Mobile 24 nt Small RNAs Direct Transcriptional Gene Silencing in the Root Meristems of Arabidopsis thaliana

Current Biology 21, 1678–1683, October 11, 2011 ª2011 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2011.08.065

Report Mobile 24 nt Small RNAs Direct Transcriptional Gene Silencing in the Root Meristems of Arabidopsis thaliana Charles W. Melnyk,1,2 Attila Molnar,1,2 Andrew Bassett,1,3 and David C. Baulcombe1,* 1Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK

Summary RNA silencing in flowering plants generates a signal that moves between cells and through the phloem [1, 2]. Nucleotide sequence specificity of the signal is conferred by 21, 22, and 24 nucleotide (nt) sRNAs that are generated by Dicer-like (DCL) proteins [3]. In the recipient cells these sRNAs bind to Argonaute (AGO) effectors of silencing and the 21 nt sRNAs mediate posttranscriptional regulation (PTGS) via mRNA cleavage [4] whereas the 24 nt sRNAs are associated with RNA-dependent DNA methylation (RdDM) [5] that may underlie transcriptional gene silencing (TGS). Intriguingly, genes involved in TGS are required for graft-transmissible gene silencing associated with PTGS [6]. However, some of the same genes were also required for spread of a PTGS silencing signal out of the veins of Arabidopsis [7], and grafting tests failed to demonstrate direct transmission of TGS signals [8–10]. It seemed likely, therefore, that mobile silencing is associated only with PTGS. To address this possibility, we grafted TGS-inducing wild-type Arabidopsis and a mutant that is compromised in 24 nt sRNA production onto a wild-type reporter line. The 21–24 nt sRNAs from the TGS construct were transmitted across a graft union but only the 24 nt sRNAs directed RdDM and TGS of a transgene promoter in meristematic cells. These data extend the significance of an RNA silencing signal to embrace epigenetics and transcriptional gene silencing and support the hypothesis that these signals transmit information to meristematic cells where they initiate persistent epigenetic changes that may influence growth, development, and heritable phenotypes. Results and Discussion To investigate signal movement and TGS, we set up grafting experiments via a two-component transgene system [11]. The target gene (T) of TGS is the GFP transgene driven by a promoter that is most active in the root and shoot meristems although there is lower level activity in other tissues [11]. The silencer (S) transgene targets the meristem-active enhancer and specifies a hairpin RNA expressed from the cauliflower mosaic virus 35S promoter (Figure 1A). In wild-type plants this dsRNA is processed into sRNAs that induce de novo methylation of the target enhancer in trans and TGS of the GFP reporter gene [11, 12]. Plants expressing GFP from the T transgene fluoresce green under UV in the meristems,

2These

authors contributed equally to this work address: MRC Functional Genomics Unit, South Parks Road, Oxford OX1 3QX, UK *Correspondence: [email protected] 3Present

