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

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: kodama@faculty.chiba-u.jp (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 population. 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 transcriptional 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 harboring a 200-bp-long fragment of promoter sequences efciently 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.

Introduction
Genetic transformation is a powerful tool of improvement of plant physiological traits, and is important to both basic and applied sciences. Successful expression of transgene sequences is always desired, and overexpression of the transgene is usually selected in a population of transgenic plants. The transgene itself is often recognized as a sequence of invasive nucleic acids and triggers RNA silencing [14]. RNA silencing, an RNAmediated 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

Abbreviations CaMV, cauliower mosaic virus; ChIP, chromatin immunoprecipitation; CHS, chalcone synthase gene; GFP, green uorescent 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 amplication 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.

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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 RNAdependent 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 rearranged transgene copies, may be a cause of synthesis of aberrant RNAs [911]. RDR6 synthesizes a complementary 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 RNAinduced silencing complex and guides the endonucleolytic 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 ve or more copies of the green uorescent 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 recognized 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 conversion 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 overexpression. This result indicates that, at least in this case, overexpression of the transgene is established via complex mechanisms, including RNA silencing.
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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 promoter [20]. Most transgenic plants showed an overexpressed phenotype, namely increased 18:3 content, and the S24 line was used as a representative of such overexpressors (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

Fig. 1. Loss of S-PTGS phenotype in sequentially transformed plants with NtFAD3 sense and NtFAD3:GUS chimeric sense transgenes. (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) Characterization 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 representative 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 transcripts in leaf tissues. The presence of the NtFAD3:GUS chimeric construct in the genome was determined by PCR amplication (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.

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NtFAD3 siRNAs [21]. Most NtFAD3 siRNAs were 21 nucleotides in length [21], and the level of 24-nucleotide 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 introduced as a secondary transgene into homozygous S44 plants, and four independent transformants (S44-SH1 4) were obtained (Fig. S1C). Unfortunately, the resultant 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 offspring 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 associated with the disappearance of NtFAD3 siRNA (Fig. 1B). Therefore, the phenotype of S44 was converted 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 endogene was recovered, and the primary NtFAD3 transgene was highly expressed in S44-SH2 leaves. Strangely, expression of the secondary NtFAD3 transgene (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 difcult to discriminate the methylation status of each promoter sequence by Southern hybridization. The El2 promoter consists of two tandemly repeated enhancer regions of the cauliower 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.

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.

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.
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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 corresponding cytosine residues (RNA-directed DNA methylation, 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 transcriptional start point (TSP); Fig. S1B], and was introduced into a homozygous S44 plant. The resulting six independent 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 promoter ()200 to +1; Fig. S1B). Two independent transgenic 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 S44end-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 counterparts 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 S44tata 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 indicated that both transcripts for the NtFAD3 transgene
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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 transformant 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.

and endogene showed an increase of about 1.4-fold in the total RNA fraction of S44-end-2 plants in comparison 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 amplication of 5 cDNA ends (RACE). The resulting RACE products amplied from both plants showed a clear single band after electrophoresis (Fig. S3, see Supporting 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 steadystate 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 generation 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

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Fig. 4. Bisulte 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 methylation at the NtFAD3 coding region of the transgene. The percentages of methylated cytosines in CpG, CpHpG and CpHpH were determined in S44 (black bars) and S44-end-2 (grey bars) plants (AC). (D) Histone modication in transgenic plants. Chromatin fractions prepared from S44, S44-end-2 and S24 leaves were immunoprecipitated (IP) using antibodies specic for H3K9me2 and H3K4me3. IgG was provided by a kit supplier and used as a control antibody. DNA was then puried from immunoprecipitants and subjected to PCR analysis. Genomic regions for the El2 promoter were amplied.

