

the genome level and in the cytoplasm, are able to combat

parasitic sequences that have an RNA genome (RNA

viruses) or a dsRNA replication intermediate (some trans-

posable elements) [53]. The host defense function of

RNA-mediated silencing is demonstrated by the increased

sensitivity ofArabidopsis PTGS mutants to some viruses

[17] and the mobilization of transposons in RNAi

mutants of C. elegans [15,16] and in the Mut6 mutant of

Chlamydomonas [40]. Methylation, possibly triggered by

RdDM, is required to subdue at least some retrotrans-

posons inArabidopsis [54].

Apart from an enhanced susceptibility to viral infection,

Arabidopsis sgs/sde mutants appear normal [17,18]. In

contrast, Arabidopsis ago1 mutants exhibit marked devel-opmental abnormalities and are infertile [19]. Similarly,

expression of HC-Pro or overexpression of rgs-CaM causes

developmental aberrations in Nicotiana species [25]

(Table 1). In C. elegans, ego1 mutants are sterile as a result

of defects in germline development [20] (Table 1).

Therefore, although the PTGS pathway appears to be as a

whole dispensable for development, the phenotypic irreg-

ularities found in a subset of cases where silencing is

blocked suggest that PTGS/RNAi and development share

common enzymes or pathways [19].

Conclusions and outlook

The convergence on dsRNA as a molecular trigger in vari-ous types of HDGS is a departure from previous models,

which frequently invoked DNADNA pairing and posited

distinct mechanisms for PTGS and TGS. This strict sepa-

ration is becoming untenable as the involvement of

dsRNA and DNA methylation in both types of silencing is

increasingly recognized. Continued use of methylation and

chromatin structure mutants will clarify how epigenetic

modifications influence the initiation and maintenance of

PTGS and RNA-mediated TGS. The identification of

additional genes unique to plants, such as SGS3 [17] will

reveal plant-specific features of RNA-based silencing.

The generation of RdDM mutants in Arabidopsis and the

identification of endogenous DNA-target sequences will


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help to establish the mechanism and natural roles of this

process. RNA helicases are potential candidates for the

RdDM machinery as it is likely that RNADNA pairing

requires a single-stranded RNA that is complementary to

target DNA [46]. RdDM might provide the key for under-

standing viroid pathogenicity if plant genes that share30 bp or more of sequence homology with viroids are

found [46].

The potential for using viral suppressors of PTGS to piece

together silencing pathways and to identify cellular com-

ponents is just beginning to be realized. On the basis of the

patterns of suppression they produce, some viral suppres-

sors affect silencing differently from either HC-Pro or PVX

p25 and are likely to define additional steps in the PTGS

pathway. For example, the obligatory nuclear localization

of the cucumber mosaic virus 2b protein [55] should help

to identify steps of PTGS that occur in the nucleus.

Determining the extent to which PTGS and RdDM con-

tribute to normal plant development, and not just host

defense, is one of the most exciting prospects for the future.

The powerful tools provided by viral suppressors of silencing

and the steadily growing collection of silencing-defective

mutants promise a continuation of the rapid progress that has

become the norm in plant gene silencing research.

UpdateThe work referred to as H Vaucheret, personal communi-

cation, has now been published [56].

AcknowledgementsWe thank B Bass, D Baulcombe, J Carrington, H Cerutti, C Cogoni,H Vaucheret and P Waterhouse for sending preprints, H Vaucheret forcommunicating unpublished results, F Mette and W Aufsatz for commentson the manuscript, and T Smith for assistance with the figure. MM and AMacknowledge financial support from the Austrian Fonds zur Frderung derwissenschaftlichen Forschung (Z21-MED) and the European Union(Contract BIO4-CT96-0253). VV acknowledges financial support from theUSDA NRI Competitive Grants Program, Plant Pathology and GeneticMechanisms Panels, and from Akkadix Corporation, La Jolla, California.

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35. Dalmay T, Hamilton A, Mueller E, Baulcombe DC: Potato virus Xamplicons in Arabidopsisme diate genetic and epigenetic genesilencing. Plant Cell2000, 12 :369-379.

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37. Stam M, de Bruin R, van Blokland R, van der Hoorn RAL, Mol JNM,Kooter JM: Distinct features of post-transcriptional gene silencingby antisense transgenes in single copy and inverted T-DNA repeatloci. Plant J2000, 21 :27-42.

38. Van Houdt H, Van Montagu M, Depicker A: Both sense and antisenseRNAs are targets for the sense transgene -induced posttranscriptionalsilencing me chanisms. Mol Gen Genet2000,26 3:995-1002.

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decay in Caenorhabditis elegans. Science2000, 28 9:1928-1930.This study revealed an intriguing overlap between RNAi and RNA surveillancepathways. Mutants in six different smg genes required for nonsense-mediateddecay in C. eleganswere tested for the ability to carry out RNAi. None of the smg

functions were required to initiate RNAi but three were required for persistence ofthe silenced state (smg2, 5 and 6). Smg-2is homologous to yeast Upf1, encod-ing an adenosine triphosphatase with RNA-binding and helicase properties,whereas the required SMG-5 and SMG-6 proteins dephosphorylate SMG-2.

