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An endogene-resembling transgene is resistant to DNA methylation and systemic silencing a
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a
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Elena Dadami , Athanasios Dalakouras , Michele Zwiebel , Gabi Krczal & Michael ab
Wassenegger a
RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany
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Centre for Organismal Studies (COS) Heidelberg; University of Heidelberg; Heidelberg, Germany Published online: 23 Jul 2014.
Click for updates To cite this article: Elena Dadami, Athanasios Dalakouras, Michele Zwiebel, Gabi Krczal & Michael Wassenegger (2014) An endogene-resembling transgene is resistant to DNA methylation and systemic silencing, RNA Biology, 11:7, 934-941, DOI: 10.4161/rna.29623 To link to this article: http://dx.doi.org/10.4161/rna.29623
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RNA Biology 11:7, 934–941; Jul 2014; © 2014 Landes Bioscience
An endogene-resembling transgene is resistant to DNA methylation and systemic silencing Elena Dadami1†, Athanasios Dalakouras1†, Michele Zwiebel1, Gabi Krczal1, Michael Wassenegger1,2,* RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany; 2Centre for Organismal Studies (COS) Heidelberg; University of Heidelberg; Heidelberg, Germany;
1
These authors contributed equally to this work.
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In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNAdirected DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFPendo). Upon agroinfiltration of a hairpin GFP (hpF) construct, transgenic Nicotiana benthamiana plants harboring GFPendo (Nb-GFPendo) were susceptible to local PTGS. Yet, in the local area, PTGS was not accompanied by RdDM of the GFPendo coding region. Importantly, hpF-agroinfiltrated Nb-GFPendo plants were resistant to systemic silencing. For reasons of comparison, transgenic N. benthamiana plants (Nb-GFPcDNA) carrying a GFP cDNA transgene (GFPcDNA) were included in the analysis. HpF-agroinfiltrated Nb-GFPcDNA plants exhibited local PTGS and RdDM. In addition, systemic silencing was established in Nb-GFPcDNA plants. In agreement with previous reports using grafted scions, in systemically silenced tissue, siRNAs mapping to the 3′ of GFP were predominantly detectable by Northern blot analysis. Yet, in contrast to other reports, in systemically silenced leaves, PTGS was also accompanied by dense RdDM comprising the entire GFPcDNA coding region. Overall, our analysis indicated that cDNA transgenes are prone to systemic PTGS and RdDM, while endogene-resembling ones are resistant to RNA silencing.
Introduction RNA silencing refers to a network of small RNA (sRNA)mediated pathways that are important for normal plant development, genome stability, defense against invading nucleic acids and response to abiotic/biotic stress.1,2 In general, there are two major pathways of RNA silencing in plants; the small interfering RNA (siRNA) and the microRNA (miRNA)mediated silencing pathways.3,4 The siRNA pathway is induced by double stranded RNA (dsRNA) that is processed by the DICERLIKE (DCL) endonucleases, DCL2, DCL3 and DCL4, into 22-, 24-, and 21-nucleotides (nt) long siRNAs, respectively.5 Mature siRNAs exhibit 3′ 2-nt overhangs and become stabilized though 3′ end methylation by HUE ENHANCER1 (HEN1).6 Depending on their 5′ terminal nucleotide, siRNAs are loaded onto specific ARGONAUTE (AGO) proteins.7 Only one siRNA strand, the guide strand, is finally selected.8 21-nt siRNAs are loaded primarily onto AGO1, and to lower extent onto AGO2, leading to the formation of RNA-induced silencing complex (RISC).9 RISC mediates post-transcriptional gene silencing (PTGS) through targeting RNA molecules that are complementary to the guide strand. PTGS results in target RNA degradation and/ or, in the case of mRNA targets, in translational inhibition.10,11 24-nt siRNAs are loaded onto AGO4/AGO6 and are supposed
to target homologous genomic DNA for de novo methylation, in a process termed RNA-directed DNA methylation (RdDM).12,13 PTGS and RdDM are both initiated by dsRNA. Among others, sources of dsRNA are replication intermediates of viruses and viroids, transcription of inverted repeats, stress-induced overlapping antisense transcripts and RNA-DIRECTED RNA POLYMERASE (RDR) transcription of aberrant RNAs (abRNAs).14-16 AbRNAs are most probably transcripts devoid of 5′ cap and/or 3′ polyadenylation tail.17,18 Such transcripts may directly be generated through abnormal DNA-DEPENDENT RNA POLYMERASE II (POL II) transcription, e.g., producing truncated and/or read-through transcripts. In addition, abRNA may derive from RISC-mediated generation of 5′ and 3′ target RNA cleavage products. Thus, while initial cleavage is conducted by primary siRNAs, RDR6 transcription of the cleavage products generates secondary dsRNA that is processed into secondary siRNAs.19,20 In contrast to primary siRNAs, that match only to a sub-region of the target RNA, secondary siRNAs may map upstream and downstream from the binding sites of the primary siRNAs. This spreading of silencing beyond initial target sites is termed transitivity.15,21 The exact nature of secondary siRNAs is not known, but it seems that at least DCL2 is involved in their biogenesis.22 Interestingly, while secondary siRNAs are cleavagecompetent, they cannot further support the biogenesis of tertiary
