Cytosine methylation is associated with RNA silencing in silenced plants but not with systemic and transitive RNA silencing through grafting

Gene 396 (2007) 321 – 331 www.elsevier.com/locate/gene

Cytosine methylation is associated with RNA silencing in silenced plants but not with systemic and transitive RNA silencing through grafting A.K.M. Nazmul Haque, Naoto Yamaoka, Masamichi Nishiguchi ⁎ The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan Received 28 October 2006; received in revised form 4 April 2007; accepted 4 April 2007 Available online 14 April 2007 Received by T. Sekiya

Abstract RNA silencing is often associated with methylation of the target gene. The DNA methylation level of transgenes was investigated in posttranscriptionally silenced or non-silenced Nicotiana benthamiana carrying either the 5′ region (200 or 400 bp) or the entire region of the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Higher levels of transgene cytosine methylation were observed in both symmetrical (CpG, CpNpG) and non-symmetrical (CpHpH) contexts (CpG N CpNpG N CpHpH) in silenced lines, but there was very lower levels or no transgene methylation in non-silenced lines. RNA silencing was induced in non-silenced scions from silenced rootstocks and spread to the 3′ region of the transgene mRNA (Haque et al., Plant Mol. Biol. 2007; 63: 35–47). In this system, transgene methylation levels were analyzed in scions at different time intervals after being grafted onto silenced or non-silenced rootstocks to investigate if transgene methylation was associated with induction or transitivity of RNA silencing. We observed that, there was no change of transgene methylation level in the initial target or in extended regions in scions. These results showed that transgene methylation was associated with RNA silencing in individual transformants, but it was not associated with systemic RNA silencing and/or transitive RNA silencing through grafting. © 2007 Published by Elsevier B.V. Keywords: RNA-dependent DNA methylation; Nicotiana benthamiana; Coat protein; Sweet potato feathery mottle virus; RNA silencing; Grafting

1. Introduction RNA silencing, a general term used to describe post-transcriptional gene silencing in plants, also called quelling in fungi and RNA interference in animals, is a highly conserved process of sequence-specific RNA degradation observed in almost all of the eukaryotes examined to date (with the exception of yeast). It is activated by a perfect or imperfect double-stranded RNAs (dsRNA) (Waterhouse et al., 1998; Bass, 2000) that are recognized and specifically cleaved by an RNase III enzyme [called Dicer or Dicer-like (DCL)] to yield 21–25 nucleotide short interfering RNA (siRNA) (Hamilton and Baulcombe, 1999). These siRNAs are then recognized, unwound and incorporated Abbreviations: CP, Coat protein gene; SPFMV, Sweet potato feathery mottle virus; RdDM, RNA-dependent DNA methylation; C, cytosine; siRNA, short interfering RNA; dsRNA, double-stranded RNA. ⁎ Corresponding author. Tel.: +81 89 946 9816; fax: +81 89 977 4364. E-mail address: [email protected] (M. Nishiguchi). 0378-1119/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.gene.2007.04.003

into a protein complex known as the RNA-induced silencing complex (RISC) and serve to direct the cleavage of target mRNA at discrete positions, probably by sequence complementarity (Elbashir et al., 2001; Martinez et al., 2002; Baumberger and Baulcombe, 2005). Molecular analyses of transgenic plants reveal a close association between RNA silencing and DNA methylation of the transgene (Ingelbrecht et al., 1994), suggesting that DNA methylation may have roles in triggering and/or maintaining the RNA silencing pathway. The first demonstration of RNAdependent DNA methylation (RdDM) came from transgenic tobacco plants that were engineered to carry RNA viroid-identical DNA sequences in their genomes. In these studies, heavy methylation of the target transgene was observed in plants in which the viroid could replicate efficiently (Wassenegger et al., 1994; Pelissier et al., 1999). The presence of dsRNA can induce sequence-specific DNA methylation (RdDM) and this RdDM has been documented in many plant systems in response to various dsRNA inducers (Cao et al., 2003). As most RNA

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viruses and viroids have no DNA intermediates in their replication cycle, it is likely to be RNA that mediates homologous DNA methylation. It has been proposed that the signal molecule for RdDM is either siRNA or precursors of siRNA (dsRNA) (Wassenegger, 2000). In vertebrate DNA, 3–6% of the total cytosines (Cs) are methylated. This value decreases with stepping down the evolutionary scale and many insects and single-cell eukaryotes have rare cytosine methylation in their DNA (Adams, 1990). As much as 30% of cytosines are methylated in higher plants (Adams and Burdon, 1985), most of which are probably involved in silencing of transposable elements and viral DNA (Finnegan et al, 1998; Yoder et al., 1997). Along with taking part in RNA silencing, DNA methylation is associated with other functions in eukaryotes: chromatin stability (Chen et al., 1998), genetic imprinting (Zeschnigk et al., 1997) and differential gene control by histone deacetylation (Razin, 1998). In mammals, DNA methylation occurs almost exclusively on Cs in the symmetric dinucleotides context CpG. In plants, CpG is also the predominant methylation context. However, in plant genomes, DNA methylation can also occur on Cs in other contexts, including the symmetric context CpNpG (where N is any base) and the asymmetric context CpHpH (where H is A, T or C), although less efficiently than Cs in CpG context (Martienssen and Colot, 2001). This difference in DNA methylation patterning is also reflected in the cytosine methyltransferases (MTase) present in plants and animals. MET1, the orthologue of mammalian Dnmt1, maintains the majority of CpG methylation (Finnegan et al., 1996; Kankel et al., 2003; Ronemus et al., 1996; Saze et al., 2003). In addition, plants have two other MTases, the chromomethyltransferase (CMT) and domain rearranged methylase (DRM) classes, which are not found in mammals (Cao et al., 2003; Finnegan and Kovac, 2000; Henikoff and Comai, 1998). CMT3 is primarily responsible for maintenance of CpNpG methylation, but it also plays roles in methylation in other contexts at some loci (Bartee et al., 2001; Cao and Jacobsen, 2002a; Lindroth et al., 2001; Papa et al., 2001). DRM1 and DRM2, which are related to the mammalian Dnmt3 group, are primarily responsible for de novo DNA methylation in all sequence contexts (Okano et al., 1999; Cao et al., 2003; Cao and Jacobsen, 2002b; Chan et al., 2004). Sweet potato feathery mottle virus (SPFMV) (accession no. D86371), a member of the potyvirus group, consists of a singlestranded sense RNA genome of 10,820 nucleotides (excluding the 3′ polyA tail) (Sakai et al., 1997). Its single open reading frame (ORF) includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins. Its coat protein gene (CP) was introduced into Nicotiana benthamiana to analyze resistance against recombinant virus carrying homologous transgenes (Sonoda et al., 1999). We reported that RNA silencing was induced and spread from 5′ to 3′ direction along the transgene mRNA from silenced rootstocks to non-silenced scions through grafting using transgenic N. benthamiana harboring the 5′ region or the entire sequence of CP (Haque et al., 2007). Previously, we observed that transgene methylation was not changed in CP-expressing scions grafted onto silenced rootstocks using methylation-sensitive

