Báo cáo khoa học: Loss of sense transgene-induced post-transcriptional gene silencing by sequential introduction of the same transgene sequences in tobacco pot

Loss of sense transgene-induced post-transcriptional gene
silencing by sequential introduction of the same transgene
sequences in tobacco
Sayaka Hirai
1
, Kouta Takahashi
2
, Tomomi Abiko
3
and Hiroaki Kodama
1
1 Graduate School of Horticulture, Chiba University, Japan
2 Graduate School of Science and Technology, Chiba University, Japan
3 Faculty of Horticulture, Chiba University, Japan
Introduction
Genetic transformation is a powerful tool of improve-
ment of plant physiological traits, and is important to
both basic and applied sciences. Successful expression
of transgene sequences is always desired, and overex-
pression of the transgene is usually selected in a popu-
lation of transgenic plants. The transgene itself is often
recognized as a sequence of invasive nucleic acids and
triggers RNA silencing [1–4]. RNA silencing, an RNA-
mediated suppression of gene activity, is a common
phenomenon in most eukaryotic organisms. Sense
transgene-induced post-transcriptional gene silencing
(S-PTGS) is a representative phenomenon of RNA
silencing targeting the sense transgene, and the
transgene and its homologous endogenous genes are
suppressed simultaneously [5,6]. S-PTGS is usually
observed in a portion of transgenic plants. In S-PTGS

Keywords
fatty acid desaturase; post-transcriptional
gene silencing; RNA-directed DNA
methylation; threshold; a-linolenic acid
Correspondence
H. Kodama, Graduate School of Horticulture,
Chiba University, 648 Matsudo, Chiba
271-8510, Japan
Fax: +81 43 290 3942
Tel: +81 43 290 3942
E-mail:
(Received 31 October 2009, revised 23
January 2010, accepted 26 January
2010)
doi:10.1111/j.1742-4658.2010.07591.x
RNA silencing is an epigenetic inhibition of gene expression and is guided
by small interfering RNAs. Sense transgene-induced post-transcriptional
gene silencing (S-PTGS) occurs in a portion of a transgenic plant popula-
tion. When a sense transgene encoding a tobacco endoplasmic reticulum
x-3 fatty acid desaturase (NtFAD3) was introduced into tobacco plants,
an S-PTGS line, S44, was obtained. Introduction of another copy of the
NtFAD3 transgene into S44 plants caused a phenotypic change from
S-PTGS to overexpression. Because this change was associated with the
methylation of the promoter sequences of the transgene, reduced transcrip-
tional activity may abolish S-PTGS and residual transcription of the sense
transgene may account for the overexpression. To clarify whether
RNA-directed DNA methylation (RdDM) can repress the transcriptional
activity of the S44 transgene locus, we introduced several RdDM
constructs targeting the transgene promoter. An RdDM construct harbor-
ing a 200-bp-long fragment of promoter sequences efficiently abrogated the

generation of NtFAD3 small interfering RNAs in S44 plants. Transcription
of the transgene was partially repressed, but the resulting NtFAD3 mRNAs
successfully accumulated and an overexpressed phenotype was established.
Our results indicate an example in which overexpression of the transgene is
established by complex epigenetic interactions among the transgenic loci.
Abbreviations
CaMV, cauliflower mosaic virus; ChIP, chromatin immunoprecipitation; CHS, chalcone synthase gene; GFP, green fluorescent protein;
GUS, b-glucuronidase; H3K4me3, histone H3 trimethylated at lysine-4; H3K9me2, histone H3 dimethylated at lysine-9; Nii, tobacco nitrite
reductase gene; Nos, nopaline synthase; nptII, neomycin phosphotransferase II gene; NtFAD3, tobacco endoplasmic reticulum x-3 fatty acid
desaturase; RACE, rapid amplification of 5¢ cDNA ends; RdDM, RNA-directed DNA methylation; RDR6, RNA-dependent RNA polymerase6;
siRNA, small interfering RNA; S-PTGS, sense transgene-induced post-transcriptional gene silencing; TSP, transcriptional start point.
FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS 1695
plants, transcription of the transgene may produce
aberrant RNAs with unusual structures, and aberrant
RNAs may promote the recruitment of an RNA-
dependent RNA polymerase6 (RDR6) [7,8]. Aberrance
has been found in transgene mRNAs lacking a cap
structure or a polyA structure, which are generated
from abortive elongation, readthrough of transgene
transcription and ⁄ or a defect in RNA processing. The
genomic insertion manner of transgenes, namely
repeated arrangement and truncated and ⁄ or rear-
ranged transgene copies, may be a cause of synthesis
of aberrant RNAs [9–11]. RDR6 synthesizes a comple-
mentary RNA using aberrant transcripts as a template,
and the resulting dsRNAs are processed into the
21–25-nucleotide-long, small interfering RNAs (called
siRNAs) by DICER-like protein4. The single-stranded
siRNA is incorporated into a multicomponent RNA-
induced silencing complex and guides the endonucleo-