*Correspondence to: Michael Wassenegger ; Email: [email protected]) Submitted: 05/05/2014; Revised: 06/11/2014; Accepted: 06/17/2014; Published Online: 07/23/2014 http://dx.doi.org/10.4161/rna.29623 934
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Results Transgenic Nb-GFPendo and Nb-GFPcDNA plants The GFPendo construct was designed to resemble the structure of one of the highly transcribed small subunit of RIBULOSE-1,5-BIPHOSPHATE CARBOXYLASE OXYGENASE (RuBisCO) genes.41 The GFPendo consists of the RuBisCO promoter/5′UTR/two introns/3′ UTR/ terminator and three (arbitrarily chosen) GFP exons (Fig. 1). RuBisCO genes are highly transcribed endogenes that are not prone to RDR6-mediated processing.15 Previously, we could show that upon agroinfiltration, GFPendo transcripts were inefficiently processed by RDR6-mediated pathway.41 In addition to transitivity, Figure 1. Schematic representation of GFPendo, hpF and GFPcDNA constructs. P35S: RDR6 is involved in the establishment of RdDM and CaMV 35S promoter; TNOS: nopaline synthase (NOS) terminator; GFP: green systemic silencing.15,42,43 We assumed that reduced RDR6fluorescent protein; PRbc: tobacco RuBisCO promoter including 5′ UTR; TRbc: processivity of the agroinfiltrated GFPendo construct tobacco RuBisCO terminator including 3′ UTR; Intron 1 and 2: tobacco RuBisCO could also have an impact on the efficiency of RdDM introns 1 and 2. The hybridization probes used for northern blot analysis (Fig. 3) and systemic silencing of a genome-integrated GFPendo and the 182 bp and 134 bp regions analyzed by bisulfite sequencing (Fig. 4, S2 and S3) are indicated by black bars. construct. Thus, transgenic N. benthamiana GFPendo plants (Nb-GFPendo) were generated. For reasons of comparison, the N. benthamiana plant line 16C carrying siRNAs. Thus, transitivity may spread to 5′ and 3′ direction, a GFP cDNA transgene44 was used. Line 16C (hereafter referred but not indefinitely.21,23 Nevertheless, transitivity results in the to as Nb-GFPcDNA) is exceptional in that it shows remarkably amplification of siRNAs constituting a self-reinforcing feedback high GFP expression without being prone to spontaneous cisloop of silencing. Silencing may spread through plasmodesmata silencing.36,39,40 Importantly, Nb-GFPendo and Nb-GFPcDNA cell-to-cell and through the vasculare system to systemic parts of plants exhibited almost similar GFP expression (Fig. 2, 3 and the plant.24,25 While cell-to-cell signals are most probably 21-nt S1). Thus, any putative divergence of responses against RNA duplex siRNAs, the nature of systemic silencing signals is still silencing of the two transgenes should reflect qualitative rather elusive.26-29 It has been proposed that 24-nt siRNAs30-32 or longer than quantitative effects. RNAs33 may be the molecules traveling through the phloem to Analysis of PTGS in locally silenced leaves metabolic sink tissues, but solid experimental evidence for either Endogenes and transgenes are prone to RNA silencing when scenario is lacking. an exogenous trigger e.g., hairpin RNA (hpRNA) is present and In general, the response of transgenes and endogenes against processed into siRNAs (trans-silencing).45,46 In order to compare RNA silencing is diverse. Both, endogenes and transgenes are trans-silencing of GFPendo and GFPcDNA, a hairpin GFP (hpF) equally susceptible to sRNA-mediated trans-PTGS. However, construct covering 139 bp of the GFP middle region (F) (Fig. 1), with a few exceptions, including TAS genes in Arabidopsis and was agroinfiltrated into Nb-GFPendo and Nb-GFPcDNA plants. For BETA-1,3-GLUCANASE genes in tobacco, (1) endogenes each treatment, three plants were agroinfiltrated (three leaves for are generally not prone to transitivity.34,35 Likewise, with the each plant) and the pooled tissue was used for subsequent analysis. exception of two endogenous INVERTED-REPEAT (IR) Both, Nb-GFPendo and Nb-GFPcDNA plants revealed local PTGS loci in Arabidopsis, (2) endogenes are not prone to systemic already 4 days post infiltration (dpi) and displayed full local GFP silencing.26,36,37 In contrast to transgenes, (3) endogenes appear silencing at 8 dpi (Fig. 2). SRNA-mediated silencing in plants to be resistant to RdDM in N. benthamiana.38 Finally, in contrast results in mRNA degradation and/or translational inhibition.10,39 to transgenes, (4) endogenes are not prone to spontaneous In order to discriminate between the two processes, RNA was silencing (cis-silencing).39,40 In order to analyze if a transgene can extracted from a pool of agroinfiltrated leaves and subjected to mimick an endogene with introns and thus, become less prone to Northern blot analysis. Our data revealed that primary GFP RNA silencing, we constructed an intron-containing endogene- siRNAs, originating from the agroinfiltrated hpF construct, resembling GFP transgene (GFPendo). We have previously shown targeted GFPendo and GFPcDNA transcripts for degradation (Fig. 3, that upon agroinfiltration, GFPendo was poorly processed into probe F). In addition to the production of primary siRNAs, siRNAs.41 In this study, we generated transgenic N. benthamiana transitivity-derived secondary siRNAs may also be generated GFPendo plants (Nb-GFPendo) and we explored the response of by RDR6/DCLs processing of abRNA. However, in samples GFPendo against RNA silencing. In summary, our data indicated of locally silenced tissue, no secondary 5′ and 3′ siRNAs were that, similar to most endogenes, GFPendo was no target for RdDM detectable under the applied conditions (Fig. 3, probes G and P). and systemic silencing. Secondary siRNAs production may be slow23 and may take longer