restriction enzymes and Southern blot analysis (Sonoda and Nishiguchi, 2000). Here, we employed bisuphite genome sequencing for comprehensive analysis of DNA methylation in silenced or non-silenced lines and in non-silenced or silenced scions grafted onto silenced or non-silenced rootstocks. We also analyzed transgene methylation at different time-points after grafting. We observed a close correlation between RNA silencing and transgene methylation (CpG N CpNpG N CpHpH) in silenced lines, but found that transgene methylation was associated neither with systemic RNA silencing nor with transitive RNA silencing through grafting. 2. Materials and methods 2.1. Plant materials Transgenic N. benthamiana lines 4.07 and 4.09, carrying the CP (including 3′ non-translated region, NTR) of SPFMV, were previously described (Sonoda et al., 1999). Line 4.07 is a nonsilenced over-expressor, and 4.09 is a silenced line, in which silencing was targeted to the 3′ region of transgene. Lines 200.1 and 200.4 carry the 5′ 200 bp region of CP and lines 400.15 and 400.89 carry the 5′ 400 bp region of CP; 200.4 and 400.15 are post-transcriptionally silenced lines and lines 200.1 and 400.89 are non-silenced over-expressors (Haque et al., 2007). 2.2. Genomic DNA extraction and methylation detection assays Genomic DNA was extracted from leaf tissues of scions approximately 6 and 12 weeks after grafting or from age-matched non-grafted plants using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Genomic DNA (400 ng) was digested with three units of methylation-sensitive restriction enzymes (Aci I, Hpa II, Msp I and Hha I) in combination with Eco RV using appropriate buffers in a total reaction volume of 30 μl at 37 °C overnight, unless stated otherwise. Eco RV had no recognition sites within the transgene, and was used to facilitate complete digestion of the genomic DNA with methylation-sensitive restriction enzymes. For digestion of genomic DNA, Hpa II or Msp I was used for lines 400.15 and 400.89, Aci I for lines 200.1 and 200.4 and Hpa II or Hha I for lines 4.07 and 4.09, respectively. After phenol-chloroform purification and ethanol precipitation, digested genomic DNA (100 ng) was used for PCR amplification of the transgene using specific primers: SPFMV.CP-F and SPFMV.CP-R200 to amplify the 5′ 200 bp region of CP for lines 200.1 and 200.4; SPFMV.CP-F and SPFMV.CP-R400 to amplify the 5′ 400 bp region of CP for lines 400.15 and 400.89; SPFMV.CP-F and SPFMV.CP-3′R (5′-GCAGAGCTCAACCATCTCCTTCGGGACT-3′) to amplify the entire CP in plant lines 4.07 and 4.09 (Haque et al., 2007). As a control for PCR, undigested DNA (100 ng) were used as templates for PCR. 2.3. Grafting procedure Approximately 8 week old plants were used for rootstocks or scions for cleft grafting as described previously (Palauqui et al., 1997; Haque et al., 2007).

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2.4. Bisulphite sequence analysis Eco RV digested genomic DNA (400 ng) was used for bisulphite treatment using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA) according to the manufacturer's instructions. A mixture of primers, containing degenerate nucleotides [either cytosine or thymine (Y) for forward primers, and adenine or guanine (R) for reverse primers] at positions corresponding to C residues in the target sequences, were used to cover all possible states of Cs, including those that had not been converted to uracils through the bisulphite treatment. Three sets of primers were used to amplify the entire CP sequence: 35S. BSP.F, 5′-ATATAAGGAAGTTYATTTYATTTGGAGAG-3′ and CP.BSP.R1, 5′-RTCRACCRRRTRTTTRCAACCTCAA3 ′ ; C P.B S P.F 2 , 5′ - A G G G Y YATTATA A AT T T Y YA AYAYTTGTYA-3′ and CP.BSP.R2, 5′-RCTRCCTTCAT CTRTAAATRTRCTTCTTTT-3′; CP.BSP.F3, 5′-TGYAAAAGAAGYAYATTTAYAGATGAAG-3′ and NOS.BSP.R, 5′AACTTTATTRCCAAATRTTTRAACRAT-3′. 35S.BSP.F and NOS.BSP.R primers were used to amplify the 5′ 200 bp or 5′ 400 bp region of CP, as these two primers cover the 3′ region of 35S promoter and the 5′ region of NOS terminator, respectively. For PCR, bisulphite-treated DNA (1 μl) was used in a 50 μl reaction mixture consisting of 5 μl of 10X PCR buffer (SigmaAldrich, St. Louis, MO, USA), 3 mM MgCl2, 0.2 mM of each dNTPs, 1 μM of each primer and 2.5 units of JumpStart Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA). PCR