lytic cleavage of transcripts of the transgene and
homologous endogenous genes. ARGONAUTE1 is a
main slicer in the RNA-induced silencing complex
[7,12,13].
Several reports have described a correlation between
the incidence of S-PTGS and high transgene copy
number [9,14]. For example, three or more copies of
the b-glucuronidase (GUS) transgene and five or more
copies of the green fluorescent protein (GFP) transgene
are required to trigger S-PTGS [15,16]. Silencing
induced by an increase in transgene copy number is
associated with the generation of transgene siRNAs
and, also, DNA methylation in the transgene locus
[16,17]. These results imply the existence of a threshold
concentration of transcripts for the onset of S-PTGS.
This concept is known as RNA threshold theory
[11,18,19]. At present, the molecular mechanism for
the recruitment of RDR6 protein in RNA threshold
theory still remains unresolved.
Although RNA threshold theory has been recog-
nized as a convincing model for the triggering of
S-PTGS, no study has reported phenotypic changes
from S-PTGS to overexpression of the transgene
manipulated by the modulation of transcriptional
activity. In this article, we report the successful conver-
sion from S-PTGS to overexpression by a decrease in
the transcriptional activity of the transgene. When
transcription of the transgene was partially repressed
by methylation of transgene promoter sequences,
siRNAs targeting the coding region of the transgene

disappeared and residual transcription of the transgene
caused a phenotypic change from S-PTGS to overex-
pression. This result indicates that, at least in this case,
overexpression of the transgene is established via com-
plex mechanisms, including RNA silencing.
Results
Recovery from S-PTGS by sequential introduction
of a same-sense transgene sequence
A tobacco microsomal x-3 fatty acid desaturase
(NtFAD3) converts linoleic acid (18:2) to a-linolenic
acid (18:3). A sense transgene, pTF1SIIn, expresses
the NtFAD3 cDNA under the control of the El2 pro-
moter [20]. Most transgenic plants showed an overex-
pressed phenotype, namely increased 18:3 content, and
the S24 line was used as a representative of such over-
expressors (Fig. 1A). A cosuppressed line, S44, which
had been transformed with pTF1SIIn, showed a large
reduction of the leaf 18:3 level, and accumulation of
A
C
B
Fig. 1. Loss of S-PTGS phenotype in sequentially transformed
plants with NtFAD3 sense and NtFAD3:GUS chimeric sense transg-
enes. (A) Characterization of the primary transgenic plants with
pTF1SIIn. The 18:3 level of each representative plant [wild-type
(WT), homozygous S24 and S44 plants] was determined, followed
by the detection of NtFAD3 mRNAs and siRNAs. (B) Characteri-
zation of the sequentially transformed plants with pTF1SIIn and
pEx9-GUS-h. The levels of 18:3 in total fatty acids and the amounts
of NtFAD3 mRNA and NtFAD3 siRNA in leaves are shown. A repre-

sentative 18:3 level of each plant is shown. The constitutively
expressed 28S rRNA and 5S rRNA are also shown to assess the
equivalence of RNA loading amounts. (C) Levels of NtFAD3 tran-
scripts in leaf tissues. The presence of the NtFAD3:GUS chimeric
construct in the genome was determined by PCR amplification (a).
The levels of NtFAD3 transcripts originating from the primary
transgene (pTF1SIIn, b), secondary transgene (pEx9-GUS-h, c) and
endogenes (d) were determined by RT-PCR analysis.
Transcriptional regulation in S-PTGS S. Hirai et al.
1696 FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS
NtFAD3 siRNAs [21]. Most NtFAD3 siRNAs were 21
nucleotides in length [21], and the level of 24-nucleo-
tide NtFAD3 siRNAs was low (Fig. 1A). To monitor
the tissue-dependent expression of the transgene in
S44 plants, we constructed a plasmid, pEx9-GUS-h, in
which a NtFAD3 cDNA was fused with GUS under
the control of the El2 promoter sequence (Fig. S1A,
see Supporting information). pEx9-GUS-h was intro-
duced as a secondary transgene into homozygous S44
plants, and four independent transformants (S44-SH1–
4) were obtained (Fig. S1C). Unfortunately, the resul-
tant fusion protein did not show any activity of either
GUS or NtFAD3 protein in wild-type plants (data not
shown). We found a unique phenotype in S44-SH
lines. After self-pollination, the offspring of three lines
(S44-SH2–4) showed increased 18:3 content in leaves
relative to that of the wild-type, and S44-SH1 off-
spring showed nearly the same 18:3 levels as those of
S44 leaves (Fig. S1D). NtFAD3 mRNAs were detected
at a high level in S44-SH2 leaves, which was associ-

ated with the disappearance of NtFAD3 siRNA
(Fig. 1B). Therefore, the phenotype of S44 was con-
verted from S-PTGS into overexpression of the
NtFAD3 transgene in S44-SH2 plants. In contrast,
NtFAD3 siRNAs were detected in S44-SH1 leaves,
indicating that S-PTGS is maintained. As expected
from the phenotype, expression of the NtFAD3 endo-
gene was recovered, and the primary NtFAD3 trans-
gene was highly expressed in S44-SH2 leaves.
Strangely, expression of the secondary NtFAD3 trans-
gene (namely NtFAD3::GUS chimeric gene) was lost in
S44-SH2 plants (Fig. 1C). In fact, northern analysis
with the GUS probe showed that NtFAD3::GUS
mRNA weakly accumulated in S44-SH1 plants, but
not in S44-SH2 plants (Fig. S1E).
We investigated the methylation status of the El2
promoter sequences in S44-SH1 and S44-SH2
(Fig. 2). Because the El2 promoter was used in both
primary and secondary transgenes, it was difficult to
discriminate the methylation status of each promoter
sequence by Southern hybridization. The El2
promoter consists of two tandemly repeated enhancer
regions of the cauliflower mosaic virus (CaMV) 35S
promoter [22] (Fig. S1B). Two main fragments, 325
and 476 bp in length, were generated from the El2
promoter sequence after digestion with AccI ⁄ MboI,
and could be visualized using the CaMV 35S
promoter sequence as a probe. These fragments were
detected in the AccI ⁄ MboI-cut S44-SH1 genome
DNA, but not in the S44-SH2 genome DNA. An