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than 8 dpi. However, progressive physiological degeneration of agroinfiltrated tissue 8 dpi did not allow analysis at later stages. Analysis of RdDM in locally silenced leaves PTGS is tightly related to RdDM. In the case of transgenes, silencing of mRNA is generally associated with RdDM of its coding region, which in turn, may contribute to maintain PTGS.46 Endogenes and transgenes are efficiently targeted for trans-PTGS. However, why only transgenes are prone to RdDM46 is still elusive. In order to investigate if the endogene-resembling GFPendo differs from GFPcDNA in view of being a target for RdDM, the methylation status of the transgene constructs was analyzed by bisulfite sequencing using pooled samples Figure 2. GFP monitoring of hpF-agroinfiltrated GFPendo and GFPcDNAN. benthamiana plants. The develfrom locally silenced leaf tissue (8 opment of RNA silencing was monitored at local levels (4 and 8 dpi) and systemic levels (21 and 46 dpi). dpi) (Fig. 2). We found that GFPcDNA was heavily methylated, not only in Analysis of RdDM in systemically silenced leaves the region targeted by the hpF silencing trigger, but also in the In grafting experiments employing transgenic plants, silenced 3′ downstream sequence (Figs. 4 and S2). This finding was in agreement with another study reporting on RdDM spreading.47 scions induced systemic PTGS and RdDM in the rootstock.30,32,50 Notably, the observed 3′ spreading of RdDM was not correlated In a reverse experiment using a silenced rootstock, the scion became with the accumulation of detectable amounts of 3′ siRNAs systemically silenced, but de novo methylation of the transgene (Fig. 3, probe P). In contrast to the GFPcDNA, GFPendo was poorly was not detectable suggesting that the signals for induction of methylated in locally silenced tissue (Figs. 4 and S3). This PTGS and RdDM are not identical.33,51,52 In contrast to grafting finding was striking, since the silencing triggers of Nb-GFPendo experiments, we induced local silencing in leaves of individual and Nb-GFPcDNA were identical (hpF). In addition, abundant Nb-GFPcDNA and Nb-GFPendo plants. DNA was extracted from a primary hpF-derived siRNAs were detectable in Nb-GFPendo and pool of newly emerged Nb-GFPcDNA (almost fully silenced) and Nb-GFPcDNA samples (Fig. 3, probe F). Thus, one would have Nb-GFPendo (non-silenced) leaves 21 dpi, respectively (Fig. 2). expected RdDM of GFPendo, at least, in the region targeted by The DNA samples were subjected to bisulfite sequencing. The hpF. These findings suggest that the mere presence of siRNAs sequencing data showed that, as expected, the GFPendo was not was not sufficient to efficiently trigger RdDM. methylated (Figs. 4 and S3). In contrast, the coding region of Systemic PTGS analysis the GFPcDNA construct was heavily methylated (Figs. 4 and S2). Endogenes and transgenes are efficiently targeted for local Importantly, RdDM patterns were not restricted to the siRNAPTGS, but only transgenes appeared to be prone to systemic mapping 3′ region but were established along the entire coding PTGS.25,37,48 Thus, we were interested to investigate if locally region (Fig. S2). Bisulfite sequencing data were validated by silenced Nb-GFPendo plants also become systemically silenced. For Southern blot analysis using the methylation sensitive restriction this reason, 12 Nb-GFPcDNA and Nb-GFPendo plants displaying endonucleases, NcoI, DdeI and PvuII (Fig. S4). In summary, our local silencing post hpF-agroinfiltration were monitored up to data suggested that in a non-grafting system, systemic PTGS 46 dpi for systemic silencing. All Nb-GFPcDNA plants exhibited of GFPcDNA is associated with efficient RdDM of the silenced systemic PTGS already 21 dpi (Fig. 2). Importantly, and in transgene. agreement with previous observations, only 3′ GFP siRNAs were detectable in systemically silenced leaves by Northern blot analysis (Fig. 3, probe P).33,49 In contrast, 21 dpi and even 46 dpi Discussion no systemic GFP silencing was observed in Nb-GFPendo plants. At 46 dpi Nb-GFPcDNA plants were fully silenced (Fig. 2). We The RdDM machinery is considered to be guided by thus conclude that, similar to most endogenes, GFPendo failed to 24-nt siRNAs.53-55 In our experiments, primary GFP siRNAs induce the production of and/or failed to respond to systemic were detected in samples of agroinfiltrated Nb-GFPendo and silencing signals. Nb-GFPcDNA leaves. However, GFPendo was inefficiently
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Figure 3. Northern blot analysis of RNA samples of hpF-agroinfiltrated GFPendo and GFPcDNA N. benthamiana plants. Large and small RNA analysis was performed at local levels (4 and 8 dpi) and systemic levels (21 dpi). The regions corresponding to hybridization probes are indicated in Figure 1. Ethidium bromide staining and miR159 accumulation served as loading controls. ‘Mock’ corresponds to 10 mM MgCl2 infiltration.