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amplification was performed with a 2 min initial denaturation at 94 °C, followed by 40 cycles of 94 °C for 30 s, 47–51 °C for 30 s (annealing temperature depending on the primers) and 72 °C for 45 s and with a final extension at 72 °C for 10 min. PCR products electrophoresed on a 2% low melting agarose (Nusieve GTG, Cambrex, USA) were purified by QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) or the excised gel slice of the expected DNA band was directly used to clone into the pGEM-T Easy Vector System (Promega, Madison, WI, USA). Individual transformants were selected by blue/white screening (Sambrook et al., 1989). Plasmid DNA was isolated from six individual clones for each PCR product using the Plasmid Miniprep Kit (Qiagen, Valencia, CA, USA). The size of the insert DNA fragment was confirmed by restriction digestion of the recombinant plasmid DNA with the appropriate restriction enzyme (Eco RI) before sequence analysis using Bigdye Terminal Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an automated sequencer (ABI 3100, Applied Biosystems, Foster City, CA, USA). 2.5. RNA gel blot analysis Total RNA was isolated from the leaf tissues with Tri reagent, according to the manufacturer's instructions (Molecular Research Center, Cincinnati, OH, USA), followed by two purification steps using equal volumes of chloroform and isoamylalcohol. Total RNA (10 μg) was size-fractionated,

Fig. 1. Analysis of transgene methylation by methylation-sensitive restriction digestion. A. Approximate locations of recognition sites for methylation-sensitive restriction endonucleases Hpa II and Msp I (H/M) in 5′ 400 bp region of CP and locations of transgene specific forward and reverse primers (unfilled and filled triangles, respectively). B. Genomic DNA (400 ng) from leaf tissue of 5′ 400 bp region of CP harboring silenced or non-silenced lines (400.15 and 400.89, respectively) or from non-silenced scions 6 weeks after being grafted onto silenced rootstocks (400.89/400.15) or from silenced scions 6 weeks after being grafted onto non-silenced rootstocks (400.15/400.89) was digested with methylation-sensitive restriction enzymes (Hpa II and Msp I). Digested DNA (100 ng) was subjected to PCR amplification with gene specific primers and separated on 2% agarose gel. C. Uncut DNA templates were used for PCR as controls. D. Approximate locations of methylation-sensitive restriction endonuclease Aci I (A) recognition sites in 5′ 200 bp region of CP and locations of transgene specific forward and reverse primers (unfilled and filled triangles, respectively). E. Genomic DNA (400 ng) isolated from leaf tissue of 5′ 200 bp region of CP harboring silenced or non-silenced lines (200.4 and 200.1, respectively) or from non-silenced scions 6 weeks after being grafted onto silenced rootstocks (200.1/200.4) or from silenced scions 6 weeks after being grafted onto non-silenced rootstocks (200.4/200.1) was digested with a methylation-sensitive restriction enzyme (Aci I). Digested DNA (100 ng) was subjected to PCR amplification with gene specific primers and separated on 2% agarose gel. F. Uncut DNA templates were used for PCR as controls.

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transferred to a nylon membrane (Hybond N+, Amersham Biosciences, Piscataway, NJ, USA) and hybridized with [α-32P] dCTP-labeled DNA probes corresponding to 5' regions (5'CP), middle regions (MCP) or 3' regions (3'CP) of CP as described previously (Haque et al., 2007). 2.6. siRNA detection Extraction of low molecular weight RNAs, gel blot analysis and hybridization of blots with [α-32P]dCTP-labeled DNA probes were performed as described previously (Haque et al., 2007). 3. Results 3.1. Methylation status of the transgene in transgenic plants Analyses of total RNA and low molecular weight RNA revealed that lines 200.4 and 400.15 are post-transcriptionally

silenced and lines 200.1 and 400.89 are non-silenced overexpressors (Haque et al., 2007). To check if RNA silencing was associated with transgene methylation, we assayed the transgene methylation using methylation-sensitive restriction digestion of genomic DNA and PCR amplification of the transgene from these four transgenic lines. If the restriction site was methylated, the methylation would prevent restriction digestion and the transgene would be amplified by PCR, whereas nonmethylated sites would allow DNA digestion and prevent PCR amplification of the gene. DNA from lines 400.15 and 400.89 was digested with Hpa II or Msp I. Both the restriction enzymes recognize the DNA sequence CCGG, but cleavage by Hpa II is blocked by methylation on either cytosine residue (C) of the recognition site; on the other hand, cleavage by Msp I is blocked by methylation on the external C residue. In this way, Hpa II and Msp I assess DNA methylation in CpG and CpNpG contexts, respectively. PCR amplification of the transgene with specific primers revealed cytosine methylation in both contexts in line

Fig. 2. Bisulphite sequence analysis of transgene methylation. A. The distribution of cytosine methylation is partially presented from the 50 to 206 bp region of CP. PCR amplification products from a bisulphite-treated DNA template were subcloned and then six independent clones were sequenced for each line (200.4-1 to 200.4-6 or 400.15-1 to 400.15-6). The locations of restriction sites for Aci I are also shown. Squares, triangles and circles indicate cytosine residues in CpG, CpNpG and CpHpH contexts, respectively. Filled and open symbols indicate methylated and non-methylated cytosine residues, respectively. B. Percent cytosine methylation in symmetrical (CpG and CpNpG) and non-symmetrical (CpHpH) contexts in the transgene coding region. Genomic DNA from silenced (200.4 and 400.15) and nonsilenced lines (200.1 and 400.89), or from non-silenced scions 6 weeks after being grafted onto silenced rootstocks (200.1/200.4 and 400.89/400.15) or from silenced scions 6 weeks after being grafting onto non-silenced rootstocks (200.4/200.1 and 400.15/400.89) was subjected to bisulphite sequencing. Percent cytosine methylation was analyzed for either the 5′ 200 bp region (for lines 200.1 and 200.4) or the 5′ 400 bp region of CP (for lines 400.15 and 400.89) and calculated from total cytosine residues present in the clones.

