Neither inverted repeat T-DNA configurations norarrangements of tandemly repeated transgenesare sufficient to trigger transgene silencing

Berthold Lechtenberg�, Daniel Schuberty,�, Alexandra Forsbachz, Mario Gils§ and Renate Schmidt�

Max-Planck-Institut fur Molekulare Pflanzenphysiologie, Am Muhlenberg 1, 14476 Golm, Germany

Received 16 December 2002; revised 1 February 2003; accepted 5 February 2003.�For correspondence (fax þ49 331 5678 408; e-mail rschmidt@mpimp-golm.mpg.de).yPresent address: University of Edinburgh, ICMB, Rutherford Building, The King’s Buildings, Edinburgh EH9 3JH, UK.zPresent address: Coley Pharmaceutical GmbH, Elisabeth-Selbert-Strasse 9, 40764 Langenfeld, Germany.§Present address: Icon Genetics GmbH, Biozentrum Halle, Weinbergweg 22, 06120 Halle (Saale), Germany.�These authors contributed equally to this work.

Summary

Transgene expression was analysed in Arabidopsis T-DNA transformants carrying defined numbers and

arrangement of different reporter genes. All transgenes were placed under the control of the strong con-

stitutive CaMV 35S promoter. High, stable transgene expression was observed in plants containing two

copies of the b-glucuronidase (GUS) gene, two or four copies of the green fluorescent protein (GFP ) gene

and two, four or six copies of the streptomycin phosphotransferase (SPT ) gene. Thus, the mere presence of

multiple promoter and/or transgene sequences did not result in gene silencing. In none of the cases

analysed were tandem repeat arrangements of transgenes and/or inverted repeat (IR) T-DNA structures

sufficient to trigger silencing of the different reporter genes. Instead, post-transcriptional gene silencing

(PTGS) correlated with the copy number of the highly expressed transgenes. Twelve copies of the SPT and

four copies of the GUS gene triggered silencing. Silencing is frequently associated with repetitive T-DNA

structures. We favour the idea that in many cases this may be attributed to the high transgene doses rather

than the repeat arrangements themselves.

Keywords: inverted repeat, post-transcriptional gene silencing, tandem repeat, T-DNA, transgene expres-

sion, Arabidopsis thaliana.

Introduction

Predictable, stable transgene expression is often a critical

parameter for the broad use of transgenic plants. Unfortu-

nately, transgene expression often varies over several

orders of magnitude, and gene silencing is frequently

observed, as has been documented for transgenic tobacco

and Arabidopsis plants, harbouring chimaeric b-glucuroni-

dase (GUS) gene constructs (Hobbs et al., 1990; Holtorf

et al., 1995).

Silencing is caused, not by an alteration of the transgene

sequence, but rather by epigenetic effects. Two different

mechanisms can be distinguished: transcriptional gene

silencing (TGS) and post-transcriptional gene silencing

(PTGS) (summarised by Vaucheret et al., 1998). TGS is

classified as an abolished transcription of the introduced

gene. It is associated with methylation of promoter

sequences of the transgene and meiotic irreversibility

(summarised by Meyer, 2000). In plants displaying PTGS

of the introduced gene, transcription is maintained, but the

transgene mRNA is degraded. Small RNAs (siRNAs) in

sense and antisense orientations and specific for the tran-

scribed transgene sequence are a hallmark of silenced

lines. PTGS is established during plant development and

spreads through the plant in a non-clonal manner. The

process is reset after meiosis. Methylation of the tran-

scribed sequence is frequently found (summarised by

Waterhouse et al., 2001).

The plant transformation methods used, such as Agro-

bacterium-mediated transformation, particle bombard-

ment and direct gene transfer, do not allow the

introduction of a defined number of transgenes into the

genome. Moreover, the integration of foreign DNA into the

genome occurs at random and, frequently, in repeat

arrangements. In numerous examples, TGS and/or PTGS

has been found to be associated with multiple inserts of the

The Plant Journal (2003) 34, 507–517

� 2003 Blackwell Publishing Ltd 507

introduced DNA. However, this phenomenon is not

restricted to the repeat structures of the transgenes. Endo-

genous plant genes forming the repeat structures may also

be subjected to gene silencing (summarised by Muskens

et al., 2000). Mechanisms based on DNA/DNA, RNA/RNA

and RNA/DNA interactions have been implicated in these

processes (summarised by Selker, 1999).

Based on the studies of transgene repeat arrays in Dro-

sophila, it was proposed that DNA/DNA pairing of the

repeats might lead to heterochromatin formation and silen-

cing (Dorer and Henikoff, 1994). Luff et al. (1999) showed

that the PAI1-PAI4 inverted repeat (IR) locus triggered

methylation of unlinked PAI sequences in Arabidopsis,

whereas methylated singlet PAI genes were not capable

of promoting methylation in unlinked targets. Moreover, a

promoter-less pai1-pai4 IR transgene construct triggered

its own methylation. These results led the authors to

conclude that methylation was communicated by a DNA/

DNA pairing mechanism.

