ORIGINAL PAPER

Transgene silencing in grapevines transformed with GFLVresistance genes: analysis of variable expressionof transgene, siRNAs production and cytosine methylation

Giorgio Gambino Æ Irene Perrone Æ Andrea Carra ÆWalter Chitarra Æ Paolo Boccacci Æ Daniela Torello Marinoni ÆMarco Barberis Æ Fatemeh Maghuly Æ Margit Laimer ÆIvana Gribaudo

Received: 30 October 2008 / Accepted: 26 May 2009

� Springer Science+Business Media B.V. 2009

Abstract Eight transgenic grapevine lines trans-

formed with the coat protein gene of Grapevine fanleaf

virus (GFLV-CP) were analyzed for a correlation

between transgene expression, siRNAs production and

DNA methylation. Bisulphite genome sequencing was

used for a comprehensive analysis of DNA methylation.

Methylated cytosine residues of CpG and CpNpG sites

were detected in the GFLV-CP transgene, in the T7

terminator and in the 35S promoter of three grapevines

without transgene expression, but no detectable level of

siRNAs was recorded in these lines. The detailed

analysis of 8 lines revealed the complex arrangements

of T-DNA and integrated binary vector sequences as

crucial factors that influence transgene expression.

After inoculation with GFLV, no change in the levels

of cytosine methylation was observed, but transgenic

and untransformed plants produced short siRNAs (21–

22 nt) indicating that the grapevine plants responded to

GFLV infection by activating a post-transcriptional

gene silencing mechanism.

Keywords Bisulphite genomic sequencing �Coat protein � DNA methylation �Transgene expression stability � Vitis

Introduction

RNA silencing, a process leading to the degradation

of homologous mRNAs, has been widely observed in

animals, plants, and fungi (Hannon 2002). A key

feature of RNA silencing is the presence of small

RNAs such as micro RNAs (miRNAs) and small

interfering RNAs (siRNAs) that are processed from

double-stranded RNA (dsRNA) by a family of the

RNase III-like enzyme known as DICER (Vaucheret

2006). RNA silencing is an important mechanism

used by plants to defend themselves against viral

infection. In this process the dsRNA are originated

during viral replication and/or from internal pairing

Electronic supplementary material The online version ofthis article (doi:10.1007/s11248-009-9289-5) containssupplementary material, which is available to authorized users.

G. Gambino (&) � W. Chitarra � P. Boccacci �I. Gribaudo

Plant Virology Institute CNR (IVV), Grugliasco Unit. Via

L. da Vinci 44, 10095 Grugliasco-TO, Italy

e-mail: g.gambino@ivv.cnr.it

I. Perrone � A. Carra � W. Chitarra � D. Torello Marinoni

Department of Arboriculture and Pomology, University of

Torino, Via L. da Vinci 44, 10095 Grugliasco-TO, Italy

F. Maghuly � M. Laimer

Plant Biotechnology Unit, Institute of Applied

Microbiology BOKU, Nussdorfer Lande 11, 1190 Vienna,

Austria

M. Barberis

S.C.D.U. Medical Genetics, A.O.U. San Giovanni

Battista, Corso Bramante 88/90, 10126 Turin, Italy

123

Transgenic Res

DOI 10.1007/s11248-009-9289-5

of long RNA molecules (Vaucheret 2006). In addi-

tion, it has been suggested that host-encoded RNA-

dependent RNA polymerases also assist in battling

viral infection by using the viral genome as a

template for the production of dsRNA, which are

then diced to siRNAs (Xie et al. 2001). Virus- and

transgene-derived siRNAs accumulate in two distinct

size classes of 21 and 24 nt in plants (Hamilton et al.

2002). The 21 nt siRNAs are sufficient to guide the

cleavage of target RNA mediated by RNA-inducd

silencing complex (RISC), while the 24 nt species are

probably involved in chromatin modifications (Ham-

ilton et al. 2002; Fusaro et al. 2006). RNA silencing

also involves RNA-directed DNA methylation

(RdDM), in which DNA homologous to a dsRNA

that triggers gene silencing is methylated de novo

(Mette et al. 2000). RdDM has been documented in

many plant systems, and requires the enzyme know

as Domains Rearranged Methylase which could be

guided by siRNAs to the target sequences (Cao et al.

2003). In mammals, DNA methylation occurs almost

exclusively on cytosines in the symmetric dinucleo-

tides CpG. In plants, cytosine methylation occurs at

both symmetric sites (CpG and CpNpG, where N is

A, T or C) and at asymmetric sites (CpNpN). In

plants, several distinct RNA silencing pathways

operate to repress gene expression at the transcrip-

tional or post-transcriptional level (Baulcombe 2004).

In both cases silencing is generally associated with

siRNAs and/or DNA methylation: siRNAs homolo-

gous to the promoter region of a target gene induces

transcriptional gene silencing (TGS), which is asso-

ciated with promoter methylation. siRNAs homolo-

gous to the coding region of the target gene induces

post-transcriptional gene silencing (PTGS), which

involves mRNA specific degradation in the cyto-

plasm and, in some case, methylation of the coding

sequences (Baulcombe 2004).

Grapevine fanleaf virus (GFLV) is the main causal

agent of grapevine fanleaf disease, one of the most

damaging and widespread viral diseases affecting

grapevine (Vitis spp.). GFLV is spread through

propagating material and the nematode vector Xiph-

inema index. Control of viral vectors in the vineyards

either remains inefficient or is being restricted

because of the detrimental effects of the pesticides

on the environment (Andret-Link et al. 2004).

Therefore the approaches to engineer resistance in

grapevine would be desirable. Resistance to GFLV in

a few lines of transgenic rootstocks expressing the

coat protein gene has been reported after a three-year

trial in a naturally infected vineyard in France (Vigne

et al. 2004).