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400.15 but not in line 400.89 (Fig. 1A–C). DNA from lines 200.1 and 200.4 were digested with Aci I. Two recognition sites of Aci I (CCGC) are located within the 5′ 200 bp region of CP (Fig. 1D) and these recognition motifs will not be recognized by the enzyme if the inner C residue is methylated, thus allows assessment of the transgene CpG methylation in these lines. PCR amplification result showed transgene methylation in line 200.4 but not in line 200.1 (Fig. 1E–F). 3.2. Bisulphite sequence analysis of transgene methylation in transgenic plants Methylation analysis using methylation-sensitive restriction enzymes allows the investigation of the methylation status of a limited number of cytosine residues and, as the transgenes in these lines are relatively short (200 and 400 bp), only a limited number of recognition sites are available to assess DNA methylation. Therefore, the bisulphite sequencing method was employed for detailed analysis of transgene methylation in these lines. Bisulphite treatment allows the conversion of all cytosines to uracils (U), except those that are methylated at the carbon-5 position of Cs. Following strand-specific PCR amplification and sequencing, all modified cytosines appear as thymidines (T), whereas methylated cytosines remain unconverted (Frommer et al., 1992). DNA samples from these four lines were treated with bisulphite and the bisulphite-treated DNA samples were subjected to PCR with primer sets designed to amplify the sense strand of the CP transgene. Six clones for each line were sequenced. Sequence analyses showed that the transgene in the non-silenced lines 200.1 and 400.89 was apparently less methylated (Figs. 2 and S1). Almost all of the transgene cytosine residues of these two lines were converted to Ts, with few unconverted Cs that were mostly in the CpG context. On the other hand, in the silenced lines 200.4 and 400.15, a high degree of DNA methylation was observed (Figs. 2 and S1). The percent cytosine methylation at CpG, CpNpG and CpHpH contexts was 90%, 74% and 48%, respectively, in line 200.4 and 92%, 77% and 49% in line 400.15. These results showed a close association of sequence-specific RNA degradation with transgene methylation in these transgenic plants.

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mRNAs were observed in the non-silenced lines of 200.1 and 400.89 grafted onto silenced lines, 200.4 and 400.15, respectively, 4 weeks after grafting (Fig. 3A). siRNAs also could be detected in these non-silenced scions (Fig. 3B). This partial reduction of transgene mRNA and accumulation of siRNAs indicated the induction of sequence-specific RNA degradation in non-silenced scions, however, it might be at an initial stage and was not at the maximum level at this time-point, as substantial levels of transgene mRNA was observed. Considerable lowering of transgene mRNA and higher accumulation of siRNAs revealed an efficient induction of sequence-specific RNA degradation in non-silenced scions from the silenced rootstocks 6 weeks after grafting (Haque et al., 2007). Thus, the silenced rootstock-derived silencing signals could cross graftjunctions and induced systemic sequence-specific RNA degradation efficiently in non-silenced scions 6 weeks after grafting. As a six-week time interval after grafting was found appropriate for an efficient induction of sequence-specific RNA degradation, we extracted DNA from scions at that time-point to analyze transgene methylation. DNA samples were digested

3.3. Transgene methylation and systemic RNA silencing through grafting RNA silencing transmits from silenced rootstocks to nonsilenced scions (Palauqui et al., 1997; Voinnet et al., 1998; Sonoda and Nishiguchi, 2000; Crete et al., 2001; Garcia-Perez et al., 2004). To elucidate whether this graft-induced RNA silencing was associated with transgene methylation, we analyzed DNA methylation status of the transgene in nonsilenced scions grafted onto silenced rootstocks. Total and small RNAs extracted from non-silenced scions grafted onto silenced rootstocks were analyzed at different time intervals to know if the sequence-specific RNA degradation was induced efficiently in the non-silenced scions from the silenced rootstocks. Reduced levels and degraded patterns of transgene

Fig. 3. Northern blot analysis 4 weeks after grafting. A. Total RNA (10 μg) from the leaves of non-silenced scions 4 weeks after grafting onto silenced rootstocks (200.1/200.4 and 400.89/400.15) was size-fractionated on a 0.8% agarose gel containing 0.66 M formaldehyde, transferred to a nylon membrane and hybridized with a 32P labeled 5′ 400 bp CP specific probe. RNA extracted from beheaded non-silenced lines, 200.1 and 400.89, and age-matched silenced rootstocks, lines 200.4 and 400.15, was used as controls. An ethidium bromide stained RNA gel photographed before transfer is shown as a control for equal loading. B. Low molecular weight RNA fractions (50 µg) were isolated from non-silenced scions, lines 200.1 and 400.89, or from beheaded non-silenced lines, 200.1 and 400.89, separated on 15% polyacrylamide gels containing 8 M urea, blotted onto a nylon membrane and hybridized with a 32P labeled 5′ 400 bp CP specific DNA probe. DNA oligomers of 25 bp were used for size control (indicated at the left side of the figure). As a control for equal loading, ethidium bromide stained tRNA fractions are shown below.