S44 and S44-end-2 plants (Fig. 4D). The El2 promoter in S24 plants was associated with histone H3 trimethylated 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, respectively [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 decient in S44-end-2 plants, but the loss of these siRNAs was not associated with any remarkable changes in chromatin modication 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 generation 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 trigger phenotypic conversion. Re-introduction of promoter sequences frequently suppresses the expression of a previously introduced transgene, which is correlated with methylation at the promoter region [28,29]. The increased methylation in the El2 promoter and the disappearance of the NtFAD3::GUS transcripts in S44SH2 plants are in good agreement with these previous observations. The methylation at the El2 promoter should coincidently occur in both primary and secondary transgenes. In this respect, the transcriptional activity of a primary transgene should decline even though its mRNA successfully accumulates by deciency of NtFAD3 siRNAs. The release of S-PTGS has been observed in several transgenic plants. Petunia C001 transgenic plants produce white owers by S-PTGS of the chalcone synthase gene (CHS) [9]. The C002 line is obtained spontaneously in the offspring of C001, and produces purple owers. 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
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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 methylated (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 overexpressor 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 representative 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 efcient methylation in the END2 region, which was followed by the abolishment of the overexpression phenotype in S24 plants. Cytosine methylation appears to direct changes in chromatin conformation to the silent chromatin region. However, the features of histone H3 modication at the El2 promoter were essentially the same in

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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 transcriptionally silenced by dense methylation of the promoter sequences, and siRNAs for the nptII gene disappear. When the expression of the transgene is almost completely 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 methylation-free promoter. One of the common features of these plants free from S-PTGS is the attenuated expression of the transgene, and therefore the threshold 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 modication, especially H3K9me2 [33]. Although pIR-END does not affect signicantly 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 methylated El2 promoter. This chromatin conformation may be interfered with by introduction of pIR-END, and transcriptional acitivity will be decreased signicantly. 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.
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Our results indicate an example in which the transgene 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 overexpression of the transgene established by the latent inuence 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 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 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 fragments 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 fragment 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 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 hygromycin-resistant offspring were used for further analysis.

Fatty acid analysis

Fatty acid composition was determined as described previously [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. Preparation 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 previously [37]. Thirty micrograms of small RNA-enriched nucleic acids were separated on acrylamide gels and transferred 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% (v v) Triton X-100, 25 mm 2-mercaptoethanol, 0.75 mm spermine, 2.5 mm spermidine) and ltered 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.

es AccI and HinfI in combination with a methylation-insensitive 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 according to the manufacturers protocol.

Genomic bisulte sequencing

Bisulte 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 bisulte, 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 nal concentration of 0.3 m. After incubation at 37 C for 5 min, ammonium acetate was added at a nal concentration of 2.7 m and the DNA was precipitated by ethanol. The primers used and the amplied regions are shown in Table S2 (see Supporting information). PCR products that originated from the transgene sequences were cloned, and 1215 individual clones were sequenced. The effectiveness of bisulte treatment was monitored by estimating the base substitution efciency of pUC119. Fragments of about 243 bp were amplied from native pUC119 and from bisulte-treated pUC119. These PCR fragments were resolved on an SSCP gel [42] to discriminate between the base-substituted fragments and the native fragments.

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 Onestep RT-PCR Kit (Qiagen, Venlo, The Netherlands). The primers used and the amplied 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 synthesized from 1 lg of DNase I-treated RNA with Primescript reverse transcriptase (TaKaRa, Kyoto, Japan) and quantied 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 amplied 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.

5 RACE
The 5 region of the NtFAD3 transgene cDNAs was amplied using 5 RACE system ver2 or a GeneRacer Kit (Invitrogen).

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was carried out according to the manufacturers protocol (EpiQuik Plant ChIP kit; Epigentek, Brooklyn, NY, USA) using mature leaves. Chromatin samples 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 amplied. Before PCR was saturated, the PCR products were electrophoresed, and detected by Southern hybridization with digoxigenin-labeled probes. The primers used are listed in Table S3 (see Supporting information).

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-

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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
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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 S44head, 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 bisulte 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 les) should be addressed to the authors.

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