The study of transcribed IR structures, which are parti-

cularly potent silencers, has provided compelling evidence

that silencing is mediated via RNA/RNA and RNA/DNA

interactions (Chuang and Meyerowitz, 2000; Hamilton

et al., 1998; Waterhouse et al., 1998). RNA, capable of

duplex formation, is processed to siRNAs, and triggers

the targeted elimination of the homologous mRNA in

the cytoplasm. This post-transcriptional process is

related to RNA interference in animals and quelling in fungi

(summarised by Zamore, 2002). RNA-directed DNA methy-

lation results in de novo methylation of cytosine residues

within a region of RNA–DNA sequence identity. When the

double-stranded RNA corresponds to the promoter

sequences, TGS can occur (Mette et al., 2000; Sijen et al.,

2001).

Read-through transcripts capable of duplex formation

were implicated in the silencing process for some trans-

genic lines harbouring IR T-DNA structures (De Buck and

Depicker, 2001; Mette et al., 1999; Muskens et al., 2000; Van

Houdt et al., 2000). However, a compilation of different IR

loci displaying gene silencing revealed that the formation of

such transcripts is unlikely for at least some of the IR

structures studied to date (summarised by Muskens et al.,

2000). To gain more insight into the role of the IR arrange-

ments on the gene expression, we determined the influence

of four IR T-DNA loci on the expression of different trans-

genes in Arabidopsis thaliana. Moreover, constructs were

developed to determine whether a tandem arrangement of

transgenes influences reporter gene expression. The

results obtained unambiguously showed that arrange-

ments of neither the tandemly repeated transgenes nor

the inverted T-DNA structures were sufficient to trigger

gene silencing. Instead, the study of the different transgenic

lines suggested a correlation between silencing and high

transgene doses.

Results

Isolation and characterisation of transgenic lines carrying

inverted repeat T-DNA structures

Agrobacterium-mediated transformation was used to intro-

duce different T-DNAs into the A. thaliana genome. All

T-DNA constructs are derivatives of the basic T-DNA-vector

pMDL-T-DNA that harbours a neomycin phosphotransfer-

ase (NPT) gene under the control of the nopaline synthase

promoter as transformation marker close to the left border

(LB) and a promoter-less copy of the hygromycin phospho-

transferase (HPT) gene in the vicinity of the right border

(RB) (Figure 1a; Forsbach et al., 2003). Chimaeric reporter

genes were inserted into pMDL-T-DNA to generate the

different constructs. The genes for GUS (Jefferson et al.,

1986), streptomycin phosphotransferase (SPT; Maliga

et al., 1988) and green fluorescent protein (GFP) were used

as reporter genes (mGFP5-ER; Haseloff et al., 1997; Siemer-

ing et al., 1996). All three genes were placed under the

control of the CaMV 35S promoter (p35S; Odell et al.,

1985) with a modified TMV O-leader (Gallie et al., 1987)

and the 30-untranslated region of the octopine synthase

gene (ocs30; De Greve et al., 1982) (Figure 1b).

T-DNA vectors were made that harboured one copy of the

chimaeric SPT gene, the chimaeric GFP gene or the chi-

maeric GUS gene. To determine the influence of direct

repeat arrays on the expression of the transgenes, con-

structs were developed that carried two and three SPT

reporter gene cassettes or two copies of the GUS transgene

in a tandem arrangement (Figure 1c).

A genetic analysis revealed whether transgenic lines

harboured one or more T-DNA loci. The transgenic lines

were characterised further by Southern blot analysis.

Genomic DNA of each of the transgenic lines was

treated with different restriction endonucleases and

hybridised with border-specific sequences to reveal the

number of integrated T-DNA copies. For each of the

transgenic lines, the results of two or more different restric-

tion endonuclease digestions were evaluated for the LB and

the RB, respectively. The goal of this analysis was the

identification of the transgenic lines that carried single-

copy T-DNA insertions or two T-DNA copies in an IR

arrangement.

For lines that harbour IR structures of two directly

adjoined complete T-DNA copies, two T-DNA flanking frag-

ments of different sizes should hybridise, if one of the

borders is used as a probe, whereas hybridisation of sin-

gle-junction fragments of predictable sizes are expected if

the other border is utilised as a probe. Three lines, 102, 127

and 159, were discovered that fulfilled these criteria. At

least seven different endonuclease digestions were evalu-

ated for each of the three transgenic lines, and in all cases,

the observed fragment sizes suggested that the two RB

508 Berthold Lechtenberg et al.

� Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 507–517

Figure 1. Maps of T-DNAs and chimaeric reporter genes.(a) Map of the T-DNA found in vector pMDL-T-DNA (Forsbach et al., 2003). A chimaeric neomycin phosphotransferase (NPT) gene is present in close proximity tothe left border (LB). The gene is under the control of the nopaline synthase promoter (pnos) and carries a modified TMVO-leader and the 30-untranslated region ofthe octopine synthase gene (ocs30). A promoter-less hygromycin phosphotransferase (HPT) gene with the 30-untranslated region of the nopaline synthase gene(nos30) is located near the right border (RB). Chimaeric reporter genes were inserted into the HindIII site of vector pMDL-T-DNA.(b) Maps of the HindIII fragments corresponding to the chimaeric reporter gene cassettes. The genes for b-glucuronidase (GUS), streptomycin phospho-transferase (SPT) and green fluorescent protein (GFP) were used as reporters. Reporter genes are driven by the CaMV 35S promoter (p35S). They harbour amodified TMVO-leader and the ocs30. Tn5 sequences which are located downstream of ocs30 sequences in each of the reporter gene cassettes are shown as whiteboxes.(c) Maps of T-DNAs introduced into Arabidopsis by Agrobacterium-mediated transformation. The different T-DNAs are designated according to the kind andnumber of the reporter genes they carry. Arrows indicate the direction of transcription. In T-DNAs of the ‘f’-type, the direction of transcription of the reporter geneis the same as that of the NPT gene, whereas the direction of transcription of the reporter gene(s) in constructs of the ‘r’-type is pointing towards the LB.(d) Structure of inverted repeat (IR) T-DNA loci. Transgenic line SPT 3xf IR 159 carries two SPT 3xf T-DNAs in an IR arrangement about the RB. Two GFP 1xr T-DNAs adjoin each other in transformant GFP 1xr IR 127. In locus GUS 1xr IR 102, two GUS 1xr T-DNAs are found in an IR arrangement about the RB. A truncated T-DNA copy adjoins a full-sized GUS 1xf T-DNA in line GUS 1xf IR 57.

� Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 507–517

Repeat structures and transgene silencing 509

fragments directly adjoined each other in an IR configura-

tion (data not shown).

Different T-DNA constructs were used to establish the

transgenic lines 102, 127 and 159. Three SPT reporter genes

in a direct repeat arrangement were found in each of the

T-DNA copies that made up the IR locus in line 159

(Figure 1d, SPT 3xf IR 159). Line 127 harboured an IR locus

with two divergently transcribed chimaeric GFP reporter

genes (Figure 1d, GFP 1xr IR 127). The reporter gene

arrangement in line 102 was similar to the one in line

127; however, the line carried the GUS transgenes instead

of the GFP genes (Figure 1d, GUS 1xr IR 102). Southern blot

experiments verified that the chimaeric reporter gene cas-

settes in the three transgenic lines had not been subjected

to rearrangements (data not shown).

The results of detailed Southern blot analyses suggested

the presence of a truncated T-DNA copy directly adjacent to

a complete copy of the T-DNA in transgenic line 57. A study

of the RB revealed sizes that are expected if two T-DNA

inserts are present in an IR configuration about the RB (data

not shown). The T-DNA construct used in this particular

transformation experiment carried a chimaeric GUS repor-

ter gene under the control of the CaMV 35S promoter

(Figure 1c, GUS 1xf). Using the CaMV 35S promoter and a

fragment of the GUS gene as probes, respectively, showed

thatthetruncatedT-DNAcopylackedbetween3.0and3.4 kbp

at the left end; nonetheless, the inverted T-DNA repeat at

this locus harboured two complete copies of the chimaeric

GUS reporter gene (Figure 1d, GUS 1xf IR 57).

Left-border flanking sequences were isolated by inverse

polymerase chain reaction (IPCR) to determine the position

of T-DNA integration in the Arabidopsis genome. Loci GFP

1xr IR 127, GUS 1xr IR 102, SPT 3xf IR 159 and GUS 1xf IR 57

mapped to chromosomes I (position 55199 in sequenced

clone F7G19, AC000106), IV (positions 49435 and 49442 in

sequence contig 28, AL161516), V (positions 15151 and

15160 in sequenced clone MOP10, AB005241) and V (posi-

tion 128162 in sequenced clone T6G21, AL589883), respec-

tively.

No evidence for silencing of the NPT gene in transgenic

lines carrying inverted repeat T-DNA structures about the

right border

Progeny of primary transformants GUS 1xf IR 57, GUS 1xr

IR 102 and GFP 1xr IR 127 showed a 3 : 1 segregation for

kanamycin resistance when plated on a kanamycin-contain-

ing medium. For all the three lines, plants homozygous for

the T-DNA were readily identified, and their progeny

showed stable expression of the NPT gene over several

generations, as judged by the resistance of seedlings to

kanamycin.

Progeny derived from the primary transformant SPT 3xf

IR 159 displayed a 1 : 1 segregation of kanamycin-resistant

versus kanamycin-sensitive plants. The same ratio of resis-

tant to sensitive seedlings was observed when plants were

grown on a streptomycin-containing medium. The high

frequency of aborted seeds in siliques of plants carrying

locus SPT 3xf IR 159 suggested a gametophytic defect.

Reciprocal crosses of this line with wild-type plants were

carried out, and the analysis of the resulting progeny

revealed a reduced transmission of the T-DNA through

the female gametophyte. As a result of the gametophytic

defect, it was not possible to recover any plant that con-

tained the T-DNA IR structure homozygously. Neverthe-

less, this transgenic line displayed stable expression of

both the NPT and SPT genes as judged by the resistance

of seedlings to kanamycin and streptomycin over several

generations.

High SPT transcript levels in transgenic lines carrying

reporter genes in repeat arrangements

A Northern blot analysis was carried out to characterise the

expression of the SPT genes in transgenic line SPT 3xf IR

159. For comparison, several other transgenic lines that

carried SPT transgenes were analysed. Single-copy T-DNA

lines were identified for constructs SPT 1xr, SPT 2xf and

SPT 3xf (Figure 1c). SPT transcript levels in 8-week-old

plants were comparably high in lines SPT 2xf 107, SPT

1xr 88, SPT 3xf 147 and SPT 3xf IR 159. Thus, transgenic

lines carrying the chimaeric SPT reporter genes showed

high expression of the transgenes, despite the fact that the

SPT genes were present in a tandem (SPT 2xf 107, SPT 3xf

147) and/or an IR arrangement (SPT 3xf IR 159) in some of

the plants analysed (Figure 2a).