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silenced scions even 12 weeks after grafting onto silenced rootstocks (Fig. 5). 3.4. Transgene methylation and transitive RNA silencing through grafting

Fig. 4. Northern blot analysis of silenced scions grafted onto non-silenced rootstocks. A. Total RNA (10 μg) extracted from the leaves of silenced scions 6 weeks after grafting onto non-silenced rootstocks (200.4/200.1 and 400.15/ 400.89) was analyzed as described in Fig. 3A. RNA extracted from beheaded silenced lines, 200.4 and 400.15, and age-matched non-silenced rootstocks, lines 200.1 and 400.89, was used as controls. An ethidium bromide stained RNA gel photographed before transfer is shown as a control for equal loading. B. Low molecular weight RNA fractions (50 µg) from silenced scions, lines 200.4 and 400.15, 6 weeks after grafting onto non-silenced rootstocks, 200.1 and 400.89, respectively, were analyzed as described in Fig. 3B. Low molecular weight RNA fractions (50 µg) extracted from non-silenced rootstocks, lines 200.1 and 400.89, were also analyzed. As a control for equal loading, ethidium bromide stained tRNA fractions are shown below.

with methylation-sensitive restriction enzymes Hpa II, Msp I or Aci I, followed by PCR amplification of the transgene as mentioned above. Results showed that hypermethylation of the transgene did not accompany the graft-induced systemic sequence-specific RNA degradation in non-silenced scions (Fig. 1). Bisulphite sequencing of DNA samples from nonsilenced scions 6 weeks after grafting onto silenced rootstocks confirmed this result (Figs. 2B and S1). Maintenance of RNA silencing and/or transgene methylation in silenced scions grafted onto non-silenced rootstocks was also analyzed. Scions from 200.4 and 400.15 lines were grafted onto 200.1 and 400.89 rootstocks, respectively, and transgene methylation was analyzed 6 weeks after grafting. These scions were found to maintain transgene silencing efficiently, as no change of RNA banding pattern was observed in these scions even after being grafted onto non-silenced rootstocks (Fig. 4A). This indicated that no rootstocks-derived silencing signals are required to maintain silencing in the RNA silenced scions. Maintenance of RNA silencing was also confirmed by the detection of siRNA in these silenced scions grafted onto non-silenced rootstocks (Fig. 4B). Transgene DNA methylation analysis showed no change of DNA methylation levels in these silenced scions 6 weeks after being grafted onto non-silenced rootstocks (Figs. 1, 2B and S1). We analyzed the silencing status and transgene methylation in non-silenced scions prolonged time (12 weeks) after grafting onto silenced rootstocks. Analysis of total RNA (Fig. S3, A) and small RNAs (Fig. S3, B) showed that induction of sequencespecific RNA degradation was maintained efficiently in nonsilenced scions 12 weeks after grafting onto silenced rootstocks. DNA extracted from non-silenced scions at that time-point was analyzed with methylation-sensitive restriction enzyme followed by PCR as described previously to assess transgene methylation. Similar results to those 6 weeks after grafting were observed, there was no induction of DNA methylation in non-

Spreading of RNA silencing to regions outside the inducer sequence, a phenomenon termed as transitive RNA silencing, has been reported in a range of plant systems (Vaistij et al., 2002; Klahre et al., 2002; Van Houdt et al., 2003; Garcia-Perez et al., 2004). Scions from a non-silenced line carrying the entire transgene (line 4.07) (Sonoda et al., 1999) were grafted onto silenced rootstocks of lines of 200.4 and 400.15 to explore whether graft-induced RNA silencing could spread along the entire transgene transcript. Total and small RNAs extracted from scions at different time intervals after grafting were analyzed with probes specific to different regions of CP (Fig. 6A) to determine if transitive RNA silencing was induced effectively. RNA silencing was found to be induced in 4.07 scions 4 weeks after grafting, as a considerable decline of transgene mRNA level (Fig. 6B) and accumulation of siRNAs (Fig. 6E) were observed in non-silenced scions when analyzed with radiolabeled probe corresponding to 5′ 400 bp of CP (5′ CP). However, such reduction of transgene mRNA and

Fig. 5. Analysis of transgene methylation 12 weeks after grafting by methylation-sensitive restriction digestion. A. Genomic DNA (400 ng) from leaf tissue of non-silenced and silenced lines (200.1 and 200.4, respectively) or from non-silenced scions 12 weeks after being grafted onto silenced rootstocks (200.1/200.4) or from silenced scions 6 weeks after being grafted onto nonsilenced rootstocks (200.4/200.1) was digested with a methylation-sensitive restriction enzyme (Aci I) at 37 °C for 4 h. Digested DNA (100 ng) was subjected to PCR amplification with gene specific primers and separated on 2% agarose gel. Uncut DNA templates were used for PCR as controls. B. Genomic DNA (400 ng) isolated from leaf tissue of post-transcriptionally silenced (400.15) or non-silenced plants (400.89) or from non-silenced scions 12 weeks after being grafted onto silenced rootstocks (400.89/400.15) or from silenced scions 12 weeks after being grafted onto non-silenced rootstocks (400.15/ 400.89) was digested with methylation-sensitive restriction enzymes (Hpa II and Msp I) at 37 °C for 4 h. Digested DNA (100 ng) was subjected to PCR amplification with gene specific primers and separated on 2% agarose gel. Uncut DNA templates were used for PCR as controls.