801-bp-long fragment was generated in S44-SH2
plants, and this fragment was in agreement with
the expected size when the AccI site was methylated.
A similar result was observed by Southern analysis
with HinfI ⁄ MboI, and a larger fragment than
the expected size was generated in S44-SH2 plants
(Fig. 2). This result suggests that transcription of
the NtFAD3 secondary transgene is severely
repressed and transcription of the primary NtFAD3
transgene is partially repressed by methylation in El2
promoter sequences in S44-SH2 plants relative to
S44 plants.
B
A
Fig. 2. Methylation status of the transgene promoter in the
sequentially transformed plants with NtFAD3 sense and NtFAD3:-
GUS chimeric sense transgenes. (A) Schematic drawing of the El2
promoter region. The AccI, HinfI and MboI sites are indicated. The
lengths of the predicted restriction fragments are also shown. (B)
Southern blot of HinfI ⁄ MboI- and AccI ⁄ MboI-digested genomic
DNAs. DNAs were isolated from S44-SH1 and S44-SH2 plants, and
hybridized with a probe for the CaMV 35S promoter. The lengths
of the observed bands are given on the left. The dot at 1228 bp
in the left panel is a hybridization artifact. The 893-bp HinfI ⁄ MboI
fragment corresponds to the long fragment including 251, 317 and
325 fragments.
S. Hirai et al. Transcriptional regulation in S-PTGS
FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS 1697
siRNAs targeting the El2 promoter abolish
S-PTGS

We examined whether a lowered transcriptional rate of
the primary transgene was associated with the evasion
of S-PTGS. siRNAs targeting the promoter sequences
can direct de novo DNA methylation on the corre-
sponding cytosine residues (RNA-directed DNA meth-
ylation, RdDM), which results in epigenetic silencing
[23–25]. Three distinct RdDM constructs targeting the
El2 promoter were introduced into S44 plants
(Fig. S1A). The regenerated double transformants were
self-pollinated, and the phenotype of the resulting
offspring was investigated. pIR-HEAD targets two
tandemly arranged regions of the El2 promoter [)743
to )554 and )418 to )219 relative to the transcrip-
tional start point (TSP); Fig. S1B], and was introduced
into a homozygous S44 plant. The resulting six inde-
pendent lines, namely S44-head-1 to S44-head-6, were
obtained (Fig. S2, see Supporting information). The
amount of NtFAD3 mRNA was nearly the same or
somewhat lower in these S44-head lines than in the
S44 parental line, and NtFAD3 siRNAs existed in
all S44-head lines (Fig. 3A). An RdDM construct,
pIR-TATA, targets the basal region of the El2 pro-
moter ()200 to +1; Fig. S1B). Two independent trans-
genic lines, S44-tata-1 and S44-tata-2, were obtained.
The phenotype of these two lines was similar to that
of S44 plants, and NtFAD3 siRNAs were detected
(Fig. 3A). One conspicuous result was obtained by the
introduction of an RdDM construct (pIR-END) that
targets two regions, from )291 to )91 (termed the
END2 region) and from )618 to )419 (termed the

END1 region), of the El2 promoter (Fig. S1B). All
seven independent transgenic lines, S44-end-1 to S44-
end-7, consistently showed higher leaf 18:3 content
than wild-type leaves (Fig. S2). A representative line
(S44-end-2) showed an increased amount of NtFAD3
mRNAs in leaves, and also a large decrease in the
NtFAD3 siRNA level (Fig. 3A). These results indicate
that RdDMs targeting two distinct 200-bp-long regions
of the El2 promoter (END1 and END2) are effective
for the evasion of S-PTGS in S44 plants.
When total RNAs prepared from leaf tissues were
used in RT-PCR analysis, the NtFAD3 mRNAs
derived from both transgene and endogenous counter-
parts were detected at a high level in S44-end-2 plants.
In contrast, nuclear nascent transcripts of the NtFAD3
transgene could be detected in S44, S44-head and S44-
tata leaves. However, in S44-end-2 leaves, the level was
reduced in comparison with that of S44 plants
(Fig. 3B). Quantitative real-time RT-PCR analysis indi-
cated that both transcripts for the NtFAD3 transgene
and endogene showed an increase of about 1.4-fold in
the total RNA fraction of S44-end-2 plants in compari-
son with S44 plants (1.43 ± 0.30 for the transgene and
1.49 ± 0.30 for the endogene). In contrast, the level of
nuclear transcripts of the NtFAD3 transgene was
reduced to 0.75 ± 0.20 of those of S44 plants
(mean ± SD, n = 3). We also analyzed TSP of the
transgene in S44 and S44-end-2 plants by the rapid
amplification of 5¢ cDNA ends (RACE). The resulting
RACE products amplified from both plants showed a