Two independent single-copy T-DNA lines carrying the

SPT 3xf T-DNA were crossed, and the progeny plants were

analysed for the presence of both loci. A line that harboured

both loci in the homozygous fashion (SPT 3xf A) was

selected for further analysis and showed a reduced SPT

transcript level. Furthermore, a truncated SPT transcript

was detected in addition to the full-length transcript

(Figure 2a). Reduced SPT transcript levels and the trun-

cated SPT transcript were also detected in independently

generated lines that were homozygous for two different

SPT3xf T-DNAloci (SPT 3xfB and SPT3xf C,data not shown).

We checked to see whether the reduction of SPT mRNA

seen in transgenic lines containing four copies of the T-DNA

SPT 3xf was as a result of the PTGS. Therefore, the trans-

genic lines were analysed for the presence of siRNAs of

the transcribed region. Using sequences corresponding

to the SPT gene as a probe, the siRNAs were detected in

transgenic lines SPT 3xf A, SPT 3xf B and SPT 3xf C, but

not in any of the transgenic lines for which high SPT

transcript levels were detected in the Northern blot analysis

(SPT 2xf 107, SPT 1xr 88, SPT 3xf 147 and SPT 3xf IR 159)

(Figure 2b).

� Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 507–517

510 Berthold Lechtenberg et al.

High and stable expression of the GFP reporter gene in

transgenic line GFP 1xr IR 127

Primary transformants GFP 1xr 129, 145 and 155 harboured

a single copy of the GFP 1xr T-DNA (Figure 1c). Lines

homozygous for the different loci showed a high GFP

transcript level comparable to the amount of RNA that

was detected in plants homozygous for the IR locus GFP

1xr IR 127 (Figure 3a). GFP fluorescence measurements in

2-, 5- and 8-week-old homozygous plants revealed that

similar values were obtained for plants of different ages,

regardless of which of the four transgenic lines was ana-

lysed (Figure 3b).

Locus GFP 1xr IR 127 conferred high and stable expres-

sion, regardless of whether it was present in the hemizy-

gous (data not shown) or homozygous state (Figure 3a,b).

Figure 2. Transgenic line SPT 3xf IR 159 shows high expression of thestreptomycin phosphotransferase (SPT) transgenes despite the presenceof the transgenes in inverted and tandem repeat arrangements.(a) Northern blot analysis of the transgenic lines carrying T-DNAs SPT 1xr,SPT 2xf or SPT 3xf (Figure 1c). With the exception of transgenic line SPT 3xfIR 159, plants homozygous for the T-DNA were analysed. Lines harbouringfour copies of the T-DNA construct SPT 3xf are marked with an asterisk. Thecopy number of the SPT reporter genes is given for each of the transgeniclines. TR and IR indicate that the reporter genes are present in a tandem or anIR arrangement, respectively. Total RNA was prepared from the leaves of 8-week-old plants, and samples containing 10 mg RNA were separated byelectrophoresis. The resulting Northern blot was hybridised with a probecorresponding to SPT sequences and subsequently with a probe derivedfrom Arabidopsis actin genes 2 and 8 (An et al., 1996).(b) Detection of small interfering RNAs (siRNAs) in silenced transgenic linescarrying four copies of T-DNA SPT 3xf. Samples of total RNA were separatedby electrophoresis. 75 ng ocs-primer (21 nucleotides) and SPT-primer (23nucleotides) were mixed with wild-type RNA and served as size standardand hybridisation control. The resulting Northern blot was hybridised with aprobe corresponding to a fragment of the SPT coding sequence.

Figure 3. Expression analysis of transgenic line GFP 1xr IR 127.(a) Northern blot analysis of homozygous transgenic lines carrying the GFP1xr T-DNA (Figure 1c; GUS 1xr IR 127, Figure 1d). Total RNA was preparedfrom leaves of 8-week-old transgenic lines homozygous for the T-DNA. TheNorthern blot was hybridised with a probe corresponding to the greenfluorescent protein (GFP) sequences and subsequently with a probe derivedfrom the Arabidopsis actin genes 2 and 8 (An et al., 1996).(b) Detection of fluorescence in homozygous transgenic lines harbouring T-DNA construct GFP 1xr. Protein extracts were prepared from leaves of 2-, 5-and 8-week-old plants. The diagram shows the mean of the four fluores-cence measurements and the standard deviation for each of the lines. Thevalues for green fluorescent protein (GFP) fluorescence are given as relativeunits of fluorescence per milligram protein.

� Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 507–517

Repeat structures and transgene silencing 511

Transgene silencing in plants homozygous for the

inverted repeat loci GUS 1xf IR 57 and GUS 1xr IR 102

Transgenic lines hemizygous or homozygous for T-DNA

loci with two copies of a GUS reporter gene (Figure 1c, GUS

2xr; Figure 1d, GUS 1xf IR 57, GUS 1xr IR 102) were ana-

lysed for GUS activity. Colorimetric assays of homozygous

plants showed high reporter gene expression in at least

some of the 2-week-old plants, but throughout the devel-

opment, the expression was severely reduced (Figure 4a).

Plants derived from different generations revealed a similar

expression pattern when GUS activity assays were per-

formed on plants of different ages (data not shown). Silen-

cing was seen, regardless of whether the transgene copies

were present in inverted (lines GUS 1xf IR 57 and GUS 1xr IR

102) or tandem repeat arrangements (GUS 2xr 114).