petioles, and root vascular tissue, whereas in plants expressing both T and S transgenes, there is TGS of the T locus and there is no GFP expression or green fluorescence (Figures 1B,1C, 2A, and S1 available online). In grafted plants with homozygous target and silencer (TTSS) shoots and target (TT) genotype roots, there was silencing of GFP in the apical meristems of primary and lateral roots as assessed by fluorescence of GFP or RT-PCR of the GFP RNA (Figures 1D and 2A; Table S1). It is likely therefore that a signal had moved from the shoots and induced TGS in the root meristems. The root meristem silencing was characterized by a gradual loss of GFP fluorescence in the root tip over 20 days (Figure S2) so that, by 3 to 4 weeks after grafting, there was no GFP fluorescence in the vascular tissue of the lateral roots (Figure 1D). In the reciprocal grafting experiments, there was no TGS of the GFP reporter gene in the shoot (Table S1), and subsequent experiments to analyze mobile TGS silencing were carried out by assaying shoot-to-root movement. The TGS silencing pathway involves 24 nt sRNAs generated by DCL3 [13]. To test whether 24 nt sRNAs are the mobile TGS signal, we grafted TTSS dcl3-5 shoots, in which the 24 nt sRNAs from the S transgene are absent [12] (Figures 2B and S3A), to TT roots. We conclude that these 24 nt sRNAs are required for the TGS silencing signal because TTSS dcl3-5 shoots did not induce TGS of the GFP reporter gene in the recipient TT root meristems as assessed by fluorescence of GFP or RT-PCR of the GFP RNA (Figures 1E, 1F, and 2A). We could detect sRNAs that had moved into the TT root tissue from TTSS shoots (Figures 2B and S3A) by northern blotting but the signal was weak, so we used the sensitivity of RNA deep sequencing to assay these low-abundance RNAs. This sequencing analysis confirmed that the TGS of the GFP reporter gene was associated with the accumulation of S locus-specific 21–24 nt sRNAs in TTSS lines (Figures 3A, 3B, S3B, and S3C). These RNAs were predominantly 21 nt, corresponded to both DNA strands, and were specific to the region of the inverted repeat in the S transgene (Figures 3A, 3B, S3B, and S3C). There were also 22–24 nt species. These sRNAs were very rare in TT roots unless they were grafted to TTSS shoots (Figures 3A, 3C, S3B, and S3D). However, these mobile sRNAs were only 1% the level of those in the TTSS shoots and they contain a much higher proportion of 24 nt sRNAs. The TTSS dcl3-5 shoot contained only the 21–23 nt sRNAs, and the unsilenced TT roots grafted to a TTSS dcl3-5 shoot similarly had the 21–23 nt but not the 24 nt species (Figures 3D, 3E, S3E, and S3F). It is clear therefore that all size classes of sRNA are mobile but that the 24 nt species were the only ones able to direct TGS of the T transgene even when they were present at a low level. 24 nt sRNAs direct de novo DNA methylation at the promoter T locus [12] (Figures 4A–4C) and at endogenous loci [14, 15]. Our grafting results are also consistent with this mechanism because the T promoter DNA that was unmethylated in the TT roots was hypermethylated in CG, CHG, and CHH contexts when grafted to TTSS but not TTSS dcl3 shoots (Figures 4A–4F and S3G). It is likely, therefore, that the sRNA-directed TGS of the T promoter is a consequence of RdDM.

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Figure 1. TGS Is Associated with a DCL3-Dependent Graft-Transmissible Signal

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(A) Schematic diagram of the silencing inducer and the target transgene constructs (adapted from [12]). The target transgene (T) contains a meristem-active enhancer (shown in gray and in white) placed upstream of a minimal promoter (black) and GFP-coding region. The silencer transgene (S) harbors an inverted DNA repeat of distal enhancer sequences (gray) under the control of the 35S promoter. The hairpin RNA transcribed from the S locus is diced into sRNAs, which induce de novo methylation of the target enhancer in trans leading to transcriptional gene silencing of the GFP reporter gene. The star represents where the primers used for sRNA northern blotting hybridize (Figure 2B). (B–F) Transmission of the TGS signal by grafting. Grafts were made between wild-type and mutant plants containing the unlinked target (T) and silencer (S) homozygous transgenes to test the movement of a transcriptional gene silencing signal (right). TTSS dcl3 plants are impaired in the production of 24 nt sRNA due to an early stop mutation in DCL3 (dcl3-5 [12]). Root fluorescence (left) and brightfield (middle) images were taken 38 days after grafting. A TT root is presented in each fluorescent panel as an exposure control (white box). The scale bar in (B) represents 0.5 cm and applies to all panels.

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However, unlike the previous analysis of the TTSS system [12], the T locus was hypermethylated and silenced in TTSS dcl3-5 shoots and roots (Figures 1E, 2A, 4A, 4E, S1C, S1D, and S3G), and we infer that 21–23 nt sRNAs or very low levels of 24 nt sRNAs mediate RdDM and TGS in intracellular but not intercellular silencing. Consistent with the former interpretation, Weigel et al. have reported a role of 21 nt sRNAs in RdDM [16]. To reconcile the previous findings [12] with our data, we propose that silencing may have restored over generations because TGS at a promoter occurs in a dcl3 mutant background [17, 18]. Alternatively, plant age or the tissues sampled may have contributed to differences in DNA methylation and GFP fluorescence. We noted previously that some 24 nt sRNA loci produce mobile sRNAs whereas others produce nonmobile species [5].

