progression of geminivirus-induced transgene silencing is associated with transgene methylation

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Research

Blackwell Science, Ltd

Progression of geminivirus-induced transgene silencing is

associated with transgene methylation

Michele K. Rodman

1

, Narendra S. Yadav

1

and Nancy N. Artus

2

1

BCS & E, Central R & D, DuPont Experimental Station, Wilmington, DE 19880, USA;

2

Biology Department, West Chester University, West Chester, PA

19383, USA

Summary

• The association of viral-induced gene silencing (VIGS) elicited by a DNA virus withDNA methylation of the silenced transgene was studied.• 35S-Green fluorescent protein (GFP) transgenic

Nicotiana benthamiana

weretreated with an inhibitor of DNA methylation, 5-azacytidine (5-Aza-C), and VIGS ofthe transgene was observed upon inoculation with tomato golden mosaic viruscarrying the GFP coding sequence.• The onset of VIGS of the 35S-GFP transgene occurred 14–16 d after inoculationin both control and 5-Aza-C-treated plants. At this stage, the silencing was observedin localized regions. Silencing was uniform by 30 d after inoculation in plants thathad methylated GFP-DNA, whereas plants that continued to display the samephenotype as seen at 14–15 d after inoculation had hypomethylated GFP-DNA.Viral expression of GFP persisted in pockets throughout the life of infected plants.• This is the first demonstration of a correlation between post transcriptional genesilencing induced by a DNA virus, and transgene methylation. The results suggestthat, while DNA methylation is not necessary for the initiation of silencing, theprogression of silencing is affected by inhibition of DNA methylation.

Key words:

5-azacytidine, DNA methylation,


, post-transcriptional gene silencing, tomato golden mosaic virus, virus-induced genesilencing.

Abbreviations

5-Aza-C, 5-azacytidine; dsRNA, double-stranded RNA; GFP, green fluorescentprotein; GFPM, mutated form of GFP; PTGS, post transcriptional gene silencing;RdDM, RNA-directed DNA methylation; RdRP, RNA-dependent RNA polymerase;siRNA, small interfering RNA; TGMV, tomato golden mosaic virus; TGS, transcrip-tional gene silencing; VIGS, viral-induced gene silencing.

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Author for correspondence:

Nancy N. Artus

Tel: +1 610 436 3546

Fax: +1 610 436 2183

Email: [email protected]

Received:

6 February 2002

Accepted:

10 May 2002

Introduction

Gene silencing is characterized by a decrease in the steadystate levels of mRNA of a specific target gene. It is believed tohave evolved as a defense mechanism against foreign geneticelements, such as viruses, viroids and transposable elements(reviewed in Vance & Vaucheret, 2001). It can be eithertranscriptional gene silencing (TGS), if transcription of the

target gene is blocked, or post-transcriptional gene silencing(PTGS), if the target gene is transcribed but its mRNA isdegraded in a sequence-specific manner before it is translated.

In plants, PTGS can spread systemically and shares featureswith RNA interference in animals (Montgomery & Fire,1998) and quelling in fungi (Cogoni & Macino, 1999). Basedon the current model, silencing in all of these systems istriggered by the formation of double-stranded RNA (dsRNA)

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that is diced into 21–25 nucleotide long pieces called smallinterfering RNAs (siRNAs) (reviewed in Bass, 2000). ThesiRNAs act as sequence-specific guides that direct an RNasecomplex to degrade homologous target RNA (Bass, 2000;Vance & Vaucheret, 2001). The dsRNA trigger for PTGS canbe: (1) the dsRNA replication intermediate in the case of silen-cing induced by plant RNA viruses and viroids; (2) comple-mentary sense and antisense transcripts from inverted repeatsor from antisense transgene RNA interacting with endogenesense mRNA; or (3) the product of RNA-dependent RNApolymerase (RdRP) on ‘aberrant’ transcripts, the nature ofwhich is unknown (Vance & Vaucheret, 2001). In plants, virus-induced gene silencing (VIGS) is a PTGS event in whicha host’s target endogenous gene or transgene is silenced uponinfection by a plant virus carrying a sequence (even if it doesnot encode a functional protein) with homology to the targetgene. It can be induced by RNA viruses ( Jones