In contrast, all plants hemizygous for the T-DNA loci GUS

1xf IR 57, GUS 1xr IR 102 and GUS 2xr 114 showed high

expression throughout the development (Figure 4a). Thus,

neither the direct nor the IR arrangement of the GUS

reporter genes was sufficient to trigger gene silencing.

GUS mRNA was detectable in 8-week-old plants homo-

zygous for the loci GUS 1xf IR 57, GUS 1xr IR 102 and GUS

2xr 114, but the levels were severely reduced when com-

pared to the amount of transcript in hemizygous plants of

the same age (Figure 4b). siRNAs specific for the tran-

scribed region of the GUS gene were only detected in

homozygous plants that showed a severely reduced GUS

mRNA level (Figure 4b). This result is consistent with a

post-transcriptional mechanism of gene silencing.

Discussion

Tandem repeat arrangements of transgenes are not

sufficient to trigger gene silencing in transgenic

Arabidopsis lines

Homology-dependent gene silencing of transgenes under

the control of the CaMV 35S promoter in cis and/or trans

was reported in several studies (e.g. Assaad et al., 1993;

Mittelsten Scheid et al., 1991; Thierry and Vaucheret, 1996).

These results provoked the statement that the repeated use

of the same promoter or of promoters sharing the homol-

ogy should be avoided in transgene constructs (De Wilde

et al., 2000). However, the stable transgene expression

seen in lines containing four copies of the GFP gene or

up to six copies of the SPT gene clearly proves that the mere

presence of multiple promoter and/or transgene copies

is not sufficient for the onset of gene silencing (Figures 2

and 3; Table 1).

The lines carrying two or three SPT transgenes in tandem

displayed SPT mRNA levels similar to those of the lines

carrying a T-DNA with a single copy of the SPT gene in the

T-DNA (Figure 2). Likewise, five independent lines contain-

ing a single copy of T-DNA GUS 2xr that harboured two

GUS transgenes in a direct repeat arrangement displayed

high and stable GUS activity (Figure 4, data not shown).

Thus, a tandem repeat arrangement of transgenes is not

capable of triggering transgene silencing (Table 1).

In contrast, Assaad et al. (1993) concluded that silencing

strictly depended on a repeat arrangement of transgenes at

Figure 4. The b-glucuronidase (GUS) transgenes are silenced in plantshomozygous for the inverted repeat (IR) T-DNA loci GUS 1xf IR 57 andGUS 1xr IR 102.(a) Colorimetric assays for GUS activity of transgenic lines GUS 1xf IR 57,GUS 1xr IR 102 (Figure 1d) and GUS 2xr 114 (Figure 1c). Plants that wereeither hemizygous or homozygous for the different T-DNA loci were ana-lysed at the age of 2 and 8 weeks. Two leaf discs per plant were incubatedovernight in phosphate buffer containing X-gluc. Blue coloration indicatesthat the plants harbour an active GUS, the intensity of staining reflects theexpression level.(b) Northern blot analysis of transgenic lines harbouring T-DNA loci GUS 1xfIR 57, GUS 1xr IR 102 and GUS 2xr 114. Total RNA was prepared from leavesof 8-week-old transgenic lines, and 16 mg samples were separated byelectrophoresis. The resulting blots were hybridised with a probe corre-sponding to the ocs30-end of the chimaeric GUS reporter gene and subse-quently with a probe derived from the Arabidopsis actin genes 2 and 8 (Anet al., 1996). Small interfering RNAs (siRNAs) were detected with a probecorresponding to the GUS reporter gene. 15 ng GUS-primer (23 nucleotides)mixed with wild-type RNA served as size standard and hybridisation control.

� Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 507–517

512 Berthold Lechtenberg et al.

a particular locus in Arabidopsis. Further analyses showed

that the repeat-induced gene silencing (RIGS) was tran-

scriptional and accompanied by an altered chromatin con-

figuration, possibly resembling heterochromatin (Ye and

Signer, 1996). Similar results were obtained by studying

arrays of the white transgene in Drosophila (Dorer and

Henikoff, 1994). In these analyses, the transgene arrays

produced phenotypes reminiscent of heterochromatin-

induced position effect variegation. Mosaic expression of

the transgene arrays was more pronounced at the sites

close to the heterochromatin in Drosophila (Dorer and

Henikoff, 1994; Sabl and Henikoff, 1996). All transgenic lines

of the SPT 2xf, SPT 3xf and GUS 2xr constructs that were

analysed in the context of this study were mapped to the

euchromatic regions of the Arabidopsis chromosome

sequence maps (data not shown). Assaad et al. (1993)

studied gene silencing of transgene arrays at one particular

locus in the Arabidopsis genome; thus, the possibility

cannot be excluded that the RIGS found was specific for

a special genomic context. All repeat arrays investigated by

Assaad et al. (1993) harboured one deletion mutant trans-

gene copy, whereas the loci we studied exclusively con-

tained complete reporter gene cassettes. This may also

account for the different behaviour of the loci with respect

to gene silencing.