clear single band after electrophoresis (Fig. S3, see Sup-
porting information), and the 5¢-termini of the NtFAD3
transgene cDNAs were distributed in the same region
of the transgene sequences, suggesting that the NtFAD3
transgene was transcribed under the control of the
same core promoter in S44 and S44-end plants.
Thus, it is likely that the transcription of the primary
NtFAD3 transgene is repressed, even though the steady-
state level of NtFAD3 mRNA was detected at a high
level in leaf tissues in S44-end-2 plants. These results
indicate that the END1 and ⁄ or END2 region of the El2
promoter should play an important role in siRNA gen-
eration of the downstream NtFAD3 transgene.
Methylation status of END regions of the El2
promoter
Cumulative methylation in all sequence contexts (CG,
CHG and CHH; H represents A, T or C) in the
A
B
Fig. 3. Loss of S-PTGS phenotype by the introduction of an RdDM
construct targeting the El2 promoter. (A) Characterization of the
transgenic plants used. The levels of 18:3 in total fatty acids and
the amounts of NtFAD3 mRNA and NtFAD3 siRNA in leaves are
shown. Two descendants of each representative double transfor-
mant were prepared. A representative 18:3 level of each plant is
shown. The equivalence of RNA loading is shown as stated in the
legend to Fig. 1. (B) Levels of NtFAD3 transcripts in leaf tissues.
Total RNA and nuclear RNA were used as templates for RT-PCR.
Transcriptional regulation in S-PTGS S. Hirai et al.
1698 FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS

END1 and END2 regions is shown in Fig. 4. In the
END1 region, most cytosine residues were highly
methylated in both S44 and S44-end plants (Fig. 4A).
The END2 region was hypermethylated in S44-end-2
plants, and almost all cytosine residues were methylat-
ed (Fig. 4B). The RdDM by pIR-END did not affect
the methylation status in the NtFAD3 coding region in
spite of a large decrease in the NtFAD3 siRNA level
(Fig. 4C). We also investigated the methylation status
of the END2 region in S24 plants, a successful over-
expressor of pTF1SIIn [21]. The END2 region was
hypomethylated in S24 plants (Fig. S4, see Supporting
information). pIR-END was then introduced into S24
plants, and three of seven transgenic plants showed a
18:3 level similar to that of the wild-type. A represen-
tative line (S24-end-4) showed an 18:3 content similar
to that of the wild-type, and all sequence contexts in
the END2 region of the El2 promoter were heavily
methylated (Fig. S4). Therefore, RdDM by pIR-END
caused efficient methylation in the END2 region,
which was followed by the abolishment of the over-
expression phenotype in S24 plants.
Cytosine methylation appears to direct changes in
chromatin conformation to the silent chromatin
region. However, the features of histone H3 modifica-
tion at the El2 promoter were essentially the same in
S44 and S44-end-2 plants (Fig. 4D). The El2 promoter
in S24 plants was associated with histone H3 trimethy-
lated at lysine-4 (H3K4me3), whereas this promoter
was free of histone H3 dimethylated at lysine-9

(H3K9me2). H3K4me3 and H3K9me2 are frequently
found in euchromatin and heterochromatin, respec-
tively [26,27]. In contrast, we detected the existence of
both H3K9me2 and H3K4me3 at the El2 promoter
region in S44 plants. The NtFAD3 siRNAs are defi-
cient in S44-end-2 plants, but the loss of these siRNAs
was not associated with any remarkable changes in
chromatin modification in the promoter region. In
summary, the END1 and END2 regions were highly
methylated in S44 plants, even though the NtFAD3
transgene was transcribed. After RdDM by pIR-END,
the END2 region was almost completely methylated,
and S-PTGS was compromised.
Discussion
Release of S-PTGS during sequential
transformation
Several reports have described a correlation between
the incidence of S-PTGS and high transgene copy
number [9,14]. Silencing induced by an increase in
transgene copy number is associated with the genera-
tion of transgene siRNAs and also DNA methylation
in the transgene locus [16,17]. Unlike these previous
results, the phenotype seen in S44-SH2 plants indicates
that duplication of the promoter sequence should trig-
ger phenotypic conversion. Re-introduction of pro-
moter sequences frequently suppresses the expression
of a previously introduced transgene, which is corre-
lated with methylation at the promoter region [28,29].
The increased methylation in the El2 promoter and the
disappearance of the NtFAD3::GUS transcripts in S44-

SH2 plants are in good agreement with these previous
observations. The methylation at the El2 promoter
should coincidently occur in both primary and second-
ary transgenes. In this respect, the transcriptional
activity of a primary transgene should decline even
though its mRNA successfully accumulates by
deficiency of NtFAD3 siRNAs.
The release of S-PTGS has been observed in several
transgenic plants. Petunia C001 transgenic plants pro-
duce white flowers by S-PTGS of the chalcone syn-
thase gene (CHS) [9]. The C002 line is obtained
spontaneously in the offspring of C001, and produces
purple flowers. In C002 plants, cytosine residues at the
transgene promoter are methylated, and transgene
expression is severely inhibited. CHS siRNAs are
absent, and the expression of endogenous CHS genes
A
D
BC
Fig. 4. Bisulfite sequence and ChIP analysis of the transgene
sequence. (A) Degree of cytosine methylation at the END1 region
of the El2 promoter. (B) Degree of cytosine methylation at the
END2 region of the El2 promoter. (C) Degree of cytosine methyla-
tion at the NtFAD3 coding region of the transgene. The percent-
ages of methylated cytosines in CpG, CpHpG and CpHpH were
determined in S44 (black bars) and S44-end-2 (grey bars) plants
(A–C). (D) Histone modification in transgenic plants. Chromatin frac-
tions prepared from S44, S44-end-2 and S24 leaves were immuno-
precipitated (IP) using antibodies specific for H3K9me2 and
H3K4me3. IgG was provided by a kit supplier and used as a control