et al

., 1999;Pelissier

et al

., 1999; Wassenegger, 2000; Waterhouse

et al

.,2001) and DNA viruses (Atkinson

et al

., 1998; Kjemtrup

et al

., 1998; Peele

et al

., 2001; Turnage

et al

., 2002), and canspread sytemically into virus-free tissues ( Jones

et al

., 1999;Peele

et al

., 2001; Waterhouse

et al

., 2001).In plants, TGS is accompanied by hypermethylation of the

promoter region of silenced genes, while PTGS is often asso-ciated with hypermethylated DNA in the coding region. The

de novo

methylation of transgenes in PTGS is RNA-mediatedand referred to as RNA-directed DNA methylation (RdDM)(Matzke

et al

., 2001). It is unclear whether the signal forRdDM is aberrant RNA, dsRNA, or siRNA, although all casesof virus-induced RdDM reported to date involve RNA virusesand viroids that have a dsRNA replication intermediate.

The relationship between methylation and silencing istightly correlated in some cases (Ingelbrecht

et al

., 1994;English

et al

., 1996; Elmayan

et al

., 1998; Jones

et al

., 1998;Dalmay

et al

., 2000, 2001; Kovarik

et al

., 2000; Morel

et al

., 2000; Mourrain

et al

., 2000) but not in others(Goodwin

et al

., 1996; Scheid

et al

., 1998; Jones

et al

., 1999;Amedeo

et al

., 2000; Sonoda & Nishiguchi, 2000; Wang& Waterhouse, 2000; Mallory

et al

., 2001). For example,the use of the demethylation agent, 5-azacytidine (5-Aza-C),resulted in demethylation of two PTGS loci but PTGS wasreleased at only one of these (Kovarik

et al

., 2000; Wang &Waterhouse, 2000). Although methylation is associatedwith VIGS of transgenes, it has been shown to be absent inthe silencing of endogenous genes ( Jones

et al

., 1999; Jones

et al

., 2001). Furthermore, methylation

per se

is not requiredfor RNA interference in invertebrates or for quelling inNeurospora (Cogoni

et al

., 1996). Clearly, there may bemultiple mechanisms for gene silencing and they may not allrequire methylation.

Virus-induced gene silencing of transgenes induced byRNA viruses can be separated into a virus-dependent initi-ation stage and a virus-independent but transgene-dependentmaintenance stage, the latter of which is associated with

RdDM (Ruiz

et al

., 1998; Jones

et al

., 1999). The maintenancestage is highly dependent on a gene silencing signal that isable to move systemically in plants to virus-free tissues( Jones

et al

., 1999; Peele

et al

., 2001; Waterhouse

et al

.,2001). The silencing signal here is likely to be RNA andincreasing evidence suggests that it may be siRNAs (Hamilton& Baulcombe, 1999; Paszkowski & Whitham, 2001). Thereis no evidence that the signal for the systemic spread of PTGSis the same as for RdDM. Initiation and maintenance stageshave not been reported for DNA viruses. Although efficientVIGS can be elicited by geminiviruses (single-stranded plantDNA viruses) (Atkinson

et al

., 1998; Kjemtrup

et al

., 1998;Peele

et al

., 2001; Turnage

et al

., 2002), it is not known ifit is associated with DNA methylation, and, if so, whethermethylation is a cause, consequence, or an amplification stepof VIGS. In this study, we address these issues by observingVIGS of a 35S-green fluorescent protein (GFP) transgene in5-Aza-C-treated


plants upon infec-tion with tomato golden mosaic virus (TGMV), a bipartitegeminivirus, carrying GFP. Our findings show that TGMV-GFP silences the 35S-GFP transgene similar to RNA virusescarrying GFP and that geminivirus-induced VIGS of thetransgene is associated with transgene methylation, andsupport the notion that RdDM of the transgene is notrequired for the initiation of VIGS but is required for theprogression of VIGS to completion.

Materials and Methods

Plant material and 5-azacytidine treatment

Seeds of wild type

N. benthamiana

were obtained fromL. Hanley-Bowdoin, North Carolina State University, Raleigh,NC, USA. Homozygous transgenic 35S-GFP

N. benthamiana

line GFP8 (Ruiz

et al

., 1998) was kindly provided byD. Baulcombe ( John Innes Centre, Norwich, UK). Plantswere grown in an environmental chamber at 26

°

C under acombination of fluorescent and incandescent lights on a 12-hphotoperiod.