Inverted repeat T-DNA configurations are not sufficient to

trigger gene silencing in transgenic Arabidopsis lines

There are numerous examples of inverted DNA repeats

associated with TGS and/or PTGS (summarised by

Muskens et al., 2000). DNA/DNA, RNA/RNA and RNA/

DNA interactions have all been implicated in these pro-

cesses (summarised by Selker, 1999). For the transgenic

lines analysed in the context of this study, it is unlikely that a

DNA/DNA pairing mechanism elicits silencing, as the stable

expression of the reporter genes in lines SPT 3xf IR 159 and

GFP 1xr IR 127 and in plants hemizygous for T-DNA loci

GUS 1xf IR 57 and GUS 1xr IR 102 unambiguously showed

that an IR arrangement as such was not sufficient to trigger

gene silencing (Table 1).

Transcribed IR structures have been found to be potent

silencers (Chuang and Meyerowitz, 2000; Hamilton et al.,

1998; Mette et al., 1999, 2000; Sijen et al., 2001; Waterhouse

et al., 1998). Thus, it is an attractive hypothesis that tran-

scription through the inverted repeats creates RNA capable

of duplex formation, consequently triggering silencing and

directing methylation (Muskens et al., 2000; Selker, 1999).

Consistent with this model, severe methylation was found

in the centre of the IR in some of the IR T-DNA loci analysed

(De Buck and Depicker, 2001; Stam et al., 1998; Van Houdt

et al., 2000).

Mette et al. (2000) showed that TGS could be triggered by

a double-stranded RNA containing promoter sequences. In

contrast, Arabidopsis plants containing an IR promoter

construct not capable of dsRNA production did not display

TGS. Thus, it is tempting to speculate that IR structures that

are not transcribed are not prone to silencing. It is inter-

esting to note that we did not see evidence for gene silen-

cing associated with IR T-DNA structures in lines unlikely to

produce read-through transcripts spanning transgenes and

the centre of the IR. In transgenic lines GUS 1xr IR 102 and

GFP 1xr IR 127, the reporter genes were transcribed in the

direction of the LB. In contrast, the direction of transcription

of the reporter genes proceeded in the direction of the

centre of the IR in transgenic lines GUS 1xf IR 57 and

SPT 3xf IR 159. Nevertheless, the production of the read-

through transcripts was very unlikely as the transgenes

Table 1 Compilation of reporter gene number, arrangement and expression in the transgenic lines studied

Transgene T-DNA locusT-DNAcopy number

Reportergene copy number

Repeatstructure

Reportergene expression

SPT SPT 1xr 882 2 2 HighSPT 2xf 1072 2 4 TR HighSPT 3xf 1472 2 6 TR HighSPT 3xf IR 1591 2 6 TR/IR HighSPT 3xf A�, SPT 3xf B�, SPT 3xf C� 4 12 TR Silencing

GFP GFP 1xr 1292, GFP 1xr 1452, GFP 1xr 1552 2 2 HighGFP 1xr IR 1272 4 4 IR High

GUS GUS 2xr 1141 1 2 TR HighGUS 1xf IR 571 2 2 IR HighGUS 1xr IR 1021 2 2 IR HighGUS 2xr 1142 2 4 TR SilencingGUS 1xf IR 572 4 4 IR SilencingGUS 1xr IR 1022 4 4 IR Silencing

�Plants harbour two different T-DNA loci in the homozygous state.1Plants hemizygous for the T-DNA locus.2Plants homozygous for the T-DNA locus.

� Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 507–517

Repeat structures and transgene silencing 513

were located at a considerable distance from the RB

(Figure 1d). Moreover, two sets of polyadenylation signals

downstream of the transgenes (ocs30 and nos30, Figure 1d)

should allow for efficient termination of transgene tran-

scription. Despite the fact that several methylation-sensi-

tive restriction endonucleases were used to characterise the

structures of the IR T-DNA loci, we did not find evidence for

methylation in the centres of the IR structures (data not

shown).

Post-transcriptional silencing in transformants carrying

12 copies of the SPT transgene or four copies of

the GUS transgene

Silencing of chimaeric reporter genes was observed in

plants harbouring four copies of the SPT 3xf T-DNA

(Figure 2, SPT 3xf A, B, C). Likewise, plants homozygous

for the GUS 2xr T-DNA (GUS 2xr 114) or the IR T-DNA loci

(GUS 1xf IR 57 or GUS 1xr IR 102; Figure 4) showed silen-

cing (Table 1). siRNAs corresponding to the transcribed

region of a silenced gene are an important hallmark of

RNA-mediated silencing (Hamilton and Baulcombe,

1999). Thus, the detection of siRNAs specific for the tran-

scribed regions of the SPT and GUS genes in silenced lines,

but not in plants expressing these reporter genes at high

levels, strongly supports a post-transcriptional mechanism

of gene silencing (Figures 2 and 4b).

Assaying plants of different ages and from various gen-

erations for GUS activity clearly showed that silencing was

established during plant development and that the silent

state was reset after meiosis. These results also support a

post-transcriptional mechanism because similar observa-

tions were made for several genes that were silenced by

PTGS (summarised by Vaucheret et al., 1998).

In all transgenic lines displaying SPT transgene silencing,

a truncated SPT transcript was found concomitantly with

the full-length mRNA (Figure 2a, data not shown). The

truncated transcript could not be attributed to reporter gene

cassettes of aberrant structure (data not shown). It is likely

that the truncated transcript is specific for silenced SPT

transgenes because the transcripts truncated by endonu-

cleolytic cleavage have been found to be associated with

several genes silenced by post-transcriptional mechanisms

(e.g. Goodwin et al., 1996; Han and Grierson, 2002; Metzlaff

et al., 1997).

