loss of sense transgene induced post-transcriptiona gene silencing by sequntial introduction of the...

8/6/2019 LOss of Sense Transgene Induced Post-transcriptiona Gene Silencing by Sequntial Introduction of the Same Transge

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Loss of sense transgene-induced post-transcriptional gene

silencing by sequential introduction of the same transgene

sequences in tobacco

Sayaka Hirai1, Kouta Takahashi2, Tomomi Abiko3 and Hiroaki Kodama1

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 oftenrecognized as a sequence of invasive nucleic acids and

triggers RNA silencing [14]. 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 aresuppressed 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: [email protected]

(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 mRNAssuccessfully 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) 16951703 2010 The Authors Journal compilation 2010 FEBS 1695


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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 transgenetranscription andor a defect in RNA processing. The

genomic insertion manner of transgenes, namely

repeated arrangement and truncated andor rear-

ranged transgene copies, may be a cause of synthesis

of aberrant RNAs [911]. RDR6 synthesizes a comple-

mentary RNA using aberrant transcripts as a template,

and the resulting dsRNAs are processed into the

2125-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 thresholdconcentration 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 andpEx9-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) 16951703 2010 The Authors Journal compilation 2010 FEBS


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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-SH24) 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 analysiswith 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 AccIMboI,

and could be visualized using the CaMV 35S

promoter sequence as a probe. These fragments were

detected in the AccIMboI-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 HinfIMboI, 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 HinfIMboI- and AccIMboI-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 HinfIMboI

fragment corresponds to the long fragment including 251, 317 and

325 fragments.

S. Hirai et al. Transcriptional regulation in S-PTGS



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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

[2325]. 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 theEND2 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 rapidamplification 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 andor 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

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.




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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 ofboth 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

B C

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

(AC). (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.




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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

RDR6on 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, andpIR-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 influenceof 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 521035 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 genewas 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 XbaIApaI antisense fragment and

XhoISacI 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 describedin 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 manufacturers protocol.




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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 1811366) 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% (vv) 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 thenprecipitated 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 actintranscripts. 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 manufacturers protocol.

Genomic bisulfite sequencing

Bisulfite treatment was carried out according to Kubota

et al. [41]. Two micrograms of total DNA and 2 lg of

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 addedto 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 1215 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-

turers 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).




8/9

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.

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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.



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