targeted for RdDM. Thus, our data suggests that either the mere presence of siRNAs is not sufficient to trigger RdDM, or molecules, other than siRNAs may target genomic DNA for de novo methylation.56,57 Establishment of RdDM is not only based on the presence of RdDM trigger molecules, but also on the transcription of RdDM targets (DNA and/or nascent transcripts). Typically, DNA-DEPENDENT RNA POLYMERASES IV and V (POLIV and POLV, respectively) are thought to transcribe RdDM targets, thus producing scaffold transcripts for the binding of trigger molecules.13,58,59 In addition, RDR2 and RDR6 transcription of target transcripts appeared to be essential for the establishment and/ or amplification of RdDM.43,47 Differential POLIV/POLV/ RDR2/RDR6 processing of GFPendo and GFPcDNA may also account for their differential methylation patterns we observed. Production of systemic silencing signals does not require DCL1, DCL2, DCL3, DCL4, RDR2, RDR6 and POLIV,29 indicating that systemic silencing is not triggered by a typical siRNA. However, upon reception, the response to systemic signals depends on RDR6, RDR2, DCL3 and POLIV.29 Our previous data showed that, when agroinfiltrated into Nb-GFPcDNA leaves, GFPendo induced diminished local and delayed systemic silencing of GFPcDNA.41 Here, we found that upon agroinfiltration of hpF, GFPendo did not respond to systemic silencing signals. Since systemic silencing requires RDR6, one may speculate that GFPendo does not produce RNA molecules that serve as a substrates for RDR6. Thus, similar to typical endogenes,36 transgenes resembling endogenes seem not to respond to systemic silencing signals.
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The diverse response of GFPendo and GFPcDNA to systemic silencing and RdDM indicated that the availability of RDR6 substrates, probably abRNA, is a critical step in these processes. RDR6 seems to prefer abRNAs that lack either 5′ cap and/or 3′ polyadenylation tail.17,18 Most likely, both GFPendo and GFPcDNA produce abRNAs. In the case of GFPcDNA, abRNAs may refer to truncated and/or read-through transcripts, lacking 5′ cap and/or 3′ polyadenylation tail, thus serving as optimal RDR6 substrates.17,18 In the case of GFPendo, abRNAs may in addition, include intron-containing mispliced and/or unspliced transcripts that were ‘overlooked’ by the spliceosome. We suggest that, while GFPcDNA abRNAs are predominantly copied by RDR6 and induce RNA silencing, GFPendo abRNAs are not processed by RDR6 but are instead eliminated via exonucleolytic RNA decay mechanism.17,60,61 GFPendo differed in several aspects from GFPcDNA (promoter, UTRs, introns, terminator, integration site). In support of our suggestion, while the presence of UTRs and introns is positively correlated with RNA decay, it is negatively correlated with RNA silencing.23,60,62-64 Spliceosome association likely excludes RDR6 recruitment.16 Thus, endogenous siRNAs matching the transcripts of intron-containing genes are underrepresented in sRNA libraries of Arabidopsis, while siRNAs derived from intronless gene transcripts are highly abundant.63 Nevertheless, a couple of endogenous transcripts are targeted by RDR6 leading to the production of endogenous siRNAs.34,65 In conclusion, we have shown that an endogene-resembling transgene is resistant to systemic silencing and DNA methylation, most probably due to its poor RDR6-mediated processing. The presence of each endogenous element (promoter, 5′ UTR,
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introns, 3′ UTR, terminator) may have its own contribution to this phenomenon, but we find of particular importance the presence of introns. Yet, investigating the role of each parameter was beyond the scope of our analysis and would have required the production of an enormous amount of independent transgene constructs comprising all free combinations of the individual RuBisCO gene elements. Moreover, such work would not include the introduction of the introns at different GFP coding region sides or the analysis of an alternative reporter gene. Our data suggest that endogene-resembling transgenes may point toward a new generation of transgenes, where high transcription rates would not be compromised by concomitant susceptibility to RNA silencing.
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Materials and Methods Generation of transgenic plants The generation of pG104-GFPendo construct has been previously described.41 Agrobacterium tumefaciens (strain ATHV) containing pG104-GFPendo was used to transform N. benthamiana via leaf disc transformation, as previously described.66 Since pG104GFPendo did not contain a selection marker, co-transformation with A. tumefaciens (strain GV3101) containing the binary vector pPCV702SM-Δ67 was conducted for kanamycin selection. Independent T1 lines were characterized by Southern and northern blot analysis for copy number and expression levels of the transgene (Fig. S5). At least two independent GFPendo lines (18 and 25) containing multiple transgene copies, displayed GFP expression levels and GFP fluorescence similar that of GFPcDNA line 16C. Segregants of the GFPendo line 25, containing 2–5 transgene copies (Fig. S5) were used for subsequent analysis. RNA extraction and northern blot analysis RNA was isolated with NucleoSpin miRNA kit (MachereyNagel, www.mn-net.com) according to the manufacturer‘s instructions. Large and small RNA fractions were separately isolated and used for mRNA and siRNA analysis. For mRNA analysis, 5 µg of large RNA fraction was analyzed in 1.2% agarose/formaldehyde gels, and