Silencing of the GUS reporter genes was observed in

plants harbouring four copies of the GUS transgene, but

not in plants carrying two copies (Figure 4). Similarly,

silencing of the SPT reporter genes was found in plants

with four copies of the SPT 3xf T-DNA, but not in lines

homozygous for a single locus of SPT 3xf (Figure 2). Thus, it

is unlikely that unintended antisense transcripts contribu-

ted to gene silencing in the lines evaluated in this study;

instead, the results were consistent with silencing triggered

by threshold concentrations of either transgene transcript

or another product of transgene expression.

The threshold hypothesis cogently explains several

observations made when studying co-suppression by

Chs transgenes. For instance, in one study, the degree of

co-suppression correlated with the number of Chs trans-

genes (Jorgensen et al., 1996). In another study, promoter

strength of the Chs transgenes was found to be an impor-

tant determinant for the extent of co-suppression (Que

et al., 1997). Additional support for the role of a threshold

in triggering silencing was provided by several studies in

which silencing was more pronounced in homozygous

than in hemizygous plants (summarised by Vaucheret

et al., 1998). Kunz et al. (1996) studied PTGS of the tobacco

class I chitinase gene CHN48. Importantly, a high incidence

of silencing was found in plants that harboured T-DNA loci

SSC2.3 or SSC2.4 in the homozygous state or both loci

hemizygously, whereas plants hemizygous for either T-

DNA locus SSC2.3 or SSC2.4 showed high expression. This

revealed that competence for chitinase gene silencing

depended on the transgene dose rather than on the inter-

action of alleles at the same locus.

Silencing of the GUS and SPT transgenes correlated with

high transgene doses, but intriguingly, the analysis of

several independent lines revealed a different copy number

threshold for the two transgenes (Table 1). The two repor-

ter genes were controlled by the same set of regulatory

elements; moreover, the same basic T-DNA vector (pMDL-

T-DNA) was used to generate the different constructs

(Figure 1). This suggests that the nature of the transcribed

region (for example, transcript stability, length or the con-

tent of guanine and cytosine residues (GC content) of the

coding sequence) may play an important role in determin-

ing the copy number threshold at which silencing is trig-

gered. The GUS and SPT transgenes differ in respect to the

length and the GC content of the coding sequence. Inter-

estingly, Petunia hybrida plants transformed with the maize

A1 cDNA showed a higher frequency of transgene inactiva-

tion than the transformants that harboured the gerbera gdfr

cDNA. Both cDNAs encoded dihydroflavonol-4-reductase

activity and were controlled by the CaMV 35S promoter, but

they differed markedly in the base composition. Based on

these results, Elomaa et al. (1995) suggested that a trans-

gene GC content very similar to that of the recipient gen-

ome would prevent the introduced gene from inactivation.

However, in the case of the GUS and SPT transgenes,

silencing is triggered more readily for the GUS than

for the SPT transgene, even though the former has a

GC content more similar to that of the Arabidopsis genome.

Decreased transcript stability correlated with a reduced

degree of Chs gene co-suppression (Que et al., 1997);

thus, it will be important to determine whether the

GUS and SPT transgenes differ in respect to transcript

stability.

� Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 507–517

514 Berthold Lechtenberg et al.

For some transgenic lines harbouring IR T-DNA struc-

tures, the read-through transcripts capable of duplex for-

mation were implicated in the silencing process

(summarised by Muskens et al., 2000). However, formation

of such transcripts is unlikely for at least some of the IR T-

DNA structures studied to date (summarised by Muskens

et al., 2000) and for the structures presented in this work.

We favour the idea that high transgene doses triggering

PTGS may, in many cases, account for the silencing asso-

ciated with repetitive T-DNA structures. Notably, the

threshold model also offers an explanation for the high

frequency of silencing that is observed for transgenes

harbouring intrinsic direct repeats (Ma and Mitra, 2002).

Experimental procedures

Standard molecular biology techniques

Standard molecular biology techniques were carried out accordingto Sambrook et al. (1989). Plant genomic DNA isolation was per-formed as described by Dellaporta et al. (1983). T-DNA flankingsequences were isolated and mapped to the chromosomesequence maps according to Forsbach et al. (2003).

T-DNA constructs

The basic T-DNA vector pMDL-T-DNA was used in all transforma-tion experiments (Figure 1a; Forsbach et al., 2003). The different T-DNA constructs were distinguished by the kind and number ofchimaeric reporter genes (Figure 1b), which were inserted intovector pMDL-T-DNA.

The chimaeric SPT reporter gene cassette was derived as a HindIIIfragment from the vector SLJ1491 (Jones et al., 1992). One to threecopies of this cassette were cloned into pMDL- T-DNA to generateconstructs SPT 1xr, SPT 2xf and SPT 3xf (Figure 1c).

The BglII/ApaI fragment from the vector SLJ1491 which con-tained part of the CaMV 35S::SPT::ocs30 reporter gene cassette wasreplaced with a BglII/ApaI fragment from SLJ4D4 (Jones et al.,1992) to yield a plasmid which contained a chimaeric GUS reportergene cassette (pMDL-GUS). The GUS reporter gene cassette wasrecovered as a HindIII fragment and cloned into pMDL-T-DNA.