In grapevines transformed with the coat protein of

GFLV (GFLV-CP) obtained previously (Gambino

et al. 2005) we did not observe a strict correlation

between number of T-DNA insertion and mRNA

accumulation levels. Moreover, in these lines multi-

copy transgene insertions with repeated sequences

and integrated binary vector sequences in complex

arrangements were observed (Gambino et al. 2009).

In these transgenic grapevines, bisulphite genome

sequencing was employed for a comprehensive

analysis of DNA methylation on lines grown under

in vivo and in vitro conditions. Besides, the correla-

tions among transgene expression, siRNA produc-

tion and DNA methylation were analyzed in three

healthy transgenic grapevines and in the same

inoculated with GFLV through grafting onto infected

rootstocks.

Materials and methods

Plant material

Transgenic plants of grapevine (Vitis spp.) were

regenerated from embryogenic calli after co-cultivation

with Agrobacterium tumefaciens LBA4404 carrying a

binary Ti vector pGA643 (An et al. 1988) with

sequences of the GFLV-CP gene in sense and antisense

orientation (Golles et al. 1998) as described previously

(Gambino et al. 2005). Transgenic lines were obtained

from V. vinifera cv Nebbiolo (NE) and Blaufrankisch

(BF) and from V. berlandieri 9 V. rupestris 110 Richter

(110R). Eight lines were chosen for the analyses as

follow: NE-A and NE-C, a transgene multicopy and a

single-copy line respectively, without vector backbone

sequences integrated; NE-E having a single-copy of

T-DNA truncated; NE-B and NE-D, two multicopy

lines with vector backbone sequences integrated; NE-F,

a multicopy line with cytosine methylation at GFLV-CP

sequence; BF-A and 110R, two multicopy lines trans-

formed with GFLV-CP gene in antisense orientation

and with vector backbone sequences integrated (Gam-

bino et al. 2005, 2009).

Transgenic lines were micropropagated in vitro for

6 years (2002–2007), by repeatedly subculturing

Transgenic Res

123

apical cuttings (3–4 cm long) on a modified Murash-

ige and Skoog (1962) medium with half strength

mineral salts and 20 g l-1 sucrose without plant

growth regulators.

In 2004 some plants of each transgenic line were

acclimatized and transferred to a contained green-

house under natural daylight conditions; scions from

these plants were wedge-grafted in 2006 onto non-

transgenic rootstocks Kober 5BB (V. berlandi-

eri 9 V. riparia) infected by GFLV. For each line

10 scions were grafted onto the infected root-

stocks and grown in the greenhouse. Untransformed

V. vinifera cv Nebbiolo plants were used as controls.

Serological assays

The reaction of the plants to GFLV infection was

investigated by visually scoring symptom develop-

ment and by serological assays. Samples of young

apical leaves from transgenic and non transgenic

scions were collected from each tested plant in June

and September for 2 years after grafting. Samples of

phloem were scraped from mature canes collected

during winter from the same tested plants. Double

antibody sandwich enzyme-linked immunosorbent

assay (DAS-ELISA) was carried out using polyclonal

antisera to GFLV purchased from Agritest (Valenz-

ano, Italy). Samples were regarded as positive if their

OD405 value was at least three times the negative

control value.

Northern blot analysis

Total RNA was extracted from micropropagated

grapevine plantlets and from young apical leaves

collected from plants grown under greenhouse con-

ditions following the protocol of Gambino et al.

(2008). Total RNA (10 lg) was separated by elec-

trophoresis on a 1.2% denaturing formaldehyde-

agarose gel and capillary-blotted in 209 SSC (3 M

NaCl, 300 mM sodium citrate) onto a nylon mem-

brane positively charged (Roche). The GFLV-CP

gene, used as probe, was amplified by PCR using the

primers indicated by Gambino et al. (2005) and

digoxigenin labeled with a PCR Dig Probe Synthe-

sis Kit (Roche) according to the manufacturer’s

instructions. Labeled probe (20 ng/ml) was added

to the DIG easy Hyb (Roche) and the hybridization

was carried out overnight at 50�C. Following

hybridization, the membranes were incubated in the

CSPD chemiluminescent substrate following the

Roche protocol and exposed to Kodak� BioMaxTM

light film (Sigma). After autoradiographs the blots

were stripped (according to the Roche protocol) and

reprobed with neomycin phosphotransferase II (nptII)

(Supplemental Table 1) and 18S rDNA probes

(Gambino et al. 2008).

Bisulphite genomic sequencing

DNA was extracted using the method of Thomas et al.

(1993) from all grapevine lines grown in vitro, and

from young apical leaves collected from healthy

plants of lines NE-B, NE-C, NE-D, NE-F and 110R,

and from GFLV-infected transgenic plants of lines

NE-C, NE-F and 110R grown under greenhouse

conditions. To investigate the methylation status of

transgene, 35S promoter and T7 terminator sequences,

the bisulphite genomic sequencing technique was

used. Bisulphite treatment allows the conversion of all

cytosines to uracils, except those that are methylated

at the carbon-5 position. Since after the bisulphite

analyses the two strands of DNA are no longer

complementary, single strands can be amplified and

sequenced. The modified cytosines appear as thymi-

dines, whereas, methylated cytosines remain uncon-

verted. Bisulphite modification and desulfonation of

approximately 2 lg genomic DNA were performed

using the EpiTect Bisulfite kit (Qiagen) according to

the manufacturer’s instructions. In addition, to con-

firm that bisulphite mutagenesis was complete a

fragment of the GFLV movement protein (GFLV-

MP) was amplified by RT-PCR (Gambino et al. 2008)

with the primers reported in Supplemental Table 2

and cloned into the pGEM-T Easy vector (Promega)

according to the manufacturer’s instructions. In all

samples 50 pg of plasmid digested with SacII

(Promega) were mixed with grapevine genomic

DNA and subjected to bisulphite modification.