Surface-sterilized seeds were planted in magenta boxes onMurashige and Skoog salt and vitamin medium (Gibco BRLLife Technologies, Grand Island, NY, USA) supplementedwith 10% sucrose and variable concentrations of 5-Aza-C(0, 20, 50, 100, 150 and 200 µ

M

). In order to protect 5-Aza-Cfrom the light, 15 g l

−

1

of carbon was added to the medium.Magenta boxes were covered with a transparent plastic dometo accommodate the size of the growing plants. Plants weretransferred to fresh media after 40 days.

Tomato golden mosaic virus constructs

Plasmids carrying partial dimers of wild-type TGMV-Aand TGMV-B genomes were kindly provided by Dr D.Robertson, North Carolina State University, Raleigh, NC, USA.


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The TGMV-GFP expression construct was derived from theTGMV-A dimer by replacing 736 bp (nucleotides 177–912,with position 1 being the

Sst

I site in the conserved hairpin)of the AR1 (coat protein) coding sequence with a 737 bpGFP coding region obtained from R. Vierstra (University ofWisconsin, Madison, WI, USA) (Davis & Vierstra, 1998).The TGMV-GFPM (GFPM is a mutated form of GFP)expression construct was the same as the TGMV-GFPconstruct except for a mutation in the GFP coding region thatinactivated viral GFP protein expression. It was constructedby cutting TGMV-GFP with

Afl

II restriction enzyme, fillingin the 5

′

overhang with Klenow, and religating the plasmid.This created a four bp insertion that disrupted the GFPcoding region. Since TGMV-B is required for the viralmovement, TGMV-B dimer plasmid was coinoculated intoplants with TGMV-A-GFP dimer plasmid (referred to asTGMV-GFP inoculation) or with TGMV-A-GFPM dimerplasmid (referred to as TGMV-GFPM inoculation).

Inoculation of plants by particle bombardment

The BIOLISTIC Particle Delivery System (Bio-Rad, Hercules,CA, USA) was used to inoculate 45-d-old-plants (approxim-ately 8 cm) with the viral constructs. Sterility of the plantswas maintained throughout the inoculation process. DNAwas fixed to gold particles by incubating 3 mg ethanol-washed gold particles with 5 µg DNA, 1

M

CaCl

2

and 16 m

M

spermidine for 3 min, washing in 70% ethanol, and finallysonicating for 10 s in 100% ethanol. One microgram each ofgold-fixed TGMV-A carrying GFP or GFPM and TGMV-Bwas delivered per plant, following the manufacturer’s recom-mended protocol.

Green fluorescent protein imaging

Green fluorescent protein fluorescence was observed withthe use of a hand-held long-wave ultraviolet light (B-100APUV lamp; Ultra Violet Products Inc., Upland, CA, USA).Photographs were taken with a Kodak 315 ProfessionalDigital Camera.

DNA extraction and gel blot analysis

The DNA was extracted from frozen plant shoot (leaves andstem) tissue 30 d after inoculation (dai) using a phenol–chloroform plant DNA extraction protocol. In cases wheresilenced and nonsilenced sectors were both present on thesame plant, DNA extractions represent a mixture of silencedand nonsilenced tissues.

Analysis of DNA methylation status

Plant genomic DNA (1 µg) was digested overnight withthe methylation sensitive restriction enzyme,

Sau

96I. A

polymerase chain reaction was performed (adapted fromKumar, 2000) with 1 µl of the 20-µl digest, 47 µl of PCRSuperMix (Gibco BRL, Life Technologies) and 2 µl of for-ward and reverse primers at 100 pmol µl