The variation between silencing loci is emphasized by comparison of the TTSS system (Figures 1, 3, and S3) with a TGS transgene TT that does not produce a mobile signal [8]. The key difference in these experimental systems is likely to involve the structure of the transgenes. In the system producing the mobile signal [12], the sRNAs are derived from the TTSS dcl3 transcribed region of the transgene whereas in the system that does not produce the signal, TTSS dcl3 the sRNAs would be derived from the promoter region [8]. Initiation of promoter silencing would prevent continued production of sRNAs and so the signal would be blocked whereas sRNA targeted to a transcribed region would not cause TGS and so production of the TTSS dcl3 sRNA silencing signal would be continuous. A similar effect is likely to apply at endogenous TT loci: those producing 24 nt siRNAs from transcribed regions are more likely to produce a mobile signal of RdDM and TGS than those in which the sRNAs are derived from the promoter. Our previous analysis of endogenous sRNAs detected preferential mobility of 24 nt rather than the smaller sRNAs [5]. However, there was similar movement of all size classes of S transgene sRNA (Figures 3 and S3) although only the 24 nt species were active in RdDM and TGS. This mobility difference, as with the property of producing a TGS signal, is probably due to locus-specific factors although we do not yet have information as to what those factors might be. One of the next challenges in mobile silencing will be to determine the cellular channels used by the mobile sRNAs. There is good evidence that movement of the signal between cells is through plasmodesmata [19] and longer distance transport is generally assumed to be through the phloem [20]. However, even in the long-distance movement, the silencing signal associated with the S transgene would pass

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with changes to gene expression in response to external stimuli. Additionally, in the shoot meristems, they might also be associated with epigenetic effects that, as with virusinduced TGS [21], could persist transgenerationally. This report of graft-transmissible TGS extends the range of mobile sRNAs and their effects. There are various types of 21 nt and 24 nt sRNAs that move from cell to cell and over long distances [4, 5, 22] and it is likely that 21 nt miRNAs are mobile [23, 24]. Future work will focus on host determinants of sRNA mobility and the biological implications of their effect.

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Figure 2. Molecular Analyses of Mobile TGS (A) GFP mRNA levels in grafted plants. Transcript levels were quantified by qPCR for two independent biological replicates that were averaged using a weighted mean based on the levels of UBC9 transcript. Shoot/root notation is used, with the tissue sampled in bold. (B) Detection of sRNAs in grafted plants by northern hybridization. Grafting combinations are indicated on the top. The primary sRNA probe detects sRNAs that are generated from the silencer locus. AtREP2 sRNAs are DCL3 dependent and this probe tested for the accumulation of endogenous 24 nt sRNAs. Hybridization with miR319 and U6 probes are shown as loading controls. s, shoot; r, root.

symplastically through several layers of cells between the vasculature and the meristem. Therefore, there are likely to be similar mechanisms in both cell-to-cell and systemic movement of sRNAs. Movement of the silencing signal associated with PTGS follows photosynthetic source-sink gradients [2, 19], and it is likely that the same property explains the movement of the TGS signal from shoots to grafted roots rather than vice versa (Table S1). This property would also explain the initiation of TGS of the GFP promoter in the root meristems (Figures 1 and S2) that would be a stronger sink than the nonmeristematic tissues adjacent to the graft union. Such an effect would be reinforced if, as seems likely, epigenetic mechanisms are more active in meristems than in other tissue types. Although we have investigated silencing signal movement into roots, there is no reason why TGS and RdDM silencing signals would not follow source-sink gradients into the shoot, as with PTGS signals [1, 2, 19]. The signal-induced TGS could be associated