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DEAH-box RNA helicase. Science2000, 29 0 :1159-1163.Mut6encodes a DEAH-box RNA helicase whose activity is required for genesilencing in Chlamydomonas. Although it is not yet clear whether this sys-tem can target homologous sequences in trans in a PTGS/RNAi phenome-non or acts in cisonly, independent of homology, the detection of sense andantisense RNAs corresponding to the silenced transgene and to reactivatedtransposons supports classical PTGS.

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42. Jones AL, Thomas CL, Maule AJ: De novome thylation and co-suppression induced by a cytoplasmically replicating plant RNAvirus. EMBO J1998, 17 :6385-6393.

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45. Plissier T, Thalmair S, Kempe D Snger HL, Wassenegger M: Heavyde novome thylation at symm etrical and non-symmetrical sites isa hallmark of R NA-directed D NA m ethylation. Nucleic Acids Res1999, 27 :1625-1643.

46. Plisser T, Wassenegger M: A DNA target of 30 bp is sufficient for RNA-directed DNA me thylation. RNA 2000, 6 :55-65.Potato spindle tuber viroid subfragments of varying sizes were used to studylength requirements for RdDM as assayed by bisulfite-assisted genomicsequencing. Although the minimal DNA target size was determined to be ~30bp, dense methylation was only observed when the sequence complemen-tarity between inducing RNA and target DNA was 60 bp or longer. The deter-mination of the lower-length limit for RdDM defines an important parameter inthe search for possible endogenous plant DNA targets of viroid RNA.

47. Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJM: Transcriptional gene silencing and promoter methylation

triggered by double stranded RNA. EMBO J2000, 1 9 :5194-5201.Conversion in plantaof a direct DNA repeat of promoter sequences into an invert-ed repeat via Crelox-mediated recombination demonstrates that trans-silencingand de novomethylation of an unlinked target promoter occurred only upon initi-ation of synthesis of promoter dsRNA, confirming its involvement in RdDM. Thepromoter dsRNA was cleaved into small RNAs 2325 nucleotides in length, sim-ilar to those observed in PTGS, demonstrating that dsRNAs with similar fates cantrigger silencing at different levels in separate cellular compartments.

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depend on D NADN A interaction or DNA m ethylation. EMBO J1996, 1 5 :3153-3163.

50. Kovarik A, Van Houdt H, Hol A, Depicker A: Drug-inducedhypomethylation of a postranscriptionally silenced transgenelocus of tobacco leads to partial release of silencing. FEBS Lett2000, 4 67 :47-51.

51. Elmayan T, Balzergue S, Bon F, Bourdon V, Daubremet J, Gunet Y,Mourrain P, Palauqui JC, Vernhettes S, Vialle T et al.: Arabidopsismutants im paired in cosuppression. Plant Cell1998, 10:1447-1457.

52. Marathe R, Smith T, Anandalakshmi R, Bowman L, Fagard M, Mourrain P, Vaucheret H, Vance V: Plant viral suppressors of post-

transcriptional gene silencing do not suppress transcriptionalgene silencing. Plant J2000, 22:51-59.

HC-Pro, originally shown to suppress PTGS, is here tested for its ability tosuppress two different trans-TGS systems that are based on promotersequence homology. Trans-silencing of target genes driven by both the35S promoter and the nopaline synthase promoter was maintained in HC-Pro-expressing plants, demonstrating that TGS is not affected by this viralsuppressor of PTGS. It remains to be seen whether methylation ofTG-silenced target promoters is also affected in the presence of HC-Pro.



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53. Matzke MA, Mette MF, Matzke AJM: Transgene silencing by the hostgenome defense: implications for the evolution of epigenetic controlmechanisms in plants and vertebrates. Plant Mol Biol2000,43 :401-415.

54. Hirochika H, Okamoto H, Kakutani T: Silencing of retrotransposonsin Arabidopsisand rea ctivation by the ddm1 mutation. Plant Cell2000, 12 :357-368.

55. Lucy A, Guo H, Li W, Ding S: Suppression of posttranscriptionalgene silencing by a plant viral protein localized in the nucleus.EMBO J2000, 1 9 :1672-1680.

56. Morel J-B, Mourrain P, Bclin C, Vaucheret H: DNA m ethylation and chromatin structure affect transcriptional and post-transcriptional

transgene silencing in Arabidopsis. Curr Biol2000, 1 0 :1591-1594.Arabidopsismutants impaired in a SWI/SNF2 chromatin remodelling pro-tein (ddm1) or the major DNA methylation transferase (met1) were crossed

with transgenic lines containing 35S-GUS reporter genes silenced by TGSor PTGS. Both ddm1 and met1 mutations efficiently released TGS and sto-chastically released PTGS during development. These results show thatDNA methylation and chromatin structure are common regulators of TGSand PTGS.

Now in pressThe work referred to as A Mallory, VB Vance, unpublished data, is now inpress:

57. Mallory AC, Ely L, Smith TH, Marathe R, Anandalakshmi R,Fagard M, Vaucheret H, Pruss G, Bowman L, Vance VB: HC-Prosuppression of trans gene silencing eliminates the small RNAsbut not transgene m ethylation or the m obile silencing signal.Plant Cell2001, in press.

curren opin genet devel 2001

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