−

1

. The reaction wascarried out as follows: 95

°

C for 60 s, 54

°

C for 60 s, 72

°

C for90 s (30 cycles) then 1 cycle at 72

°

C for 7 min.The sequences of polymerase chain reaction (PCR) primers

used in this study and shown in Fig. 1 are as follows:Primer 1: 5

′

-GAATCAAAGGCCATGGAGTCAAAG-3

′

Primer 2: 5

′

-GTCGGCAGAGGCATCTTCAACGA-3

′

Primer 3: 5

′

-AACTCGCCGTAACTGGCGAA-3

′

Primer 4: 5

′

-GCAAGTGGATTGATGTGATATC-3

′

Primer 5: 5

′

-TTCATCCATGCCATGTGTAATCCCAG-3

′

Primer 6: 5

′

-GAAGTTGAGAAATGCCTTGTG-3

′

Primer 7: 5

′

-GTTCCACGTCTCATCATTTACTAAC-3

′

Results

The 5-Aza-C treatment reduced VIGS in some plants

Seeds from 35S-GFP transgenic

N. benthamiana

plants weregerminated aseptically in the presence of 0, 20, 50, 100, 150and 200 µ

M

5-Aza-C to select the highest level that was notvisibly phytotoxic. Seedlings germinated on 20 µ

M

5-Aza-Cwere phenotypically indistinguishable from the untreatedcontrol, while those grown on either 50 or 100 µ

M

5-Aza-Cshowed no visible sign of plant toxicity, except for someinhibition of root growth (not shown). Seeds failed togerminate or died after a few days of germination on 150 or200 µ

M

5-Aza-C.To induce silencing of the 35S-GFP transgene, we inocu-

lated 45 d-old 35S-GFP transgenic plants that had beentreated with 0, 20, 50, and 100 µ

M

5-Aza-C with the TGMVcarrying either a functional GFP gene (TGMV-GFP) or amutant form of GFP (TGMV-GFPM). The mutant GFP wasmade (see the Materials and Methods section) to eliminateviral GFP expression and to ensure that only the 35S-GFPtransgene expression was observed during silencing. Over thenext 30 d, plants were observed for viral symptoms (primarilyleaf curling), silencing of the 35S-GFP transgene and viralGFP expression in the case of TGMV-GFP inoculation.

Fig. 1 Physical map of the 35S-green fluorescent protein (GFP) transgene in Nicotiana benthamiana line GFP8 showing the positions of Sau96I sites (arrows), polymerase chain reaction (PCR) primers 1–5, and the expected PCR product sizes.



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Geminivirus-induced VIGS of 35S-GFP transgene

At approximately 7 dai, TGMV viral symptoms were seenon all inoculated plants. Speckled viral GFP expressionwas apparent on all plants inoculated with TGMV-GFP. ViralGFP expression was easily distinguished from the transgeneGFP expression in that the viral GFP was brighter and hada speckled distribution (not shown). Because viral symptomsand the degree of viral GFP expression appeared similarbetween treated and untreated plants, it is unlikely that 5-Aza-C had a significant effect on viral replication and spread.

Viral symptoms peaked between 10 dai and 12 dai. At thispoint, no silencing of the 35S-GFP transgene was observed.By 14–16 dai, transgene GFP silencing became visible onall plants inoculated with both TGMV-GFP and TGMV-GFPM, regardless of 5-Aza-C treatment. The leaves displayeda patchy pattern, with 35S-GFP expression silenced in someareas and strong in others.

At 30 dai, 35S-GFP transgene expression had silenceduniformly throughout the leaves in all four control plants(two of which are shown in Fig. 2c,d), in all four plants treatedwith 20 µ

M

5-Aza-C (data not shown), in three out of fourplants treated with 50 µ

M

5-Aza-C (data not shown) and in 11of 14 plants treated with 100 µ

M

5-Aza-C (two of which areshown in Fig. 2e,h. The remaining plants – one treatedwith 50 µ

M

5-Aza-C (Fig. 2f ) and three treated with 100 µ

M

5-Aza-C (Fig. 2g, 2i,j) – exhibited a transgene silencingphenotype at 30 dai that was similar to that seen at 14 dai.Thus, 50 µ

M

and 100 µ

M

5-Aza-C concentrations wereeffective in suppressing the GFP-silencing phenotype in somebut not all treated plants.

As expected from a previous study with an RNA virus(Thomas

et al

., 2001), the nonfunctional viral GFP didnot interfere with transgene silencing (VIGS) and transgenesilencing induced by TGMV-GFP was identical to that forTGMV-GFPM (Fig. 2d compared to 2c).