For bisulphite-PCR analysis of methylation differ-

ent primer pairs were used to amplify three fragments

of the sense strand of 35S promoter, GFLV-CP and

T7 terminator sequence (Fig. 1 ; Supplemental

Table 2). A mixture of primers with different nucle-

otides [either cytosine or thymine (Y) for the forward

primers, and adenine or guanine (R) for the reverse

primers], at positions corresponding to cytosine

residues in the target sequence, were used to cover

Transgenic Res

123

the possible states of methylation of cytosines,

including those bases that had not been converted

to uracils through the bisulphite reaction. Primers

designed using the software Primer 3 (Rozen and

Skaletsky 2000; http://frodo.wi.mit.edu/cgi-bin/primer3/

primer3_www.cgi) are listed in Supplemental Table 2.

The PCR reaction mix (20 ll) contained 200 ng of

bisulphite-treated DNA, 0.2 mM dNTPs, 2 lM each

primer, 2 mM MgSO4 and 0.5 unit Taq polymerase

(AccuPrimeTM Taq DNA Polymerase High Fidelity,

Invitrogen Life Technologies).

Cycling conditions for all primer pairs consisted of

initial denaturation at 94�C for 30 s, followed by 35

cycles at 94�C for 30 s, 50�C for 30 s and 68�C for

2 min. Reaction products were analyzed by electro-

phoresis on 1% agarose gels buffered in TBE

(45 mM Tris–borate, 1 mM EDTA) and visualised

by UV-light after staining with ethidium bromide.

Amplified PCR products were gel purified by Wiz-

ard� SV Gel and PCR Clean-Up System (Promega)

and cloned into the pGEM-T Easy vector (Promega).

The plasmid DNA was isolated by Wizard Plus SV

Minipreps DNA Purification Systems (Promega)

following the Promega protocol and sequenced using

M13 forward and reverse primers.

Five independent clones from each PCR product

were sequenced using a Big-Dye Terminator v1.1

Cycle Sequencing kit (Applied Biosystems) follow-

ing the manufacturer’s instructions. PCR products

were purified using an AutoSeq G-50 Dye Terminator

Removal kit (GE Healthcare) and analysed using a

3130 Genetic Analyzer capillary sequencer (Applied

Biosystems).

The sequences of individual clones were aligned

with the original references using the ClustalW

program 1.83 (Chenna et al. 2003; http://www.ebi.ac.uk/

c110Rstalw/). These alignment data were processed

by CyMATE program (Cytosine Methylation Analy-

sis Tool for Everyone; Hetzl et al. 2007; http://www.

gmi.oeaw.ac.at/CyMATE) for in silico analysis of

DNA sequences after bisulphite conversion of plant

DNA. The program allows to distinguish methylation

at CpG, CpNpG and CpNpN sites.

siRNAs detection

Small RNAs were extracted from young apical leaves

collected from healthy and GFLV-infected transgenic

plants (NE-C, NE-F and 110R) grown under green-

house conditions, following the protocol of Carra

Fig. 1 Schematic

representation of GFLV-CP

gene in a sense (GFLV-CP)

and b antisense (GFLV-AS)

orientation. Arrows indicate

direction of primers used

for bisulphite-PCR analysis

of methylation. The three

primer pairs were used to

amplify in three fragments

the filament sense of

transgene. Primers

sequences and locations

within the plasmid pGA643

and the GFLV-CP gene are

reported in Supplemental

Table 2. P 35S: CaMV 35S

promoter; T7:

Agrobacterium gene 7

transcription termination

region

Transgenic Res

123

et al. (2007). Low molecular weight RNA (3 lg per

lane) was separated on a 15% (v/w) polyacrylamide

gel containing 8 M urea. Gels were stained with

ethidium bromide and the RNA was transferred to

nylon membranes (Roche) with a Trans-blot semi-dry

transfer cell (Bio-Rad) at 2.5 mA/cm2 for 30 min.

RNA was cross-linked by exposing each side of the

membranes for 2.5 min to UV on a transilluminator,

followed by baking at 120�C for 30 min. Membranes

were analyzed both with 32P-a-UTP labelled probes

following the protocol of Szittya et al. (2002) and

with digoxigenin-labelled (DIG-dUTP) probes. For

DIG hybridization the membranes were prehybrid-

ized with PerfectHybTM Plus (Sigma) and hybridized

with DNA probe at 50�C overnight. Filters were

washed twice with 29 SSC-0.2% SDS and twice with

19 SSC-0.1% SDS at 50�C. The membranes were

then incubated in the CSPD chemiluminescent sub-

strate following the Roche protocol and exposed to

Kodak� BioMaxTM light film (Sigma). After autora-

diographs the blots were stripped (according to the

Roche protocol) and reprobed. Two regions of the

GFLV-CP gene were used as probes for this study: 50

region (50CP) and 30 region (30CP), amplified with the

primers reported in Supplemental Table 1.