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Fig. 6. Analysis of transitive RNA silencing in non-silenced scions 4 weeks after grafting onto silenced rootstocks. A. Schematic representation of different regions of CP used as probes. B, C and D. Total RNA (10 μg) from non-silenced scions 4 weeks after grafting onto silenced rootstocks (4.07/200.4 and 40.7/400.15), were analyzed by Northern hybridization with 32P labeled probes specific to 5′CP (B), MCP (C) and 3′CP (D). RNA, extracted from beheaded non-silenced plants, was used as controls (4.07). An ethidium bromide stained RNA gel photographed before transfer is shown as a control for equal loading. E, F and G. Low molecular weight RNA fractions (50 μg) were analyzed by Northern hybridization with 32P labeled probes specific to 5′CP (E), MCP (F) and 3′CP (G). DNA oligomers were used for size control (size indicated in nucleotides). As a control for equal loading, ethidium bromide stained tRNA fractions are shown below.

accumulation of siRNAs were not observed when the membranes were re-hybridized with probes corresponding to the middle region of CP (MCP) or the 3′ region of CP (3′CP) (Fig. 6C, D, F and G). These results showed that even though RNA silencing was induced considerably in the non-silenced scions 4 weeks after grafting, it could not yet, or little if any, spread to the adjacent regions of transgene mRNA effectively at this time-point. An efficient induction of RNA silencing as well as spreading from 5′ to 3′ direction along the entire transgene transcript in scions either from line 4.07 or from line 4.09 (entire CP harboring silenced line, but silencing is targeted solely to the 3′ region of the transgene) was observed when grafted onto silenced rootstocks of lines 200.4 and 400.15 (Haque et al., 2007) 6 weeks after grafting. To reconcile our above finding that systemic RNA silencing through grafting was not associated with transgene DNA methylation, we assessed transgene DNA methylation in scions of these two lines at this time-point. DNA samples digested with methylation-sensitive restriction enzymes Hha I or Hpa II were subjected to PCR amplification of the transgene. Hha I is sensitive to methylation on the inner C residue of its recognition site (GCGC). Two recognition sites of Hha I and one recognition site of Hpa II are located at the 3′ and 5′ region of CP, respectively (Fig. 7A), which facilitated

analysis of DNA methylation at both the 5′ and 3′ regions of the transgene. Transgene methylation of lines 4.07 and 4.09 was determined at first as a control. Almost no transgene methylation was detected at 5′ regions in both the lines, 4.07 and 4.09, as no PCR amplification products were observed using the restriction enzyme Hpa II (Fig. 7B). PCR fragments were less amplified in line 4.07 compared to that in line 4.09 using the restriction enzyme Hha I (Fig. 7B), revealed that transgene was partially and highly methylated at 3′ regions in lines 4.07 and 4.09, respectively. This reflected the percentage of cytosine methylation at two Hha I sites, 67% in 4.07 contrasted 96% in 4.09 (Fig. S2). The percent cytosine methylation of CP at CpG, CpNpG and CpHpH contexts was 35%, 18% and 9%, respectively, in line 4.07 and 63%, 34% and 24% in line 4.09. In scions from lines 4.07 and 4.09 grafted onto silenced rootstocks of lines 200.4 and 400.15, no change in DNA methylation level was observed over the transgene (Fig. 7C, D and S2). CP was not methylated at 5′ regions in both the scions, but partially and highly methylated at 3′ regions in 4.07 and 4.09 scions, respectively. Transgene methylation is postulated to require the interaction of systemic silencing signals with transgene in nuclei, if they are provided continuously but in low quantity from silenced roottocks, it might need longer time for the interaction. Therefore,

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transgene methylation as well as silencing status in these scions was analyzed 12 weeks after grafting onto silenced rootstocks. Analysis of total and low molecular weight RNAs, and transgene methylation with methylation-sensitive restriction enzyme followed by PCR were performed as mentioned above. Similar results, as found 6 weeks after grafting, were observed, showing spreading of RNA silencing from 5′ to 3′ direction along transgene mRNA (Fig. S4) but no change of transgene methylation (Fig. 8) 12 weeks after grafting. These results showed that induction of systemic RNA silencing as well as

Fig. 8. Analysis of transgene methylation 12 weeks after grafting. A. Genomic DNA (400 ng) from 4.07 or 4.09 scions 12 weeks after grafting onto silenced rootstocks (200.4, 400.15 and 4.09) was digested with a methylation-sensitive restriction enzyme at 37 °C for 4 h. Digested DNA (100 ng) was subjected to PCR amplification as described in Fig. 7. B. Uncut DNA templates were used for PCR as control.

transitive RNA silencing did not accompanied transgene methylation up to 12 weeks after grafting. 4. Discussion

Fig. 7. Analysis of transgene methylation by methylation-sensitive restriction digestion. A. Approximate locations of recognition sites for methylationsensitive restriction endonucleases Hpa II and Hha I in CP (including 3′ NTR) and locations of transgene specific forward and reverse primers (unfilled and filled triangles, respectively). B. Genomic DNA (100 ng) isolated from leaf tissue of transgenic lines (4.07 and 4.09), digested with a methylation-sensitive restriction enzyme, was subjected to PCR amplification with gene specific primers and separated on a 0.8% agarose gel. Uncut DNA templates were used for PCR as controls. C. Genomic DNA (100 ng) isolated from 4.07 or 4.09 scions 6 weeks after grafting onto silenced rootstocks (200.4 or 400.15) was digested with a methylation-sensitive restriction enzyme and subjected to PCR amplification. Uncut DNA templates were used for PCR as control. D. Percent cytosine methylation in symmetrical (CpG and CpNpG) and non-symmetrical (CpHpH) contexts in transgene coding region. Genomic DNA extracted from lines 4.07 or 4.09 or from scions of these two lines 6 weeks after grafting onto different rootstocks was subjected to bisulphite sequencing. Percent cytosine methylation was analyzed for the entire CP transgene (including NTR) and calculated from total cytosine residues present in the clones.