blotted onto positively charged nylon membranes BioBond Plus (Sigma-Aldrich, www. sigmaaldrich.com) and UV312nm-cross-linked (300 mJ/cm2). The PerfectHyb Plus 1x (Sigma-Aldrich) was used for overnight hybridization at 64 °C. Membranes were washed at 64 °C with buffer 1 (2x SSC, 0.1% SDS (w/v)) for 30 min and with buffer 2 (0.5x SSC, 0.1% SDS (w/v)) for 15 min. For siRNA analysis, 5 µg of small RNA fraction was analyzed in 15% TBE-Urea gels and blotted onto positively charged nylon membranes Bright Star (Life Technologies, www.lifetechnologies.com) by electroblotting at 300mA for 1 h and UV312nm-cross-linked (300 mJ/ cm2). The PerfectHyb Plus 1x (Sigma-Aldrich) was used for overnight hybridization at 42 °C and membranes were washed twice with buffer 1 (2x SSC, 0.1% SDS (w/v)) at 42 °C for 30 min. For the generation of probes, templates obtained by PCR on GFP sequence were used in a random labeling reaction with DecaLabel DNA labeling kit (ThermoScientific, www. thermoscientific.com) with a-32P dATP. For probe G (291 bp,
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Figure 4. Histogram representing bisulfite sequencing data from GFPcDNA and GFPendo. Non-agroinfiltrated, 8 dpi local and 21 dpi systemic tissue was used for analysis. 10–15 clones were sequenced and data were analyzed with CyMate software.68 Histogram represents the data presented in Fig. S2 and S3. In non-agroinfiltrated GFPcDNA, transgenerationally maintained CG methylation was detected, in agreement with previous observations.41,70
62% A/T context), the primers 5′-ATG AAG ACT AAT CTT TTT CT-3′ and 5′-ATC TGG GTA TCT TGA AAA GC-3′ were used. For probe F (144 bp, 47% A/T context), the primers 5′-CAT ATG AAG CGG CAC GAC TT-3′ and 5′-CTC GAT CCT GTT GAC GAG GG-3′ were used. For probe P (336 bp, 53% A/T context), the primers 5′-CTT AAG GGA ATC GAT TTC AA-3′ and 5′-TAG TTC ATC CAT GCC ATG TG-3′ were used. Probes G, F and P are continuous and cover the full GFP sequence. For the detection of miR159, the oligonucleotide 5′-AAG AGC TCC CTT CAA TCC AAA-3′ was used in an end-labeling reaction using T4 polynucleotide kinase (New England Biolabs, www.neb.com) and γ-32P ATP. DNA extraction and Southern blot analysis To 1 g of grinded fresh leaf material, 4 ml extraction buffer (2% (w/v) CTAB, 20 mM EDTA, 1.4 M NaCl, 100 mM TRISHCl, pH = 8), 40 µl proteinase K solution and 8 µl RNase A were added, the mixture was incubated at 65 °C for 1 h then briefly cooled on ice. Four ml of chloroform:octanol (24:1) was added and the sample was centrifuged (6000 rpm, 15 min, 4 °C) on Rotanda 360R. To the upper phase, 400 µl CTAB/NaCl solution (10% (w/v) CTAB, 0.7 M NaCl) and 4 ml chloroform:octanol (24:1) were added and the sample was centrifuged (6000 rpm, 15 min, 4 °C). To the upper phase 4 ml precipitation buffer (1% (w/v) CTAB, 10 mM EDTA, 50 mM TRIS-HCl, pH = 8) were added and the mixture was kept overnight at 37 °C. The DNA was precipitated (6000 rpm, 15 min, 4 °C) and the pellet was resuspended in 1 ml high salt TE solution (1 M NaCl, 0.1 mM EDTA, 10 mM TRIS-HCl, pH = 8) at 65 °C for 30 min. The DNA was re-precipitated (6000 rpm, 15 min, 4 °C) with 1 ml isoporopanol and washed twice (centrifugation at 6000 rpm, 15 min, 4 °C) with 1 ml ethanol 70% (v/v). Finally, the DNA pellet was air-dried for 5 min and resuspended in water. For Southern blot analysis, 10 µg DNA were digested with the corresponding restriction endonucleases, separated onto 1% agarose gels and treated for 30 min with denaturation buffer (1 M NaCl, 0.4 N
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NaOH). DNA was capillary transferred overnight onto positively charged nylon membranes (BioBond Plus, Sigma-Aldrich, www. sigmaaldrich.com) with denaturation buffer. After transfer, the membranes were treated with equilibration buffer (1 M NaCl, 1 M TRIS-HCl, pH = 7), air-dried and UV312nm-cross-linked (100 mJ/cm2). For the generation of probe 35S, templates obtained by PCR (primers 5′-CCG GAA ACC TCC TCG GAT TCC3′ and 5′-GAT GAT CCC CCT CTC CAA ATG-3′) on 35S promoter sequence were used in a random labeling reaction with DecaLabel DNA labeling kit (ThermoScientific, www. thermoscientific.com) and a-32P dATP. The PerfectHyb Plus 1x (Sigma-Aldrich, www.sigmaaldrich.com) was used for overnight hybridization at 64 °C. Membranes were washed at 64 °C with buffer 1 (2x SSC, 0.1% (w/v) SDS) for 30 min and with buffer 2 (1x SSC, 0.1% (w/v) SDS) for 15 min. Membranes were exposed to FujiFilm Imaging Plates (Fuji, www.fujifilm.com) for 2 d and scanned using PharosFX Plus PhosphorImager (BioRad, www. bio-rad.com). Western blot analysis Protein was extracted from Nb-GFPendo and Nb-GFPcDNA and subjected to western blot analysis with GFP antibody as previously described.67 Bisulfite sequencing For bisulfite sequencing, 500 ng DNA were pre-digested with DraI and subjected to bisulfite treatment using the EZ DNA Methylation-Lightning Kit (ZYMO Research, www. zymoresearch.com) according to the manufacturer‘s instructions. The recovered material was amplified by PCR with Zymo Taq DNA Polymerase (ZYMO Research, www.zymoresearch.com) with the primer pair 5′-TTT GTT YYA AAA AAA AAA GAA GAA GAA G-3′ and 5′-RRA RTT RTA RTT RTA TTC CAA References 1. Martínez de Alba AE, Elvira-Matelot E, Vaucheret H. Gene silencing in plants: a diversity of pathways. Biochim Biophys Acta 2013; 1829:1300-8; PMID:24185199; http://dx.doi.org/10.1016/j. bbagrm.2013.10.005 2. Pumplin N, Voinnet O. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nat Rev Microbiol 2013; 11:745-60; PMID:24129510; http://dx.doi. org/10.1038/nrmicro3120 3. Voinnet O. Use, tolerance and avoidance of amplified RNA silencing by plants. Trends Plant Sci 2008; 13:317-28; PMID:18565786; http://dx.doi. org/10.1016/j.tplants.2008.05.004 4. Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell 2009; 136:669-87; PMID:19239888; http://dx.doi.org/10.1016/j. cell.2009.01.046 5. Fusaro AF, Matthew L, Smith NA, Curtin SJ, Dedic-Hagan J, Ellacott GA, Watson JM, Wang MB, Brosnan C, Carroll BJ, et al. RNA interferenceinducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep 2006; 7:116875; PMID:17039251; http://dx.doi.org/10.1038/ sj.embor.7400837 6. Yang Z, Ebright YW, Yu B, Chen X. HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide. Nucleic Acids Res 2006; 34:667-75; PMID:16449203; http://dx.doi.org/10.1093/nar/ gkj474