Results

Transgene expression

The expression levels of the GFLV-CP gene were

studied in transgenic lines by Northern blot on in

vitro and in vivo plant material. The Northern assays

carried out on 6-year-old in vitro cultures showed a

stable GFLV-CP expression in 3/8 analyzed trans-

genic grapevines (Table 1; Fig. 2): high in NE-A,

slightly lower in NE-C and NE-B, while the mRNA

transcript was not detected in NE-E and 110R as well

as in the untransformed control plant. In three lines

(NE-D, NE-F and BF-A) a reduction of transgene

expression after the long-term in vitro culture was

observed, as transcripts of the GFLV-CP gene were

not detected by Northern hybridization. Previously a

low signal in Northern blot was detected for lines

NE-F and BF-A, while the GFLV-CP expression in

line NE-D was high with a hybridization signal

similar to that observed in NE-A (Table 1; Fig. 2)

(Gambino et al. 2005). In grapevines transferred to

the greenhouse the expression analyses were carried

out on young apical leaves (1-4 from the top)

collected in spring. The transgene expression in

plants grown in vivo conditions (Table 1; Fig. 2) is

not substantially different from that observed in the

same lines cultivated in vitro, except for NE-B, where

a slight reduction of GFLV-CP expression was

observed.

The gene for nptII, that was transferred with

GFLV-CP gene for selection of transgenic plants,

showed a high and stable expression in all grape-

vines, both in vitro and in vivo, including the lines

NE-D, NE-E, NE-F, BF-A and 110R that showed

GFLV-CP silencing (Table 1; Fig. 2).

Methylation status of the transgene

in grapevine plants

The methylation status of transgene, 35S promoter

and T7 terminator sequences were analyzed by

bisulphite genomic sequencing technique. In the

GFLV-MP gene used as unmethylated control of

the reaction, a high conversion rate of cytosines to

thymidines was obtained: the percentage of methyl-

ation varied from 0 to 1.1% for all samples. The

correlation between transgene expression and meth-

ylation was analyzed in all the 8 lines cultivated in

vitro (Table 1; Fig. 3). Comparisons of the methyl-

ation status in the non-methylated line NE-C and in

the highly methylated lines 110R for the 35S

promoter are reported in Supplemental Fig. 1. The

transgenic grapevine lines in which GFLV-CP tran-

script was not detected (NE-D, NE-F and 110R)

consistently exhibited cytosine methylation at the

CpG and CpNpG sites (Fig. 3) which were distrib-

uted in the GFLV-CP gene and in the T7 terminator

sequence. Cytosine methylation in asymmetrical

sequences (CpNpN) was also observed throughout

these regions, but at a lower frequency.

Furthermore, in lines NE-D and 110R high

frequencies of methylation were observed in sym-

metrical sites within the 35S promoter sequence

(Fig. 3), while in the asymmetrical cytosines the

methylation was observed at some residues of both

lines (Supplemental Fig. 1). 50-CTA-30 and 50-CAA-

30 cytosine sites had a much more prominent

tendency for methylation than other types of asym-

metrical cytosine sequence: cytosines at the positions

-76 (from the transcription start site) adjacent to

Transgenic Res

123

TATA-box, -315, -331 and -429 were preferentially

methylated. In addition the cytosine at -206, a central

residue of eight consecutive asymmetrical cytosines

containing one adenine gap, was preferentially

methylated, whereas, the other seven cytosines were

rarely methylated (Supplemental Fig. 1). The ten-

dency to cytosine methylation in the asymmetrical

sites 50-CTA-30 and 50-CAA-30 was also observed in

the T7 terminator sequences, while in the GFLV-CP

sequence these sites did not exhibit a significant

methylation compared to other asymmetrical sites.

The GFLV-CP sequence in lines NE-D, NE-F and

110R was divided in two regions of 800 bp at 50 and

30 of the gene and the two regions were analyzed

separately. A slight increase of methylation was

observed in the symmetrical context at 30 half of the

GFLV-CP sequence in lines NE-F and 110R, while

the opposite occurred in line NE-D (Supplemental

Fig. 2). However, these variations in the methylation

percentages between the 50 and 30 regions of GFLV-

CP are limited, and in general the methylated

cytosines seem to be distributed quite uniformly in

the sequence.

In the line NE-E, that has a single copy of T-DNA

truncated with a partial deletion of 30 region of

GFLV-CP gene (Gambino et al. 2009), very few

cytosine methylations were observed. In line BF-A a

limited methylation was observed only in the 35S

promoter and in the GFLV-CP sequence, where about

8% and 4% of cytosines were methylated, respec-

tively (Table 1). In non-silenced grapevines NE-A,

NE-B and NE-C hardly any cytosine methylation was

observed in the transgenic sequences (Fig. 3).

In two lines with high expression levels (NE-B and

NE-C) and in three without GFLV-CP expression

(NE-D, NE-F and 110R) a comparison of methylation

Table 1 Molecular characterization, analyses of transgenes expression and cytosine methylation of eight transgenic grapevine lines

Line T-DNA copy

numberaVBSb TR/IRc GFLV-CP expression nptII

expressioneCytosine methylation (%)

2004d, e In vitroe In vivoe 35S promoter GFLV-CP T7 terminator

NE-A 3 - - ??? ??? ??? ??? 1.4 2.1 1.8

NE-B 2 ? ? ?? ?? ? ??? 1.5 2.2 1.4

NE-C 1 - - ?? ?? ?? ??? 2.2 0.7 1.1

NE-D 3 ? ? ??? - - ??? 18.6 34.6 41.7

NE-E 1 - - - - - ??? 1 2.1 -f

NE-F 3 ? ? ? - - ??? 10.7 39.4 50.6

BF-A 1 ? ? ? - - ??? 7.8 4.1 2.8

110R 4 ? ? - - - ??? 34.5 36.2 34.7

a T-DNA copy number determined by Gambino et al. (2005)b Presence of vector backbone sequences (VBS) linked to the LB and RB of the T-DNA (Gambino et al. 2009)c Presence of tandem repeat (TR) and/or inverted repeat (IR) structures of the T-DNA (Gambino et al. 2009)d Expression of GFLV-CP determined in 2004 by Northern hybridization on in vitro plantlets (Gambino et al. 2005)e Symbols represent the levels of gene expression determined by Northern hybridization after normalization to 18S rRNA: ???