Plant Material, Grafting, and GFP Imaging The Arabidopsis lines TT, TTSS, and TTSS dcl3-5 have been described previously [11, 12]. Arabidopsis plants were grown under 10 hr supplemental lighting (fluorescent lights) at a constant temperature of 20 C on vertically mounted plates of 1.2% agar, 0.5 MS media (pH 5.7). Plants were grafted 7 days after seed germination using grafting techniques previously described [6, 25]. Five weeks after grafting, plant tissue was harvested. GFP fluorescence in Arabidopsis shoots and roots was taken using a Leica DFC310 FX camera attached to a Leica M165FC dissecting microscope. Nucleic Acid Isolation, Genotyping, and sRNA Cloning Total nucleic acid (TNA), a mixture of DNA and RNA, was isolated from root and shoots using a modified protocol by White and Kaper [26] that can be downloaded from our website http://www.plantsci.cam.ac.uk/research/ baulcombe/smallrnacloning.html. TNA samples were used for genotyping, sRNA northerns, and the methylation analysis. To isolate pure DNA for genotyping and bisulphite sequencing, 3 mg of TNA was digested with RNase A and then the DNA was purified by phenol-chloroform extraction. After precipitation and resuspension, DNA was diluted 1003 and PCR amplified using 45 cycles for genotyping, or converted for bisulphite sequencing (see below). The sensitivity of the PCR genotyping could detect contamination of less than 1%, and in all of the samples used, no contamination was detected. The primer combinations used can be found in Table S3. 1 mg of TNA was used to clone sRNAs by the Illumina v1.5 sRNA cloning kit. The sRNA libraries generated from biological duplicates were multiplexed and sequenced on the same lane of the flow cell. Information regarding the sRNA libraries is presented in Table S2. Northern Blotting, Bisulphite Sequencing, and qPCRs sRNA northern blotting was performed as previously described [27]. The concentration of the purified DNA was estimated by quantitative PCR (Biorad CFX) against a standard curve of known genomic DNA concentrations. 400 ng of DNA was treated with sodium bisulphite using the EZDNA methylation Gold kit (Zymo Research) according to the manufacturer’s instructions. DNA was amplified by PCR with Taq DNA polymerase with a 62 C elongation temperature. PCR products were gel extracted and cloned into pGEM-T Easy (Promega) and clones were sequenced with BigDye 3.1 (Applied Biosystems). Complete conversion of the DNA was confirmed by testing methylation at the PHAVOLUTA locus which lacks DNA methylation [28, 29]. Data were analyzed by CyMATE software [30]. Conversion efficiency at the PHAVOLUTA locus was greater than 95% for each bisulphate-treated sample (data not shown). Original data for the target (T) and Tag2 loci are shown in Figure S4. RNA was cleaned of contaminating DNA using the Ambion Turbo DNA-free. 1 mg of RNA was added to random hexamer primers and cDNA synthesized using Superscript III (Invitrogen) and the manufacturer’s instructions. After cDNA synthesis, the reaction was RNase treated. Semiquantitative PCR reactions were performed with a Biorad CFX thermocycler and SYBR Green JumpStart Taq ReadyMix (Sigma). Primer sequences are presented in Table S3. Accession Numbers sRNA sequencing raw data are available from Gene Expression Omnibus (GSE 31651). Supplemental Information Supplemental Information includes four figures and three tables and can be found with this article online at doi:10.1016/j.cub.2011.08.065.

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Figure 3. Analysis of sRNAs Associated with Mobile TGS by Deep Sequencing sRNA libraries were generated from grafted Arabidopsis and aligned at the first nucleotide to 500 base pairs of the T enhancer where it is targeted by the S transgene. The positive or negative y axis shows the number of reads at each position on either the plus or minus strand. The line below the x axis represents portions of the T enhancer (white) and the region targeted by the S transgene (black). Shoot/root notation is used, with the tissue sampled in bold. See Figure S3 for an independent biological replicate. Table S2 summarizes the sRNA library details. (A) TT shoot grafted onto TT root. (B) TTSS shoot grafted onto TTSS root. (C) TTSS shoot grafted onto TT root. (D) TTSSd3 shoot grafted onto TTSSd3 root. (E) TTSSd3 shoot grafted onto TT root.

Acknowledgments

2. Voinnet, O., and Baulcombe, D.C. (1997). Systemic signalling in gene silencing. Nature 389, 553.

We thank Marjori Matzke, Lucia Daxinger, and Tatsuo Kanno for TT, TTSS, and TTSS dcl3-5 seeds and Marjori Matzke and Taku Sasaki for assistance with bisulphite sequencing analyses. We also thank Krys Kelly and Vera Pancaldi for discussion and advice about computational analysis and Risha Narayan for technical assistance. C.W.M. was supported by the Cambridge Commonwealth trust and the Natural Sciences and Engineering Research Council of Canada. This work was supported by the Gatsby Charitable Foundation and European Research Council Advanced investigator grant 233325 REVOLUTION. D.C.B. is supported as a Royal Society Research Professor. A.M., C.W.M., and D.C.B. designed the research. C.W.M., A.M., and A.B. performed the experiments. C.W.M., A.M., A.B., and D.C.B. analyzed the data. C.W.M., A.M., and D.C.B. wrote the paper.

3. Baulcombe, D. (2004). RNA silencing in plants. Nature 431, 356–363.

Received: March 8, 2011 Revised: August 1, 2011 Accepted: August 31, 2011 Published online: September 29, 2011

7. Smith, L.M., Pontes, O., Searle, I., Yelina, N., Yousafzai, F.K., Herr, A.J., Pikaard, C.S., and Baulcombe, D.C. (2007). An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell 19, 1507–1521.

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