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CTT-3′ for GFPendo and the primer pair 5′-CTT TRA TRC CRT TCT TTT RCT TRT C-3′ and 5′-TTY AAG GAY GAY GGG AAY TAY AAG A-3′ for GFPcDNA at the following conditions: 10 min at 95 °C, 44 amplification cycles (95 °C for 30 s, gradient 45–47–51 °C for 30 s and 72 °C for 30 s), and 72 °C for 10 min. Gradient amplicons were gel excised with Qiagen gel extraction kit (Qiagen, www.qiagen.com), pooled and ligated into pGEM T Easy (Promega, www.promega.com). 10–15 clones were sequenced for each experiment. For interpretation of bisulfite sequencing data the CyMate software was used.68 Agroinfiltration assay Agrobacterium tumefaciens (strain GV3101) containing the binary vector pPCV702SM-GpG (hpF) 67 was used for N. benthamiana leaf agroinfiltration at an optical density (OD) of 1.0, as previously described.69 GFP monitoring Pharos FX molecular scanning (Biorad, www.bio-rad-com) was used for scanning of GFP fluorescence, as recommended by the manufacturer‘s instructions. Leaves were scanned by CY3 and FITC filter channels and the merged picture depicted GFP fluorescence. Acknowledgments
This project was supported by the German Research Foundation (DFG) (grants: Wa1019/8–1). We thank Dr. Mirko Moser and Nora Schwind for their contribution in the production of transgenic plants. Supplemental Materials
Supplemental materials may be found here: www.landesbioscience.com/journals/rnabiology/article/29623/
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49. McHale M, Eamens AL, Finnegan EJ, Waterhouse PM. A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis. Plant J 2013; 76:519-29; PMID:23937661; http://dx.doi. org/10.1111/tpj.12306 50. Bai S, Kasai A, Yamada K, Li T, Harada T. A mobile signal transported over a long distance induces systemic transcriptional gene silencing in a grafted partner. J Exp Bot 2011; 62:4561-70; PMID:21652532; http://dx.doi.org/10.1093/jxb/ err163 51. Haque AK, Yamaoka N, Nishiguchi M. Cytosine methylation is associated with RNA silencing in silenced plants but not with systemic and transitive RNA silencing through grafting. Gene 2007; 396:321-31; PMID:17521830; http://dx.doi. org/10.1016/j.gene.2007.04.003 52. Mallory AC, Mlotshwa S, Bowman LH, Vance VB. The capacity of transgenic tobacco to send a systemic RNA silencing signal depends on the nature of the inducing transgene locus. Plant J 2003; 35:82-92; PMID:12834404; http://dx.doi. org/10.1046/j.1365-313X.2003.01785.x 53. Bender J. RNA-directed DNA methylation: getting a grip on mechanism. Curr Biol 2012; 22:R4001; PMID:22625855; http://dx.doi.org/10.1016/j. cub.2012.04.010 54. Haag JR, Pikaard CS. Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nat Rev Mol Cell Biol 2011; 12:483-92; PMID:21779025; http://dx.doi. org/10.1038/nrm3152 55. Law JA, Jacobsen SE. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 2010; 11:20420; PMID:20142834; http://dx.doi.org/10.1038/ nrg2719 56. Dalakouras A, Dadami E, Wassenegger M. Viroidinduced DNA methylation in plants. BioMol Concepts 2013; 4:557-65 57. Dalakouras A, Wassenegger M. Revisiting RNAdirected DNA methylation. RNA Biol 2013; 10:4535; PMID:23324611; http://dx.doi.org/10.4161/ rna.23542 58. Wierzbicki AT, Haag JR, Pikaard CS. Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 2008; 135:635-48; PMID:19013275; http://dx.doi.org/10.1016/j. cell.2008.09.035 59. Wierzbicki AT, Ream TS, Haag JR, Pikaard CS. RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat Genet 2009; 41:630-4; PMID:19377477; http://dx.doi.org/10.1038/ng.365 60. Christie M, Brosnan CA, Rothnagel JA, Carroll BJ. RNA decay and RNA silencing in plants: competition or collaboration? Front Plant Sci 2011; 2:99; PMID:22639621; http://dx.doi.org/10.3389/ fpls.2011.00099 61. Gy I, Gasciolli V, Lauressergues D, Morel JB, Gombert J, Proux F, Proux C, Vaucheret H, Mallory AC. Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors. Plant Cell 2007; 19:3451-61; PMID:17993620; http://dx.doi. org/10.1105/tpc.107.055319 62. Christie M, Carroll BJ. SERRATE is required for intron suppression of RNA silencing in Arabidopsis. Plant Signal Behav 2011; 6:2035-7; PMID:22112452; http://dx.doi.org/10.4161/psb.6.12.18238 63. Christie M, Croft LJ, Carroll BJ. Intron splicing suppresses RNA silencing in Arabidopsis. Plant J 2011; 68:159-67; PMID:21689169; http://dx.doi. org/10.1111/j.1365-313X.2011.04676.x
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An endogene-resembling transgene is resistant to DNA methylation and systemic silencing a
a
a
a
Elena Dadami , Athanasios Dalakouras , Michele Zwiebel , Gabi Krczal & Michael ab
Wassenegger a
RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany
b
Centre for Organismal Studies (COS) Heidelberg; University of Heidelberg; Heidelberg, Germany Published online: 23 Jul 2014.