high expression, ?? medium expression, ?low expression, -no expressionf In line NE-E was inserted a single copy of T-DNA with a partial deletion of GFLV-CP gene (30 region) and T7 terminator

(Gambino et al. 2005, 2009)

Fig. 2 Northern blot analysis of transgene expression in leaf

tissues collected from transgenic grapevines grown under in

vitro and greenhouse conditions. Total RNA was probed with

GFLV-CP gene labeled with digoxigenin. The blots were

stripped and reprobed with nptII and 18S rDNA digoxigenin-

labeled probes. The control DNA (C) is from an untransformed

‘Nebbiolo’ plant

Transgenic Res

123

levels of GFLV-CP and its regulatory sequences was

made between plants grown in vitro and in vivo

(Fig. 4). The methylation levels in the 35S promoter

and T7 terminator regions were stable without

significant differences. In line NE-B was observed

an increase of CpG methylation in the GFLV-CP

sequence in the plants grown in vivo, as in line NE-F

was observed an increase of CpNpG and CpNpN

methylation. In lines NE-D and 110R no difference

was observed. In line NE-B the high level of CpG

methylation could explain the reduction in GFLV-CP

expression.

Finally, the methylation of GFLV-CP and its

regulatory sequences were compared in healthy and

GFLV-infected lines NE-C, NE-F and 110R. After

grafting onto infected rootstocks, neither the trans-

genic lines nor the non transgenic control plants

showed visible symptoms of GFLV infection. DAS-

ELISA showed GFLV infection in all untransformed

controls as well as in all transgenic lines. The

serological data were stable over time in tests carried

out for 2 years collecting leaves during vegetative

season and woody cuttings during winter. The meth-

ylation status was studied to highlight possible differ-

ences between the two silenced lines (NE-F and 110R)

and the non-silenced line (NE-C) after virus inocula-

tion, although all of them did not show resistance to

GFLV. For all lines the methylation levels in 35S

promoter, GFLV-CP sequence and T7 terminator were

stable without significant differences between healthy

and infected plants (Supplemental Fig. 3).

siRNAs production

The presence of siRNAs sequence specific to the

GFLV-CP gene was analyzed by RNA gel blot in leaf

samples from one line with high transgene expression

(NE-C), in two silenced lines (NE-F and 110R) and in

the same lines inoculated with GFLV. In healthy

transgenic grapevines siRNAs specific to GFLV-CP

were not detected, while these lines as well as

untransformed control grapevines produced siRNAs

specific to the GFLV-CP gene when inoculated with

the virus (Fig. 5). In the inoculated lines a similar

pattern of expression was observed and two hybrid-

ization bands were detected: one of 21-nt with a

strong signal and a second weak band of 22-nt

(Fig. 5). In this study the GFLV-CP gene was divided

into two regions which were used as probes (50CP and

30CP): no difference was observed in the hybridiza-

tion signals between the 50 and 30 regions of the

GFLV-CP gene and consequently the production of

siRNAs seems to be distributed uniformly in the

sequence. These results obtained by DIG hybridiza-

tion were confirmed by using 32P-a-UTP labelled

probes. It was not possible to establish a correlation

between transgene silencing and siRNAs production,

since both the multicopy, silenced and methylated

lines (NE-F and 110R), and a single copy line with

stable transgene expression (NE-C) did not produce

detectable levels of siRNAs specific to GFLV-CP.

Fig. 3 Percent cytosine methylation in symmetrical (CpG and

CpNpG) and asymmetrical (CpNpN) context in the 35S

promoter (a), transgene GFLV-CP (b) and T7 terminator (c).

In line NE-E a single copy of T-DNA was inserted with

deletion of the T7 terminator. DNA was extracted from

transgenic lines cultivated in vitro and was subjected to

bisulphite sequencing. Percent cytosine methylation and

standard deviation were analyzed by CyMATE program and

calculated from total residues present in the five clones

analyzed

Transgenic Res

123

Discussion

The insertion of foreign DNA into a plant genome may

lead to alterations in its structure, which may have

effects on transgene expression. Several potential

silencing mechanisms seems to be involved in the

epigenetic gene inactivation in plants. Although

Fladung et al. (1997) suggested that gene silencing is

relatively rare in woody trees, transgene silencing was

observed in the transgenic grapevines analysed. Even

if the possibility of TGS cannot be ruled out entirely,

in this study PTGS was induced in 5 out of 8 transgenic

lines (NE-D, NE-E, NE-F, BF-A and 110R), since a

low level of GFLV-CP mRNA was detected by RT-

PCR (Gambino et al. 2005) and the transcripts were

not detected by Northern hybridization.

The transgenic grapevine lines were monitored for

stability of GFLV-CP expression under in vitro and in

vivo conditions. After 6 years of continuous in vitro

culture, in 3 lines (NE-D, NE-F and BF-A) a

reduction of transgene expression was observed

compared to previous results (Gambino et al. 2005)

obtained after 2 years of in vitro culture. This

reduction could be explained as the result of varia-

tions within plantlets due to some physiological

adaptations. The lines NE-A and NE-C showed a

stable level of transgene expression following the

transfer to greenhouse, while in lines NE-D, NE-E,

NE-F, BF-A, 110R, GFLV-CP transcript was not

detected in vivo compared to in vitro conditions. In

line NE-B the GFLV-CP expression was reduced

following the transfer in vivo, but the transgene

continued to be expressed. Analysis of methylation in

line NE-B after the transfer to greenhouse conditions

revealed an increase of symmetric cytosine (CpG)

methylation which could be linked to reduction of

transgene expression. Such results are in agreement

with other studies (Kumar and Fladung 2001;

Fig. 4 Percent cytosine methylation in symmetrical (CpG and

CpNpG) and asymmetrical (CpNpN) context in the 35S

promoter (a), transgene GFLV-CP (b) and T7 terminator (c).