Fig. 2 Green fluorescent protein (GFP) expression in Nicotiana benthamiana plants. (a) Untreated, non-inoculated wild type; (b) untreated, uninoculated 35S-GFP transgenic plant. (c–j) Inoculated 35S-GFP transgenic plants, 30 d after inoculation: (c) untreated, inoculated with tomato golden mosaic virus (TGMV)-GFP; (d) untreated, inoculated with TGMV-GFPM (GFPM, mutated form of GFP); (e) 100 µM 5-Aza-C-treated, inoculated with TGMV-GFP showing complete transgene silencing; (f) 50 µM 5-Aza-C-treated, inoculated with TGMV-GFP showing incomplete transgene silencing; (g) 100 µM 5-Aza-C-treated plant inoculated with TGMV-GFP showing incomplete transgene silencing; (h) 100 µM 5-Aza-C-treated, inoculated with TGMV-GFPM showing complete transgene silencing; (i,j) 100 µM 5-Aza-C-treated, inoculated with TGMV-GFPM showing incomplete GFP silencing.

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Inoculation of TGMV-GFP in 35S-GFP transgenic

N.benthamiana

plants resulted in viral GFP expression thatpersisted as small areas of green fluorescence throughout thelife of the plant (at least 3 months), even when the 35S-GFP trans-gene was completely silenced (Fig. 2c,e). Similar viral GFPexpression and its persistence were observed by TGMV-GFPinoculation into wild-type

N. benthamiana

plants (not shown).

Hypomethylation correlates with reduced transgene silencing

The methylation status of the 35S-GFP transgene wasdetermined by PCR on

Sau

96I-digested genomic DNAisolated from shoots of plants treated with 0 or 100 µ

M

5-Aza-C at 30 dai. Since

Sau

96I enzyme is sensitive to cytosinemethylation, the absence or the presence of the correct-sized PCR product indicated the lack or the presence ofmethylation, respectively, at the Sau96I sites in the transgene( Jones et al., 2001). A control PCR was performed on thedigested DNA to confirm complete Sau96I digestion usingprimers 1 and 2 (Fig. 1) that flank a Sau96I site in the 35Spromoter region. Since the promoter region is not methylatedin PTGS (Wassenegger, 2000), absence of the 331 bp PCRproduct confirmed complete digestion of the DNA. Oncecomplete Sau96I digestion was confirmed, PCR was per-

formed on the digested genomic DNAs to determine themethylation status of 35S-GFP transgene. For this, primers 4,corresponding to the 3′ end of the 35S promoter, and primer5, corresponding to the 3′ end of the GFP coding sequence,were used. Because TGMV is a DNA virus, it was necessarythat primer 4 be located in the 35S promoter region to ensurethat the amplified PCR product was from the plant’s GFPtransgene and not from the viral GFP coding region. A851 bp PCR product (Fig. 1) resulted when the Sau96I sitesin the coding region of the transgene were methylated. ThePCR results are shown in Fig. 3a. Lack of methylation at oneor more of the Sau96I sites in the GFP coding sequencewas indicated by the lack of the 851 bp PCR product inDNA samples from non-inoculated control plants that wereeither treated with 100 µM 5-Aza-C (Fig. 3a, plant no. 2) oruntreated (Fig. 3a, plant no. 1) with 5-Aza-C. These plantsserved as negative controls.

Methylation of the GFP coding sequence was indicated bythe presence of the 851 bp PCR product in samples from fullysilenced plants that were either untreated with 5-Aza-C andinoculated with TGMV-GFP (Fig. 3a, plant nos. 9–12) orTGMV-GFPM (Fig. 3a, plant nos. 19–21) or treated with100 µM 5-Aza-C and inoculated with TGMV-GFP (Fig. 3a,plant nos. 3 and 6–8) or TGMV-GFPM (Fig. 3a, plant nos.13, and 15–17).