antibody. DNA was then purified from immunoprecipitants and sub-
jected to PCR analysis. Genomic regions for the El2 promoter were
amplified.
S. Hirai et al. Transcriptional regulation in S-PTGS
FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS 1699
is restored in C002 plants [30,31]. Similarly, S-PTGS
of the neomycin phosphotransferase II gene (nptII)is
eliminated during regeneration from in vitro-cultured
cells [32]. Expression of the transgene is transcription-
ally silenced by dense methylation of the promoter
sequences, and siRNAs for the nptII gene disappear.
When the expression of the transgene is almost com-
pletely inhibited by DNA methylation, the generation
of siRNAs should be repressed because of the absence
of RDR6 templates. Elimination of S-PTGS is also
observed when expression of the transgene is partially
inhibited. S-PTGS of the tobacco nitrite reductase gene
(Nii) is abolished in the regenerated plants from
in vitro-cultured tissues [19]. The promoter for the Nii
transgene is methylated in cosuppressed plants, but is
free from methylation in plants escaping S-PTGS.
Curiously, Nii transgene expression is weakened, even
though its transcription is controlled by the methyla-
tion-free promoter. One of the common features of
these plants free from S-PTGS is the attenuated
expression of the transgene, and therefore the thresh-
old theory has been proposed as a trigger of S-PTGS.
In this respect, recovery from S-PTGS in S44-SH2
plants may be accounted for by the threshold theory.
Promoter activity determines the recruitment of

RDR6 on transcripts of the NtFAD3 transgene
The introduction of pIR-END into S24 plants induces
a dense methylation and eliminates the overexpression
of the NtFAD3 transgene (Fig. S4). In contrast, the
El2 promoter is highly methylated in S44 plants, and
pIR-END induces only a slight increase in methylation
in S44-end-2 plants. Transcriptional inactivation by
RdDM requires both DNA methylation and histone
modification, especially H3K9me2 [33]. Although
pIR-END does not affect significantly H3K9me2 and
H3K4me3 at the El2 promoter sequences, as seen in
S44-end-2 plants (Fig. 4D), another epigenetic change
in chromatin structure is regulated by RdDM. In S44
plants, there should be a chromatin conformation that
allows active transcription, driven by a densely methy-
lated El2 promoter. This chromatin conformation may
be interfered with by introduction of pIR-END, and
transcriptional acitivity will be decreased significantly.
Considered together, promoter activity determines the
recognition of downstream NtFAD3 transcripts by
RDR6. However, the initial trigger of recognition of
transgene transcripts by RDR6 is still unknown. The
low transcription may avoid the incidence of irregular
processing of transcripts, or RDR6 may not be able to
recognize such a low quantity of transgene mRNA in
S44-end plants.
Our results indicate an example in which the trans-
gene is overexpressed as a result of the interaction of
RNA silencing. The instability of transgene expression
has often been observed in offspring and, in some

cases, overexpression and silencing of the transgene are
segregated. Attention should be paid to the overexpres-
sion of the transgene established by the latent influence
of silencing mechanisms.
Materials and methods
Plasmid construction and plant transformation
The sense transgene construct, pTF1SIIn, contains NtFAD3
cDNA under the control of the El2 sequence. A NtFAD3
cDNA fragment (nucleotide position 52–1035 of GenBank
Acc. No. D26509) was fused to the N-terminus of the GUS
gene and inserted into pTF1SIIn to replace the NtFAD3
sequence. Then, a cassette containing a nopaline synthase
(Nos) promoter and hygromycin phosphotransferase B gene
was inserted into the EcoRI site to generate pEx9-GUS-h.
The CaMV 35S promoter of pSH-hp-RDR6 [34] was
replaced with a Nos promoter, and the inverted repeat
sequences were replaced with the sense and antisense frag-
ments of the El2 promoter as follows. XbaI and ApaI sites
were added to the 5¢ and 3¢ ends of a partial El2 promoter
fragment by means of PCR with primers harboring these
restriction enzyme sites. Similarly, XhoI and SacI sites were
created at the 5¢ and 3¢ ends of the corresponding El2 frag-
ment by PCR. The XbaI–ApaI antisense fragment and
XhoI–SacI sense fragment were inserted into the same sites
of the RNAi cassette. The target region of each RdDM
construct (pIR-HEAD, pIR-END, pIR-TATA) is described
in the text (Fig. S1). These binary vectors were introduced
into homozygous S44 plants by Agrobacterium-mediated
transformation. pIR-END was also introduced into
homozygous S24 plants. The regenerated, sequentially