Plasmid pBIN mgfp5-ER (J. Haseloff, University of Cambridge,UK) was cut with XbaI and SstI to isolate the mgfp5-ER codingsequences. After an end-filling reaction with the Klenow-fragmentof DNA polymerase I, this fragment was cloned into vector pMDL-GUS, which had been restricted with NcoI/BamHI, and subse-quently treated with mungbean nuclease. The resulting plasmidpMDL-mgfp5-ER contained a chimaeric GFP reporter gene cassettethat was cloned as a HindIII fragment into the HindIII site of pMDL-T-DNA.

Agrobacterium-mediated transformation of Arabidopsis

thaliana

The T-DNA vectors were transformed (Hofgen and Willmitzer,1988) into Agrobacterium tumefaciens strain LBA4404 and usedfor Agrobacterium-mediated transformation. Root explant trans-formation was performed as described by Valvekens et al. (1988),and the protocols developed by Bechtold et al. (1993) or Cloughand Bent (1998) were used for in planta transformation.

Genetic analysis of transgenic lines

All derivatives of the pMDL-T-DNA vector that were used in thetransformation experiments contained a chimaeric NPT geneconferring kanamycin resistance. Seeds were plated on kanamy-cin-containing medium (50 mg l�1 kanamycin) to determine thesegregation data for this dominant selectable marker gene.

Transgenic lines harbouring one or more chimaeric SPT geneswere germinated on an agar medium containing streptomycin(200 mg l�1) to assay the introduced transgene(s) for activity.

Plant growth conditions

Seeds were germinated on kanamycin-containing agar mediumand kept for 2 weeks in sterile culture (16 h light at 208C, 8 h dark at188C, 70% relative humidity) before they were transferred to thesoil. Growth in soil took place under short-day conditions (9 h lightat 208C, 15 h dark at 188C, 70% relative humidity).

RNA preparation and Northern blot analysis

Total RNA was prepared from the Arabidopsis leaves using thePeqGOLD-RNAPureTM Kit (PeqLab Biotechnologie, Erlangen, Ger-many). Spectrophotometric measurements were carried out todetermine the concentration of the RNA solutions. Total RNAwas then separated on 1.2 or 1.5% agarose gels in the presenceof formaldehyde. Transfer onto nylon membranes was performedaccording to the manufacturer’s instructions (Hybond Nþ Amer-sham, Braunschweig, Germany). Hybridisation was carried outusing a buffer described by Church and Gilbert (1984). The blotswere pre-hybridised for 30 min at 688C, and hybridisations wereperformed overnight at 588C. Blots were washed at room tem-perature with 6� SSPE/0.5% SDS, 4� SSPE/0.5% SDS and 2�SSPE/0.5% SDS for 5 min each. Subsequently, Northern blots wereincubated for 30 min at 608C with 2� SSPE/0.1% SDS.

For the isolation of siRNAs, the total RNA was precipitated for20 min at �208C, and after centrifugation, the pellet was re-sus-pended in DEPC-treated dH2O. Electrophoresis was performed on2 or 2.5% agarose gels in the presence of formaldehyde. Blots ofsiRNAs were pre-hybridised at 588C, and hybridisations wereperformed overnight at 428C. Washing was carried out at roomtemperature for 5 min each with 6� SSPE/0.5% SDS, 4� SSPE/0.5% SDS and 2� SSPE/0.5% SDS, then the blots were incubatedfor 15 min at room temperature with 2� SSPE/0.1% SDS.

Colorimetric b-glucuronidase activity assay

The transgenic lines were analysed for GUS activity using histo-chemical staining (Jefferson, 1987). Leaf discs were incubatedovernight in 96-microwell plates at 378C, with phosphate buffer(50 mM sodium phosphate pH 7.0, 1 mM EDTA, 0.1% Triton-X-100) containing 1 mM X-Gluc.

Assay for the detection of GFP fluorescence

For each transgenic line, two extracts were prepared from 10 to 15small rosette leaves derived from up to 10 plants in 1 ml, 1 M Tris(pH 8.0). Protein concentrations were determined for two aliquotsof each of the plant extracts using the Bio-Rad Protein Assay(Bio-Rad, Richmond, USA), based on the method of Bradford.After 10-fold dilution in Tris-buffer (1 M, pH 8.0), two aliquots ofeach of the extracts were used for fluorescence measurements.Fluorescence was measured for each sample with excitation at480 nm, emission at 510 nm at the Versa-FluorTM fluorimeter

� Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 507–517

Repeat structures and transgene silencing 515

(Bio-Rad, Richmond, USA), relative to the 1 M Tris-buffer (pH 8.0).For each of the transgenic lines analysed, the mean of the fourfluorescence measurements and the standard deviation weredetermined. GFP fluorescence is given as relative fluorescenceunits per milligram protein.

Acknowledgements

The research was initiated at the Max-Delbruck-Laboratorium inder Max-Planck-Gesellschaft (Koln, Germany) with a grant fromthe Bundesministerium fur Bildung und Forschung (BMBF, grantno. 0311107). D. S. and B. L. were supported by fellowships fromthe Max Planck Society. We thank the greenhouse staff at the Max-Delbruck-Laboratorium and the Max Planck Institute of MolecularPlant Physiology for taking care of the plants, and S. Stegemannfor excellent technical assistance. J. Jones (Sainsbury Laboratory,Norwich, UK) and J. Haseloff (University of Cambridge, UK) aregratefully acknowledged for providing plasmids. We thank M.McKenzie for carefully editing the manuscript.

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