Fig. 3 Ethidium bromide-stained agarose gel of polymerase chain reaction (PCR) products demonstrating the methylation status of the 35S-green fluorescent protein (GFP) transgene in Sau96I-digested genomic DNA from wild-type (w1 and w2) and 35S-GFP transgenic (nos. 1–22) Nicotiana benthamiana plants. 5-Aza-C treatment, inoculation by tomato golden mosaic virus (TGMV)-GFP or TGMV-GFPM (GFPM, mutated form of GFP), and post-transcriptional gene silencing (PTGS) of transgene are indicated by +. The corresponding plants shown in Fig. 2 are indicated by letters. Plant numbers in red indicate plants with incomplete transgene silencing. (a) Presence of an 851 bp PCR product using PCR primers 4 and 5 is indicative of transgene methylation. (b) Presence of a 285 bp PCR product using PCR primers 2 and 3 on selected plants to confirm genomic DNA quality.


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Little or no methylation of the GFP coding sequence wasindicated by the absence or near absence of the 851 bp PCRproduct in samples from four plants that were only partlysilenced upon treatment with 50 µM or 100 µM 5-Aza-C andinfected with TGMV-GFP (Fig. 3a, plant nos. 4 and 5) orTGMV-GFPM (Fig. 3a, plant nos. 14 and 18). The qualityof the digested DNA in these plants was confirmed by PCRusing primers 2 and 3 that flanked a region in the 35S promoterregion that did not contain a Sau96I site (Figs 1 and 3b).To confirm that these partly silenced plants were all infectedwith the viruses, a PCR reaction was performed using primers6 and 7 that were located in the AL1 coding region ofTGMV. The presence of the expected 778 bp PCR productconfirmed that all four of the plants were inoculated withTGMV (Fig. 4).

To summarize the silencing and methylation results, when5-Aza-C treatment inhibited methylation of the transgenecoding region, viral-induced silencing was incomplete at 30 dai.In these plants, the silencing pattern resembled the silencingpattern seen at 14–16 dai and silencing did not progressfurther. When methylation of 35S-GFP transgene occurredin the inoculated plants, the plants were completely anduniformly silenced by 30 dai. This included untreated controlsas well as 11 of 14 plants treated with 100 µM 5-Aza-C.

Discussion

Geminivirus-induced VIGS is associated with transgene methylation

We have shown that inoculation of 35S-GFP transgenicN. benthamiana by TGMV-GFP initiates silencing of thetransgene by 14–16 dai and completes silencing by 30 dai.This timing of silencing is similar to that induced by potatovirus X carrying GFP ( Jones et al., 1998; M. K. Rodmanet al., unpublished).

Although geminiviruses were previously reported tosilence host endogenous and transgenes, the methylationstatus of the silenced genes was not reported (Atkinson et al.,1998; Kjemtrup et al., 1998; Peele et al., 2001). This is thefirst report correlating hypermethylation with VIGS by ageminivirus. Thus far, RdRM has been associated withreplicating RNA viruses and viroids, and transcribed invertedrepeats that all involve a dsRNA. Since geminivirusesreplicate through a dsDNA intermediate, this implies thateither the dsRNA replication intermediate is not essentialfor VIGS and RdRM or dsRNA is made during geminivirusreplication.

In 35S-GFP transgenic plants infected with TGMV-GFP,the viral GFP was clearly distinguished from the host GFPby its brightness and pattern of expression. Viral GFP expres-sion is similar in TGMV-GFP-inoculated wild-type (data notshown) and 35S-GFP transgenic plants. In both cases, viralGFP expression persisted in small patches throughout thelife of the plants (i.e. even when the 35S-GFP transgene iscompletely silenced in the background). Thus, TGMV-GFPexpression is different from that of potato virus X-GFP, whichsilences completely with the 35S-GFP transgene ( Jones et al.,1998; M. K. Rodman et al., unpublished). It is possible thatthe ability of TGMV-GFP to suppress/escape gene silencingin pockets is related to the presence in TGMV genomesA and/or B of a silencing suppressor. Tomato golden mosaicvirus-A encodes AL2, a homolog of AC2 that is a silencingsuppressor protein in another geminivirus, African cassavamosaic virus (Voinnet et al., 1999).