transformed plants were self-pollinated, and the hygromy-
cin-resistant offspring were used for further analysis.
Fatty acid analysis
Fatty acid composition was determined as described previ-
ously [35].
RNA gel blot analysis
Total RNA was isolated using TRIzol reagent (Invitrogen,
Carlsbad, CA, USA). Twenty micrograms of total RNAs
were denatured and separated on a 1% agarose gel. Prepa-
ration of the blot was performed as described previously
[36]. Blots were hybridized with a digoxigenin-labeled DNA
probe for the NtFAD3 and GUS fragments according to
the manufacturer’s protocol.
Transcriptional regulation in S-PTGS S. Hirai et al.
1700 FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS
Small RNA was extracted from leaves as described previ-
ously [37]. Thirty micrograms of small RNA-enriched
nucleic acids were separated on acrylamide gels and trans-
ferred onto nylon membranes. Polyacrylamide gels were
stained by ethidium bromide. The digoxigenin-labeled
NtFAD3 riboprobe (nucleotide position 181–1366) was
hybridized to small RNAs as described previously [38].
Preparation of nuclear RNA
Nuclei were isolated according to the method of van
Blokland et al. [39]. Ten grams of frozen leaves were
ground in liquid nitrogen, suspended in 60 mL of nucleus
isolation buffer A (1.25 m sucrose, 25 mm NaCl, 50 mm
Mes, pH 6.0, 25 mm EDTA, 3% (v ⁄ v) Triton X-100,
25 mm 2-mercaptoethanol, 0.75 mm spermine, 2.5 mm sper-
midine) and filtered through four layers of gauze. The

nuclei were then pelleted by centrifugation for 10 min at
830 g, resuspended in 2 mL of nucleus isolation buffer A
and transferred to Eppendorf tubes. The nuclei were then
precipitated by centrifugation at 400 g for 10 min. Nuclear
RNA was extracted from this crude nucleus fraction using
TRIzol reagent.
RT-PCR analysis
For RT-PCR analysis, total and nuclear RNAs were
treated with DNase I (Nippon Gene, Tokyo, Japan). About
100 ng of DNase-treated RNA was analyzed with a One-
step RT-PCR Kit (Qiagen, Venlo, The Netherlands). The
primers used and the amplified regions are shown in
Table S1 (see Supporting information).
Quantitative RT-PCR
Total and nuclear RNAs were prepared from S44 and
S44-end-2 plants grown at different times. cDNA was syn-
thesized from 1 lg of DNase I-treated RNA with Prime-
script reverse transcriptase (TaKaRa, Kyoto, Japan) and
quantified by a Rotor-Gene Q 2plex system (Qiagen) with
SYBR Green Realtime PCR Master Mix (ToYoBo,
Osaka, Japan). The cycling conditions were as follows:
5 min at 95 °C, followed by 45 cycles of 30 s at 95 °C,
30 s at 55 °C and 60 s at 72 °C. The primers used and the
amplified regions are shown in Table S1. The levels of
NtFAD3 transcripts were normalized to the level of actin
transcripts. The relative values for S44 plants are indicated
in the text.
DNA gel blot analysis
Total DNA was prepared from tobacco leaves as described
previously [40]. Ten micrograms of total DNA were

digested with methylation-sensitive restriction endonucleas-
es AccI and HinfI in combination with a methylation-insen-
sitive enzyme MboI. The digoxigenin-labeled riboprobe
covering a partial CaMV 35S promoter region (nucleotide
position )744 to )94) was prepared. The digested total
DNA was separated by electrophoresis on a 1% agarose
gel and blotted to a positively charged nylon membrane.
Membranes were hybridized with riboprobes at 50 °C.
Visualization of hybridized probes was carried out accord-
ing to the manufacturer’s protocol.
Genomic bisulfite sequencing
Bisulfite treatment was carried out according to Kubota
et al. [41]. Two micrograms of total DNA and 2 lgof
pUC119 cloning plasmid were mixed, and denatured in 2 m
NaOH at 37 °C for 10 min. The mixture was incubated in
a total volume of 500 lL with freshly prepared 3.6 m
sodium bisulfite, 10 mm hydroquinone, pH 5.0, for 22 h.
DNA samples were desalted with a Wizard DNA Clean-up
System (Promega, Madison, WI, USA). NaOH was added
to the desalted DNA solution at a final concentration of
0.3 m. After incubation at 37 °C for 5 min, ammonium ace-
tate was added at a final concentration of 2.7 m and the
DNA was precipitated by ethanol. The primers used and
the amplified regions are shown in Table S2 (see Support-
ing information). PCR products that originated from the
transgene sequences were cloned, and 12–15 individual
clones were sequenced. The effectiveness of bisulfite treat-
ment was monitored by estimating the base substitution
efficiency of pUC119. Fragments of about 243 bp were
amplified from native pUC119 and from bisulfite-treated

pUC119. These PCR fragments were resolved on an SSCP
gel [42] to discriminate between the base-substituted frag-
ments and the native fragments.
5¢ RACE
The 5¢ region of the NtFAD3 transgene cDNAs was ampli-
fied using 5¢ RACE system ver2 or a GeneRacer Kit (Invi-
trogen).
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was carried out according to the manufac-
turer’s protocol (EpiQuik Plant ChIP kit; Epigentek,
Brooklyn, NY, USA) using mature leaves. Chromatin sam-
ples were immunoprecipitated with control antibody (IgG;
Epigentek) and antibodies against H3K9me2 and
H3K4me3 (Epigentek). The 470-bp El2 promoter ()434 to
+36 from TSP of El2 promoter) was amplified. Before
PCR was saturated, the PCR products were electrophore-
sed, and detected by Southern hybridization with digoxige-
nin-labeled probes. The primers used are listed in Table S3
(see Supporting information).
S. Hirai et al. Transcriptional regulation in S-PTGS
FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS 1701
Acknowledgements
We wish to thank Dr Takeo Kubota (University of
Yamanashi) for technical advice. We thank Yoshiko
Murohashi and Fumie Fukushima for the production
of double transformants. This research was supported
by Grants-in-Aid for Challenging Exploratory Research
(21657012) from the Ministry of Education, Science
and Culture, Japan. S.H. is a recipient of a scholarship
from the Japan Society for the Promotion of Science.