Click for updates To cite this article: Elena Dadami, Athanasios Dalakouras, Michele Zwiebel, Gabi Krczal & Michael Wassenegger (2014) An endogene-resembling transgene is resistant to DNA methylation and systemic silencing, RNA Biology, 11:7, 934-941, DOI: 10.4161/rna.29623 To link to this article: http://dx.doi.org/10.4161/rna.29623
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RNA Biology 11:7, 934–941; Jul 2014; © 2014 Landes Bioscience
An endogene-resembling transgene is resistant to DNA methylation and systemic silencing Elena Dadami1†, Athanasios Dalakouras1†, Michele Zwiebel1, Gabi Krczal1, Michael Wassenegger1,2,* RLP AgroScience GmbH; AlPlanta-Institute for Plant Research; Neustadt, Germany; 2Centre for Organismal Studies (COS) Heidelberg; University of Heidelberg; Heidelberg, Germany;
1
These authors contributed equally to this work.
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†
In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNAdirected DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFPendo). Upon agroinfiltration of a hairpin GFP (hpF) construct, transgenic Nicotiana benthamiana plants harboring GFPendo (Nb-GFPendo) were susceptible to local PTGS. Yet, in the local area, PTGS was not accompanied by RdDM of the GFPendo coding region. Importantly, hpF-agroinfiltrated Nb-GFPendo plants were resistant to systemic silencing. For reasons of comparison, transgenic N. benthamiana plants (Nb-GFPcDNA) carrying a GFP cDNA transgene (GFPcDNA) were included in the analysis. HpF-agroinfiltrated Nb-GFPcDNA plants exhibited local PTGS and RdDM. In addition, systemic silencing was established in Nb-GFPcDNA plants. In agreement with previous reports using grafted scions, in systemically silenced tissue, siRNAs mapping to the 3′ of GFP were predominantly detectable by Northern blot analysis. Yet, in contrast to other reports, in systemically silenced leaves, PTGS was also accompanied by dense RdDM comprising the entire GFPcDNA coding region. Overall, our analysis indicated that cDNA transgenes are prone to systemic PTGS and RdDM, while endogene-resembling ones are resistant to RNA silencing.
Introduction RNA silencing refers to a network of small RNA (sRNA)mediated pathways that are important for normal plant development, genome stability, defense against invading nucleic acids and response to abiotic/biotic stress.1,2 In general, there are two major pathways of RNA silencing in plants; the small interfering RNA (siRNA) and the microRNA (miRNA)mediated silencing pathways.3,4 The siRNA pathway is induced by double stranded RNA (dsRNA) that is processed by the DICERLIKE (DCL) endonucleases, DCL2, DCL3 and DCL4, into 22-, 24-, and 21-nucleotides (nt) long siRNAs, respectively.5 Mature siRNAs exhibit 3′ 2-nt overhangs and become stabilized though 3′ end methylation by HUE ENHANCER1 (HEN1).6 Depending on their 5′ terminal nucleotide, siRNAs are loaded onto specific ARGONAUTE (AGO) proteins.7 Only one siRNA strand, the guide strand, is finally selected.8 21-nt siRNAs are loaded primarily onto AGO1, and to lower extent onto AGO2, leading to the formation of RNA-induced silencing complex (RISC).9 RISC mediates post-transcriptional gene silencing (PTGS) through targeting RNA molecules that are complementary to the guide strand. PTGS results in target RNA degradation and/ or, in the case of mRNA targets, in translational inhibition.10,11 24-nt siRNAs are loaded onto AGO4/AGO6 and are supposed
to target homologous genomic DNA for de novo methylation, in a process termed RNA-directed DNA methylation (RdDM).12,13 PTGS and RdDM are both initiated by dsRNA. Among others, sources of dsRNA are replication intermediates of viruses and viroids, transcription of inverted repeats, stress-induced overlapping antisense transcripts and RNA-DIRECTED RNA POLYMERASE (RDR) transcription of aberrant RNAs (abRNAs).14-16 AbRNAs are most probably transcripts devoid of 5′ cap and/or 3′ polyadenylation tail.17,18 Such transcripts may directly be generated through abnormal DNA-DEPENDENT RNA POLYMERASE II (POL II) transcription, e.g., producing truncated and/or read-through transcripts. In addition, abRNA may derive from RISC-mediated generation of 5′ and 3′ target RNA cleavage products. Thus, while initial cleavage is conducted by primary siRNAs, RDR6 transcription of the cleavage products generates secondary dsRNA that is processed into secondary siRNAs.19,20 In contrast to primary siRNAs, that match only to a sub-region of the target RNA, secondary siRNAs may map upstream and downstream from the binding sites of the primary siRNAs. This spreading of silencing beyond initial target sites is termed transitivity.15,21 The exact nature of secondary siRNAs is not known, but it seems that at least DCL2 is involved in their biogenesis.22 Interestingly, while secondary siRNAs are cleavagecompetent, they cannot further support the biogenesis of tertiary