In this paper, we showed the levels of transgene cytosine methylation in post-transcriptionally silenced and non-silenced plants, elucidated that transgene methylation was associated with sequence-specific RNA degradation, but not with systemic induction of sequence-specific RNA degradation through grafting. Transgene methylation was studied by methylation-sensitive restriction endonuclease digestion and bisulphite genomic sequencing methods. In post-transcriptionally silenced lines carrying 5′ 200 or 400 bp regions of CP, a high level of DNA methylation was observed in the transgene coding sequence (Figs. 1, 2 and S1). A sharp separation of methylated transgene coding region from the unmethylated 3′ terminal promoter region was observed in these silenced lines. Only one clone showed a rare methylation in the 3′ promoter region. Such methylation might reflect a general methylation noise and probably did not influence promoter activity (Fojtova et al., 2006). In silenced lines, 200.4 and 400.15, transgene methylation was observed in both symmetrical (CpG and CpNpG) and asymmetrical (CpHpH) contexts (Figs. 1 and 2). A very low level of DNA methylation, mostly in the CpG context, was observed in non-silenced lines (Figs. 2 and S1). Thus, transgene methylation in lines carrying the 5′ region of CP showed a correlation with sequence-specific RNA degradation, which coincides with previous reports (Ingelbrecht et al., 1994; Wassenegger et al., 1994a; Pelissier et al., 1999). These two silenced lines carry two copies of the transgene (Haque et al., 2007), so these results thereby agree with a phenomenon that a repeated DNA structure is a major factor that identifies foreign DNA as a target for methylation (Linn et al., 1990).

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However, this correlation of DNA methylation with RNA silencing or with multiple copies of the transgene was not in agreement with DNA methylation patterns observed in lines 4.07 and 4.09 (Figs. 7 and S2). Both lines carry one copy of CP (including the 3′ NTR) of SPFMV (Sonoda et al., 1999). Lines 4.09 and 4.07 are post-transcriptionally silenced and nonsilenced, respectively. In the former one, silencing is targeted to the 3′ 400 bp region of transgene (Sonoda et al., 1999). Methylation was observed at lower levels at 5′ regions (data not shown) and higher levels at middle or 3′ regions of transgene in line 4.09 (Fig. S2). In line 4.07, methylation was also observed at the 3′ half of the transgene sequence, though at lower levels compared to line 4.09 (Fig. S2). In both lines, methylation was observed in both symmetrical and non-symmetrical contexts (Figs. 7D and S2), which is a notable feature of RNA-dependent DNA methylation (RdDM) in plants (Aufsatz et al., 2002; Jones et al., 1999; Melquist and Bender, 2003, 2004; Pelissier et al., 1999; Wang et al., 2001). Methylation in symmetrical CpG and CpNpG contexts in line 4.09 was 1.8 and 2.1 fold higher than that in line 4.07, while that in asymmetrical CpHpH context was by 2.7 folds. Methylation at 3′ regions of the transgene in line 4.09 seems consistent with the RdDM phenomenon, as siRNAs corresponding to 3′ 400 bp region of CP was detected. However, no siRNA corresponding to the middle or 5′ regions of CP could be detected in this line (Haque et al., 2007). The full-length transgene transcript was observed and no siRNAs could be detected in non-silenced line 4.07 using conventional polyacrylamide gel electrophoresis (Sonoda et al., 1999; Haque et al., 2007). It is proposed that RNA signaling molecules, which might be under the detection level, were responsible for the DNA methylation patterns observed in lines 4.07 and 4.09. Similar results were also observed in Arabidopsis, in which tryoptophan-biosynthetic gene PA1 was methylated at both symmetrical and non-symmetrical contexts, but no PA1 siRNAs could be detected (Melquist and Bender, 2003, 2004). Sonoada et al. (1999) showed hypermethylation at 5′ regions of transgene in a non-silenced transgenic line, 7.05. These results indicate that RdDM can be activated by levels of RNA signaling molecules less than the threshold level of those needed for RNA silencing (also reviewed in Mathieu and Bender, 2004) and/or transgene methylation is not sufficient to induce transgene silencing (Meza et al., 2002). DNA methylation was at the highest levels at 3′ regions of the transgene and was extended further to the NOS terminal region in all of the silenced lines studied, with a notable exception in line 4.07, in which DNA methylation was not observed in the 3′ terminal 80 bp region of CP or in the NOS terminal region. Insertion of a truncated transgene might be responsible for such an irregular pattern of transgene methylation in line 4.07, even though this line is reported to carry a single copy of transgene (Sonoda et al., 1999). Similarly, insertion of truncated T-DNA copies led to transgene methylation but not RNA silencing in transgenic Arabidopsis (Meza et al., 2002). Our RNA gel blot analysis shows that sequence-specific RNA degradation as well as transitive RNA silencing was not induced efficiently 4 weeks after grafting onto silenced