References
1 Meyer P, Niedenhof I & ten Lohuis M (1994) Evi-
dence for cytosine methylation of non-symmetrical
sequences in transgenic Petunia hybrida. EMBO J 13,
2084–2088.
2 Depicker A & Van Montagu M (1997) Post-transcrip-
tional gene silencing in plants. Curr Opin Cell Biol 9,
373–382.
3 Stam M, Mol JN & Kooter JM (1997) The silence of
genes in transgenic plants. Ann Bot 79, 3–12.
4 Que Q, Wang H & Jorgensen RA (1998) Distinct pat-
terns of pigment suppression are produced by allelic
sense and antisense chalcone synthase transgenes in
petunia flowers. Plant J 13, 401–409.
5 Napoli C, Lemieux C & Jorgensen R (1990) Introduc-
tion of a chimeric chalcone synthase gene into Petunia
results in reversible co-suppression of homologous genes
in trans. Plant Cell 2, 279–289.
6 van der Krol AR, Mur LA, Beld M, Mol JNM &
Stuitje AR (1990) Flavonoid genes in petunia: addition
of a limited number of gene copies may lead to a sup-
pression of a gene expression. Plant Cell 2, 291–299.
7 Baulcombe D (2004) RNA silencing in plants. Nature
431, 356–363.
8 Luo Z & Chen Z (2007) Improperly terminated,
unpolyadenylated mRNA of sense transgenes is targeted
by RDR6-mediated RNA silencing in Arabidopsis. Plant
Cell 19, 943–958.
9 Jorgensen RA, Cluster PD, English J, Que Q & Napoli
CA (1996) Chalcone synthase cosuppression phenotypes

in petunia flowers: comparison of sense vs. antisense
constructs and single-copy vs. complex T-DNA
sequences. Plant Mol Biol 31, 957–973.
10 Muskens MW, Vissers AP, Mol JN & Kooter JM
(2000) Role of inverted DNA repeats in transcriptional
and post-transcriptional gene silencing. Plant Mol Biol
43, 243–260.
11 Ma C & Mitra A (2002) Intrinsic direct repeats generate
consistent post-transcriptional gene silencing in tobacco.
Plant J 31, 37–49.
12 Tang G, Reinhart BJ, Bartel DP & Zamore PD (2003)
A biochemical framework for RNA silencing in plants.
Genes Dev 17, 49–63.
13 Brodersen P & Voinnet O (2006) The diversity of
RNA silencing pathways in plants. Trends Genet 22,
268–280.
14 Hobbs SLA, Kpodar P & DeLong CMO (1990) The
effect of the T-DNA copy number, position and methyl-
ation on reporter gene expression in tobacco transfor-
mants. Plant Mol Biol 15, 851–864.
15 Lechtenberg B, Schubert D, Forsbach A, Gils M &
Schmidt R (2003) Neither inverted repeat T-DNA con-
figurations nor arrangements of tandemly repeated
transgenes are sufficient to trigger transgene silencing.
Plant J 34, 507–517.
16 Schubert D, Lechtenberg B, Forsbach A, Gils M, Baha-
dur S & Schmidt R (2004) Silencing in Arabidopsis T-
DNA transformants: the predominant role of a gene-
specific RNA sensing mechanism versus position effects.
Plant Cell 16, 2561–2572.

17 Lawrence R & Pikaard CS (2003) Transgene-induced
RNA interference: a strategy for overcoming gene
redundancy in polyploids to generate loss-of-function
mutations. Plant J 36, 114–121.
18 Lindbo JA, Silva-Rosales L, Proebsting WM &
Dougherty WG (1993) Induction of a highly specific
antiviral state in transgenic plants: implications for reg-
ulation of gene expression and virus resistance. Plant
Cell 5, 1749–1759.
19 Cre
´
te
´
P & Vaucheret H (1999) Expression and sequence
requirements for nitrite reductase co-suppression. Plant
Mol Biol 41 , 105–114.
20 Hamada T, Kodama H, Takeshita K, Utsumi H & Iba
K (1998) Characterization of transgenic tobacco with
an increased a-linolenic acid level. Plant Physiol 118,
591–598.
21 Tomita R, Hamada T, Horiguchi G, Iba K & Kod-
ama H (2004) Transgene overexpression with cognate
small interfering RNA in tobacco. FEBS Lett 573,
117–120.
22 Mitsuhara I, Ugaki M, Hirochika H, Ohshima M,
Murakami T, Gotoh Y, Katayose Y, Nakamura S,
Honkura R, Nishimiya S et al. (1996) Efficient pro-
moter cassettes for enhanced expression of foreign genes
in dicotyledonous and monocotyledonous plants. Plant
Cell Physiol 37, 49–59.