DNA was extracted from transgenic lines NE-B, NE-C, NE-D,

NE-F and 110R cultivated in vitro and in greenhouse and was

subjected to bisulphite sequencing. Percent cytosine methyla-

tion and standard deviation were analyzed by CyMATE

program and calculated from total residues present in the five

clones analyzed

Fig. 5 RNA gel blot analysis for detection of siRNAs. Nucleic

acid preparation were separated on 15% polyacrylamide gel

and hybridized with DNA probes labeled with digoxigenin

corresponding to 50 region (b) and 30 region (c) of GFLV-CP.

H: untransformed healthy grapevine plant; 110R (I), NE-F(I),

NE-C(I): transgenic grapevines GFLV-infected; I: untrans-

formed GFLV-infected grapevine plant. Position of the 21-, 24-

nt DNA markers are at the right. Ethidium bromide staining of

5S rRNA and tRNA was applied as loading controls (a)

Transgenic Res

123

Maghuly et al. 2007), which reported that stress and

other changes in environmental conditions as well as

the developmental stage of the plant could affect the

expression level of transgenes. The explants for

expression analyses were sampled from young leaves

in spring, according to previous works that reported

that highest transgene activity was usually observed

in young rather than in mature leaves (Maghuly et al.

2007). These seasonal variations are most probably

correlated with the physiological activity of the plant

material under study. The expression of the nptII

gene inserted in the T-DNA next to GFLV-CP was

stable both in vitro and in vivo, in all lines. The

kanamycin resistance gene is regulated by the nos

promoter, while the GFLV-CP gene is driven by the

35S, a strong promoter that appears more susceptible

to inactivation (McCabe et al. 1999).

The methylation status of cytosines present in the

transgene coding sequence was previously analyzed

by Southern hybridization using methylation-sensi-

tive restriction enzymes: cytosine methylation was

detected only in the GFLV-CP region in the trans-

genic line NE-F (Gambino et al. 2005). Those

methylation-sensitive restriction enzymes however,

allowed the investigation of the methylation status of

a limited number of cytosine residues because only a

limited number of recognition sites are available to

assess DNA methylation. Therefore in this study

bisulphite genomic sequencing was employed which

allows the conversion of all cytosines to uracils,

except those that are methylated at the carbon-5

position of C. The methylation status of GFLV-MP

gene cloned into the pGEM-T Easy vector was

determined to ascertain the complete chemical con-

version of the DNA. The low percentages (0–1.1%)

of methylation observed in this gene might indicate a

random base exchanges during PCR steps or an

incomplete conversion of non-methylated cytosines

after treatment with bisulphite. These technical

problems are not uncommon in the bisulphite geno-

mic sequencing approach. However, the high vari-

ability observed in the methylation levels of the

grapevines transgene sequences can be considered

reliable.

In three silenced lines (NE-D, NE-F and 110R)

transgene methylation was observed in both symmet-

rical and asymmetrical (at lower levels) contexts, and

a very low level of DNA methylation was observed in

non-silenced lines. In the 35S promoter sequence, the

50-CTA-30 and 50-CAA-30 cytosine sites have shown a

much more prominent tendency to methylation than

other types of asymmetrical cytosine. These results

coincide with the methylation observed in the SUP

locus in METI antisense transformants of Arabidopsis

(Kishimoto et al. 2001) and in the 35S promoter of

transgenic gentian (Mishiba et al. 2005). Inverted

repeats have been considered to trigger transgene

silencing and to be susceptible to de novo methylation

(Selker 1999). In this study the lines NE-F and 110R,

which contain inverted T-DNAs (Gambino et al.

2009), were silenced and had high methylation levels

in the 35S promoter, GFLV-CP and T7 terminator

sequences. However, the correlation between DNA

methylation and multiple copies of the integrated

transgene was not in agreement with cytosine meth-

ylation patterns observed in all analyzed lines. In

grapevine NE-B, that carries two T-DNA copies

linked to each other in tandem configuration with two

vector backbone sequences that are connected to the

left (LB) and right border (RB; Gambino et al. 2009),

no silencing and no methylation was observed. In line

BF-A, which carries one T-DNA copy linked by

vector backbone sequences to a second copy of

truncated T-DNA (Gambino et al. 2009), the trans-

gene silencing was associated with very low percent-

ages of cytosines methylation in the 35S promoter and

in the GFLV-CP transgene. The levels of transgene

cytosine methylation in silenced and non-silenced

grapevines indicated that transgene silencing was not

always associated with DNA methylation.

Methylation in CpG, CpNpG and CpNpN sites is a

notable feature of RNA-dependent DNA methylation

(RdDM) in plants, as reported by several authors in

different species (Aufsatz et al. 2002; Haque et al.