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rootstocks (Figs. 3 and 6), but both the processes were effectively induced 6 weeks after grafting (Haque et al., 2007). Such a requirement of a longer time-span (5–7 weeks) (Crete et al., 2001; Mallory et al., 2003) compared to a relatively shorter one (2–4 weeks) (Palauqui et al., 1997; Sonoda and Nishiguchi, 2000; Garcia-Perez et al., 2004; Schwach et al., 2005; Tournier et al., 2006) for an efficient induction of RNA silencing through grafting has already been reported. These differences might reflect the nature of transgenes, and/or competency of silencing rootstocks for the production and transportation of silencing signals to the non-silenced scions through graft-junctions. The trigger molecule for RdDM has not yet been discovered, it might be siRNA, a precursor of siRNA or complementary RNA (Wassenegger, 2000; Dougherty and Parks, 1995; Sijen et al., 1996; Depicker and Van Montagu, 1997; Wassenegger and Pelissier, 1998). Recently, siRNA of a longer class (24–25 nt) is proposed to be the trigger molecule for RdDM (Hamilton et al., 2002). Longer class of siRNAs was also detected in our silenced lines (200.4, 400.15 and 4.09) (Haque et al., 2007). However, this correlation of RNA silencing or accumulation of longer class siRNAs with transgene methylation was not clear in our graft experiments. No change in transgene methylation levels was observed in non-silenced scions after grafting onto silenced rootstocks (Figs. 1, 2, 5, 7, 8, S1 and S2), despite the induction of systemic RNA silencing and accumulation of transgene siRNAs (Fig. 4, S3, S4 and Haque et al., 2007). Similarly, transgene methylation and systemic signaling has also been reported not to correlate with the accumulation of siRNAs or even degradation of the transgene transcript in transgenic plants (Mallory et al., 2003; Sonoda and Nishiguchi, 2000). This implies that induction of systemic RNA silencing or transitive RNA silencing through grafting does not accompany transgene methylation. Although no correlation between systemic RNA silencing and transgene methylation was observed in our graft experiments, induction of DNA methylation in association with RNA silencing is reported in localized introduction of transgene by agro-infiltration (Jones et al., 1999) or by inoculation with recombinant virus (Wassenegger et al., 1994; Pelissier et al., 1999; Vaistij et al., 2002). This difference of transgene methylation in response to systemic silencing signals may reflect the strength (extent) of interaction between silencing signals and transgene (in nuclei) in different experimental systems used to induce systemic silencing (agro-infiltration/virus inoculation versus grafting). In these former two systems, silencing signals interact efficiently with transgene mRNA in cytoplasm and with transgene DNA in nuclei to induce mRNA degradation and DNA methylation, respectively. On the contrary, in graft-induced silencing, silencing signals are provided continuously from rootstocks to scions, but possibly in low quantity. It might be more efficient to induce mRNA degradation in cytoplasm than to perform transgene methylation after transportation into nuclei. Both compartmentation and quantitative aspects needs to be considered for transgene methylation. Either larger amount of silencing signals or much longer time (essentially over 12 weeks, Fig. 8) after grafting might be needed for the extent of interaction sufficient for transgene methylation (above threshold level). Considering the quantitative aspects of

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silencing signals, it is reasonable to understand the transgene methylation by viral vectors (many cycles of viral RNA multiplication even in cytoplasm) or by agro-infiltration (large number of transcripts in the nuclei). Acknowledgements The authors are grateful for the grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Ministry of Agriculture, Forestry and Fisheries of Japan. They are also grateful to T. Tamda and Andika I.B. for the bisulphite sequencing method and S. Sonoda for critical reading of the manuscript. A.K.M.N. Haque is a recipient of the fellowship of Ministry of Education, Science, Culture and Sports of Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gene.2007.04.003. References Adams, R.L., 1990. DNA methylation. The effect of minor bases on DNA– protein interactions. Biochem. J. 265, 309–320. Adams, R.L.P., Burdon, R.H., 1985. Molecular Biology of DNA Methylation. Springer Verlag, New York, NY. Aufsatz, W., Mette, M.F., van der Winden, J., Matzke, A.J., Matzke, M., 2002. RNA-directed DNA methylation in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 99, 16499–16506. Bartee, L., Malagnac, F., Bender, J., 2001. Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes Dev. 15, 1753–1758. Bass, B.L., 2000. Double-stranded RNA as a template for gene silencing. Cell 101, 235–238. Baumberger, N., Baulcombe, D.C., 2005. Arabidopsis Argonaute1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl. Acad. Sci. U. S. A. 102, 11928–11933. Cao, X., Jacobsen, S.E., 2002a. Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc. Natl. Acad. Sci. U. S. A. 99, 16491–16498. Cao, X., Jacobsen, S.E., 2002b. Role of the Arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr. Biol. 12, 1138–1144. Cao, X., et al., 2003. Role of the DRM and CMT3 methyltransferases in RNAdirected DNA methylation. Curr. Biol. 13, 2212–2217. Chan, S.W., Zilberman, D., Xie, Z., Johansen, L.K., Carrington, J.C., Jacobsen, S.E., 2004. RNA silencing genes control de novo DNA methylation. Science 303, 1336. Chen, R.Z., Pettersson, U., Beard, C., Jackson-Grusby, L., Jaenisch, R., 1998. DNA hypomethylation leads to elevated mutation rates. Nature 395, 89–93. Crete, P., et al., 2001. Graft transmission of induced and spontaneous posttranscriptional silencing of chitinase genes. Plant J. 28 (5), 493–501. Depicker, A., Van Montagu, M., 1997. Post-transcriptional gene silencing in plants. Curr. Opin. Cell Biol. 9, 373–382. Dougherty, W.G., Parks, T.D., 1995. Transgenes and gene suppression telling us something new. Curr. Opin. Cell Biol. 7, 399–405. Elbashir, S.M., Lendeckel, W., Tuschl, T., 2001. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200. Finnegan, E.J., Kovac, K.A., 2000. Plant DNA methyltransferases. Plant Mol. Biol. 43, 189–201. Finnegan, E.J., Peacock, W.J., Dennis, E.S., 1996. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc. Natl. Acad. Sci. U. S. A. 93, 8449–8454.

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