23 Chan SW, Henderson IR & Jacobsen SE (2005)
Gardening the genome: DNA methylation in Arabidop-
sis thaliana. Nat Rev Genet 6, 351–360.
24 Matzke MA & Birchler JA (2005) RNAi-mediated
pathways in the nucleus. Nat Rev Genet 6, 24–35.
25 Matzke M, Kanno T, Huettel B, Daxinger L & Matzke
AJ (2007) Targets of RNA-directed DNA methylation.
Curr Opin Plant Biol 10 , 512–519.
26 Johnson LM, Cao X & Jacobsen SE (2002) Interplay
between two epigenetic marks: DNA methylation and
histone H3 lysine 9 methylation. Curr Biol 12, 1360–
1367.
Transcriptional regulation in S-PTGS S. Hirai et al.
1702 FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS
27 Lorincz MC, Dickerson DR, Schmitt M & Groudine M
(2004) Intragenic DNA methylation alters chromatin
structure and elongation efficiency in mammalian cells.
Nat Struct Mol Biol 11, 1068–1075.
28 Matzke MA, Primig M, Trnovsky J & Matzke AJM
(1989) Reversible methylation and inactivation of mar-
ker genes in sequentially transformed tobacco plants.
EMBO J 8, 643–649.
29 Gorning DR, Thomson L & Rothstein ST (1991)
Transformation of a partial nopaline synthase gene
into tobacco suppresses the expression of a resident
wild-type gene. Proc Natl Acad Sci USA 88, 1770–
1774.
30 Metzlaff M, O’Dell M, Cluster PD & Flavell RB (1997)
RNA-mediated RNA degradation and chalcone syn-
thase A silencing in petunia. Cell 88, 845–854.

31 Kanazawa A, O’Dell M & Hellens RP (2007) Epigenetic
inactivation of chalcone synthase-A transgene transcrip-
tion in petunia leads to a reversion of the post-
transcriptional gene silencing phenotype. Plant Cell
Physiol 48, 638–647.
32 Fojtova
´
M, Bleys A, Bedr
ˇ
ichova
´
J, Van Houdt H, Kr
ˇ
ı
´
-
zˇ ova
´
K, Depicker A & Kovar
ˇ
ı
´
k A (2006) The trans-
silencing capacity of invertedly repeated transgenes
depends on their epigenetic state in tobacco. Nucleic
Acids Res 34, 2280–2293.
33 Okano Y, Miki D & Shimamoto K (2008) Small inter-
fering RNA (siRNA) targeting of endogenous promot-
ers induces DNA methylation, but not necessarily gene
silencing, in rice. Plant J 53, 65–77.

34 Oka S, Hirai S, Choi KS & Kodama H (2008) Preferen-
tial spreading of RNA silencing into the 3¢ downstream
region of the transgene in tobacco. J Plant Biol 51,
227–232.
35 Kodama H, Hamada T, Horiguchi T, Nishimura M &
Iba K (1994) Genetic enhancement of cold tolerance by
expression of a gene for chloroplast x-3 fatty acid
desaturase in transgenic tobacco. Plant Physiol 105,
601–605.
36 Kodama H, Ito M, Ohnishi N, Suzuka I &
Komamine A (1991) Molecular cloning of the gene
for plant proliferating-cell nuclear antigen and expres-
sion of this gene during the cell cycle in synchronized
cultures of Catharanthus roseus cells. Eur J Biochem
197, 495–503.
37 Goto K, Kanazawa A, Kusaba M & Masuta C (2003)
A simple and rapid method to detect plant siRNA using
nonradioactive probes. Plant Mol Biol Rep 21, 51–58.
38 Hirai S, Oka S, Adachi E & Kodama H (2007) The
effects of spacer sequences on silencing efficiency of
plant RNAi vectors. Plant Cell Rep 26, 651–659.
39 van Blokland R, Van der Geest N, Mol JNM & Kooter
JM (1994) Transgene-mediated suppression of chalcone
synthase expression in Petunia hybrida results from an
increase in RNA turnover. Plant J 6, 861–877.
40 Adachi E, Shimamura K, Wakamatsu S & Kodama H
(2004) Amplification of plant genomic DNA by Phi29
DNA polymerase for use in physical mapping of the
hypermethylated genomic region. Plant Cell Rep 23 ,
144–147.

41 Kubota T, Das S, Christian SL, Baylin SB, Harman JG
& Ledbetter DH (1997) Methylation-specific PCR
simplifies imprinting analysis. Nat Genet 16, 16–17.
42 Celotto AM & Graveley BR (2001) Alternative splicing
of the Drosophila Dscam pre-mRNA is both temporally
and spatially regulated. Genetics 159, 599–608.
Supporting information
The following supplementary material is available:
Fig. S1. Transgene constructs and characterization of
the S44-SH transgenic lines.
Fig. S2. Levels of leaf 18:3 in total fatty acids of S44-
head, S44-end and S44-tata plants.
Fig. S3. TSP of transcripts of the NtFAD3 transgene
in S44 and S44-end-2 plants.
Fig. S4. RdDM by pIR-END in S24 plants.
Table S1. Primers used in RT-PCR analysis.
Table S2. Primers used in bisulfite sequence analysis.
Table S3. Primers used in ChIP assay.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
S. Hirai et al. Transcriptional regulation in S-PTGS
FEBS Journal 277 (2010) 1695–1703 ª 2010 The Authors Journal compilation ª 2010 FEBS 1703