2007). Hamilton et al. (2002) proposed that siRNAs of

the longer class (24-25 nt) could be the trigger

molecule for RdDM. The short class of siRNAs (21

nt) is part of natural plant defense mechanism against

viruses (Voinnet 2005) and was proposed to guide the

RISC in the degrading specific mRNAs in herbaceous

plants (Hamilton et al. 2002). The accumulation of

different siRNAs size classes could be the result of

different inducers, virus infection or transgenes. In

plants, transgene- and virus-induced RNA silencing

pathways are overlapping but not identical (Voinnet

2005). The virus seems to act both as inducer and

target in this process, as observed in grapevine (this

study), in Prunus (Hily et al. 2005), in peach

Transgenic Res

123

inoculated with Peach latent mosaic viroid and in

chrysanthemum inoculated with Chrysanthemum clo-

rotic mottle viroid (Martinez de Alba et al. 2002). Hily

et al. (2005) demonstrated the accumulation of two

classes of siRNAs in transgenic Prunus resistant to

Plum pox virus and showed that those siRNAs are

constitutively present only in the resistant clone. The

resistance mounted by the shorter siRNAs is appar-

ently overcome and the longer siRNAs are responsible

for high levels of resistance against Plum pox virus. In

our grapevine lines the correlation between accumu-

lation of longer class of siRNAs, transgene methyl-

ation and RNA silencing could not be confirmed. In

the GFLV-CP transcript no small RNAs could be

detected both in methylated and non methylated lines,

while short siRNAs were detected only in transgenic

and non transgenic grapevines following GFLV

infection. These small RNAs originate from virus

replication and not from the GFLV-CP transgene.

However, it is possible that RNA signalling molecules

were responsible for the DNA methylation patterns

observed in these lines, even if siRNAs were below

the detection level. Similar results were observed

in Arabidospis, in which gene PA1 was methylated

at both symmetrical and asymmetrical contexts, but

no PA1 siRNAs cold be detected (Melquist and

Bender 2004); likewise, no siRNAs could be detected

in methylated CP gene of SPFMV in transgenic

N. benthamiana (Haque et al. 2007).

In this study eight transgenic grapevine lines

expressing GFLV-CP gene were tested for virus

resistance in greenhouse by grafting onto infected

rootstocks. All inoculated transgenic plants were

positive in ELISA, even when not showing GFLV

symptoms. The severity of symptoms following a

GFLV infection may be linked to environment and to

plant physiological conditions. The growth in pots

and in greenhouse could cause the lack of symp-

toms—at least for the first 2 years after inoculation—

both in the transgenic and in the control infected

grapevines; however, the serological results indicated

that the transgenic grapevines analyzed were suscep-

tible to GFLV transmitted by grafting onto infected

rootstocks. On the contrary Nicotiana benthamiana,

transformed with the same constructs and expressing

the full-length GFLV-CP gene, showed protection

against virus inoculation (Golles et al. 2000). Vigne

et al. (2004) showed that 3/18 transgenic grapevine

lines were resistant in the vineyard to natural GFLV

transmission by nematode vectors. Therefore, the

susceptibility to GFLV observed in the analyzed

transgenic grapevines could be due to high viral

inoculum from the rootstock applied to relatively

young and small plants, since Sonoda et al. (1999)

suggested a dose-dependent resistance. Probably

under these conditions the transgenic plants are

unable to suppress GFLV replication. Future tests

might include the use of natural vectors of the virus to

keep the viral pressure comparable to field condi-

tions. Further analyses will be necessary to investi-

gate thoroughly the complex relationships between

GFLV and silencing process in order to increase our

knowledge of the molecular mechanisms related to

transgene silencing and virus resistance in grapevine

as well as other woody species.

Acknowledgments We thank Federico Ghilino for excellent

support in the grafting, Danila Cuozzo and Tiziano Strano for

their technical assistance in micropropagation of transgenic

grapevines and in greenhouse management.

References

An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In:

Gelvin SB, Schilperoort RA (eds) Plant molecular biology

manual. Kluwer, Dortrecht p A3/1-19

Andret-Link P, Laporte C, Valat L et al (2004) Grapevine

fanleaf virus: still a major threat to the grapevine industry.

J Plant Pathol 86:183–195

Aufsatz W, Mette MF, van der Winden J et al (2002) RNA-

directed DNA methylation in Arabidopsis. Proc Natl Acad

Sci USA 99:16499–16506. doi:10.1073/pnas.162371499

Baulcombe D (2004) RNA silencing in plants. Nature

431:356–363. doi:10.1038/nature02874

Cao XF, Aufsatz W, Zilberman D et al (2003) Role of the

DRM and CMT3 Methyltransferases in RNA-directed

DNA methylation. Curr Biol 13:2212–2217. doi:10.1016/

j.cub.2003.11.052

Carra A, Gambino G, Schubert A (2007) A Cetyltrimethyl

ammonium bromide -based method to extract low molec-

ular weight RNA from polysaccharide-rich plant tissues.

Anal Biochem 360:318–320. doi:10.1016/j.ab.2006.09.022

Chenna R, Sugawara H, Koike T et al (2003) Multiple sequence

alignment with the Clustal series of programs. Nucleic

Acids Res 31:3497–3500. doi:10.1093/nar/gkg500

Fladung M, Kumar S, Ahuja R (1997) Genetic transformation

of Populus genotypes with different chimaeric gene con-

structs: transformation efficiency and molecular analysis.

Transgenic Res 6:111–121. doi:10.1023/A:101842162

0040

Fusaro AF, Matthew L, Smith NA et al (2006) RNA interfer-

ence-inducing hairpin RNAs in plants act through the

viral defence pathway. EMBO Rep 7:1168–1175. doi:

10.1038/sj.embor.7400837

Transgenic Res

123

Gambino G, Gribaudo I, Leopold S et al (2005) Molecular

characterization of grapevine plants transformed with

GFLV resistance genes: I. Plant Cell Rep 24:655–662.

doi:10.1007/s00299-005-0006-4

Gambino G, Perrone I, Gribaudo I (2008) A rapid and effective

method for RNA extraction from different tissues of

grapevine and other woody plants. Phytochem Anal

19:520–525. doi:10.1002/pca.1078

Gambino G, Chitarra W, Maghuly F, et al (2009) Character-

ization of T-DNA insertions in transgenic grapevines

obtained by Agrobacterium-mediated transformation. Mol

Breed. doi:10.1007/s11032-009-9293-8

Golles R, da Camara Machado A, Tsolova V et al (1998)

Transformation of somatic embryos of Vitis sp. with dif-

ferent constructs containing nucleotide sequences from

nepovirus coat protein genes. Acta Hortic 447:265–270

Golles R, Moser M, Puhringer H et al (2000) Transgenic

grapevines expressing coat protein gene sequences of

Grapevine fanleaf virus, Arabis mosaic virus, Grape-vine virus A and Grapevine virus B. Acta Hortic

528:305–311

Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two

classes of short interfering RNA in RNA silencing.