Role of transgene methylation in progression of VIGS

Although DNA methylation is associated with VIGS oftransgenes, it was unclear if it is a cause or a consequenceof VIGS, or if it is independent of VIGS. We have usedVIGS in 5-Aza-C-treated transgenic N. benthamianaplants to study the relationship between VIGS and de novoDNA methylation. Since 5-Aza-C treatment did not affectviral symptoms, viral replication by PCR (Fig. 4), viralGFP expression (in the case of TGMV-GFP) or the onsetof gene silencing at 14–16 dai, it is unlikely that 5-Aza-Ctreatment had a significant effect on viral replication andspread.

Of the concentrations of 5-Aza-C used in this study,100 µM was the highest concentration that was not obviouslyphytotoxic. The effectiveness of 50 µM and 100 µM 5-Aza-Cconcentrations in inhibiting transgene methylation varied. Itis likely that this is due to the biological variation in the uptakeand/or translocation of 5-Aza-C in different plants or to itschemical instability. To be able to comment with confidenceon the efficacy of the 5-Aza-C treatments, an examinationof global methylation by southern analysis of genomic DNAusing a methylation-sensitive restriction enzyme would berequired. This was not done in this study because growing the

Fig. 4 Ethidium bromide-stained agarose gel showing polymerase chain reaction (PCR) products to detect tomato golden mosaic virus (TGMV) infection in leaves of 35S-GFP transgenic Nicotiana benthamiana plants showing incomplete transgene silencing upon inoculated with TGMV-green fluorescent protein (GFP) (Fig. 3, plants 4 and 5) or TGMV-GFPM (GFPM, mutated form of GFP) (Fig. 3, plants 14 and 18) or wild type N. benthamiana inoculated with TGMV-GFP (Fig. 3, plant w1). A 778 bp PCR product using PCR primers 6 and 7 is indicative of the presence of TGMV virus.


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plants under aseptic conditions in a confined environmentrestricted plant growth and hence the yields of DNAobtained. Instead, we used a PCR assay ( Jones et al., 2001) ongenomic DNA treated with the methylation restrictionenzyme, Sau96I, to determine the methylation status of theGFP transgene. Although the PCR method used to detectmethylation was not quantitative, the results show that GFPsilencing in plants with reduced methylation of GFP DNA didnot progress to completion as seen in plants with methylatedDNA (Figs 2 and 3). Thus, although the effectiveness of5-Aza-C varied, there was a consistent correlation betweeninhibition of 35S-GFP transgene methylation at the Sau96Isites and incomplete DNA virus-induced transgene silencing.These results suggest a methylation-dependent progression oftransgene silencing. However, the possibility that methylationoccurred as a consequence of silencing rather than as a causecannot be ruled out since it is possible that 5-Aza-C had anunanticipated effect on the progression of silencing.

Plants show different types of methylation: they methylatecytosines at CG symmetric sites, as in animals, and alsouniquely at symmetric CNG and asymmetric (non-CG) sites(Paszkowski & Whitham, 2001). Plants have a variety ofDNA methyltransferase enzymes (Paszkowski & Whitham,2001) and it is possible that at least some of them are respon-sible for the different types of methylation. For example,the methyltransferase encoded by the Arabidopsis MET1 geneencodes a methylase transferase for CG methylation. Thereis also evidence that different types of methylation contributeto different types of silencing. For example, while TGS wasreleased in the met1 mutant, suggesting that symmetricmethylation plays a role in TGS, RNA virus-induced VIGSinvolved only asymmetric methylations ( Jones et al., 2001).In the current study, all of the methylation sites analysed inthe GFP coding region were asymmetric. Hypomethylationof these sites prevented the progression of PTGS to the main-tenance stage, consistent with the suggestion that asymmetricmethylations are important in VIGS induced by both RNAand DNA viruses.

In conclusion, our results indicate that DNA methylationof the coding region of a transgene occurs when VIGS isinduced by the TGMV geminivirus carrying a GFP sequence.Furthermore, it appears that methylation is not necessary forthe initiation of silencing, but may be a necessary step for theprogression of VIGS to completion.

Acknowledgements

We thank Dr David Baulcombe and colleagues, SainsburyLaboratory, Norwich Research Park, Colney, Norwich, UK,and the Gatsby Charitable Foundation for providing uswith the seeds of 35S-GFP transgenic N. benthamiana andDr D. Robertson, North Carolina State University, USA, forplasmid clones of the wild-type TGMV-A and TGMV-Bgenomes.

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