EMBO J 21:4671–4679. doi:10.1093/emboj/cdf464

Hannon GJ (2002) RNA interference. Nature 418:244–251.

doi:10.1038/418244a

Haque AKMN, Yamaoka N, Nishiguchi M (2007) Cytosine

methylation is associated with RNA silencing in silenced

plants but not with systemic and transitive RNA silencing

through grafting. Gene 396:321–331. doi:10.1016/j.gene.

2007.04.003

Hetzl J, Foerster AM, Raidl G, Scheid OM (2007) CyMATE: a

new tool for methylation analysis of plant genornic DNA

after bisulphite sequencing. Plant J 51:526–536. doi:

10.1111/j.1365-313X.2007.03152.x

Hily JM, Scorza R, Webb K, Ravelonandro M (2005) Accu-

mulation of the long class of siRNA is associated with

resistance to Plum pox virus in a transgenic woody

perennial plum tree. Mol Plant Microbe Interact 18:794–

799. doi:10.1094/MPMI-18-0794

Kishimoto N, Sakai H, Jackson J et al (2001) Site specificity of

the Arabidopsis METI DNA methyltransferase demon-

strated through hypermethylation of the superman locus.

Plant Mol Biol 46:171–183. doi:10.1023/A:10106362

22327

Kumar S, Fladung M (2001) Gene stability in transgenic aspen

(Populus). II. Molecular characterization of variable

expression of transgene in wild and hybrid aspen. Planta

213:731–740. doi:10.1007/s004250100535

Maghuly F, Machado A, Leopold S et al (2007) Long-term

stability of marker gene expression in Prunus subhirtella:

a model fruit tree species. J Biotechnol 127:310–321. doi:

10.1016/j.jbiotec.2006.06.016

Martinez de Alba AE, Flores R, Hernandez C (2002) Two

chloroplastic viroids induce the accumulation of small

RNAs associated with posttranscriptional gene silencing. J

Virol 76:13094–13096. doi:10.1128/JVI.76.24.13094-130

96.2002

McCabe MS, Schepers F, van der Arend A et al (1999)

Increased stable inheritance of herbicide resistance in

transgenic lettuce carrying a petE promoter-bar gene

compared with a CaMV 35S-bar gene. Theor Appl Genet

99:587–592. doi:10.1007/s001220051272

Melquist S, Bender J (2004) An internal rearrangement in an

Arabidopsis inverted repeat locus impairs DNA methyl-

ation triggered by the locus. Genetics 16:437–448. doi:

10.1534/genetics.166.1.437

Mette MF, Aufsatz W, van der Winden J et al (2000) Tran-

scriptional silencing and promoter methylation triggered

by double-stranded RNA. EMBO J 19:5194–5201. doi:

10.1093/emboj/19.19.5194

Mishiba K, Nishihara M, Nakatsuka T et al (2005) Consistent

transcriptional silencing of 35S-driven transgenes in

gentian. Plant J 44:541–556. doi:10.1111/j.1365-313X.

2005.02556.x

Murashige T, Skoog F (1962) A revised medium for rapid growth

and bio assays with tobacco tissue cultures. Physiol Plant

15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x

Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for

general users and for biologist programmers. In: Krawetz

S, Misener S (eds) Bioinformatics methods and protocols:

methods in molecular biology. Humana Press, Totowa,

pp 365–386

Selker EU (1999) Gene silencing: repeats that count. Cell

97:157–160. doi:10.1016/S0092-8674(00)80725-4

Sonoda S, Mori M, Nishiguchi M (1999) Homology-dependent

virus resistance in transgenic plants with the coat protein

gene of Sweet potato feathery mottle potyvirus: target

specificity and transgene methylation. Phytopathology

89:385–391. doi:10.1094/PHYTO.1999.89.5.385

Szittya G, Molnar A, Silhavy D et al (2002) Short defective

interfering RNAs of tombusviruses are not targeted but

trigger post-transcriptional gene silencing against their

helper virus. Plant Cell 14:359–372. doi:10.1105/tpc.

010366

Thomas MR, Matsumoto S, Cain P, Scott NS (1993) Repetitive

DNA of grapevine: classes present and sequences suitable

for cultivar identification. Theor Appl Genet 86:173–180

Vaucheret H (2006) Post-transcriptional small RNA pathways

in plants: mechanisms and regulations. Genes Dev

20:759–771. doi:10.1101/gad.1410506

Vigne E, Komar V, Fuchs M (2004) Field safety assessment of

recombination in transgenic grapevines expressing the coat

protein gene of Grapevine fanleaf virus. Transgenic Res

13:165–179. doi:10.1023/B:TRAG.0000026075.79097.c9

Voinnet O (2005) Induction and suppression of RNA silencing:

insights from viral infections. Nat Rev Genet 6:206–220.

doi:10.1038/nrg1555

Xie Z, Fan B, Chen C, Chen Z (2001) An important role of an

inducible RNA-dependent RNA polymerase in plant

antiviral defense. Proc Natl Acad Sci USA 98:6516. doi:

10.1073/pnas.111440998

Transgenic Res

123