*Correspondence to: Michael Wassenegger ; Email: [email protected]) Submitted: 05/05/2014; Revised: 06/11/2014; Accepted: 06/17/2014; Published Online: 07/23/2014 http://dx.doi.org/10.4161/rna.29623 934
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Results Transgenic Nb-GFPendo and Nb-GFPcDNA plants The GFPendo construct was designed to resemble the structure of one of the highly transcribed small subunit of RIBULOSE-1,5-BIPHOSPHATE CARBOXYLASE OXYGENASE (RuBisCO) genes.41 The GFPendo consists of the RuBisCO promoter/5′UTR/two introns/3′ UTR/ terminator and three (arbitrarily chosen) GFP exons (Fig. 1). RuBisCO genes are highly transcribed endogenes that are not prone to RDR6-mediated processing.15 Previously, we could show that upon agroinfiltration, GFPendo transcripts were inefficiently processed by RDR6-mediated pathway.41 In addition to transitivity, Figure 1. Schematic representation of GFPendo, hpF and GFPcDNA constructs. P35S: RDR6 is involved in the establishment of RdDM and CaMV 35S promoter; TNOS: nopaline synthase (NOS) terminator; GFP: green systemic silencing.15,42,43 We assumed that reduced RDR6fluorescent protein; PRbc: tobacco RuBisCO promoter including 5′ UTR; TRbc: processivity of the agroinfiltrated GFPendo construct tobacco RuBisCO terminator including 3′ UTR; Intron 1 and 2: tobacco RuBisCO could also have an impact on the efficiency of RdDM introns 1 and 2. The hybridization probes used for northern blot analysis (Fig. 3) and systemic silencing of a genome-integrated GFPendo and the 182 bp and 134 bp regions analyzed by bisulfite sequencing (Fig. 4, S2 and S3) are indicated by black bars. construct. Thus, transgenic N. benthamiana GFPendo plants (Nb-GFPendo) were generated. For reasons of comparison, the N. benthamiana plant line 16C carrying siRNAs. Thus, transitivity may spread to 5′ and 3′ direction, a GFP cDNA transgene44 was used. Line 16C (hereafter referred but not indefinitely.21,23 Nevertheless, transitivity results in the to as Nb-GFPcDNA) is exceptional in that it shows remarkably amplification of siRNAs constituting a self-reinforcing feedback high GFP expression without being prone to spontaneous cisloop of silencing. Silencing may spread through plasmodesmata silencing.36,39,40 Importantly, Nb-GFPendo and Nb-GFPcDNA cell-to-cell and through the vasculare system to systemic parts of plants exhibited almost similar GFP expression (Fig. 2, 3 and the plant.24,25 While cell-to-cell signals are most probably 21-nt S1). Thus, any putative divergence of responses against RNA duplex siRNAs, the nature of systemic silencing signals is still silencing of the two transgenes should reflect qualitative rather elusive.26-29 It has been proposed that 24-nt siRNAs30-32 or longer than quantitative effects. RNAs33 may be the molecules traveling through the phloem to Analysis of PTGS in locally silenced leaves metabolic sink tissues, but solid experimental evidence for either Endogenes and transgenes are prone to RNA silencing when scenario is lacking. an exogenous trigger e.g., hairpin RNA (hpRNA) is present and In general, the response of transgenes and endogenes against processed into siRNAs (trans-silencing).45,46 In order to compare RNA silencing is diverse. Both, endogenes and transgenes are trans-silencing of GFPendo and GFPcDNA, a hairpin GFP (hpF) equally susceptible to sRNA-mediated trans-PTGS. However, construct covering 139 bp of the GFP middle region (F) (Fig. 1), with a few exceptions, including TAS genes in Arabidopsis and was agroinfiltrated into Nb-GFPendo and Nb-GFPcDNA plants. For BETA-1,3-GLUCANASE genes in tobacco, (1) endogenes each treatment, three plants were agroinfiltrated (three leaves for are generally not prone to transitivity.34,35 Likewise, with the each plant) and the pooled tissue was used for subsequent analysis. exception of two endogenous INVERTED-REPEAT (IR) Both, Nb-GFPendo and Nb-GFPcDNA plants revealed local PTGS loci in Arabidopsis, (2) endogenes are not prone to systemic already 4 days post infiltration (dpi) and displayed full local GFP silencing.26,36,37 In contrast to transgenes, (3) endogenes appear silencing at 8 dpi (Fig. 2). SRNA-mediated silencing in plants to be resistant to RdDM in N. benthamiana.38 Finally, in contrast results in mRNA degradation and/or translational inhibition.10,39 to transgenes, (4) endogenes are not prone to spontaneous In order to discriminate between the two processes, RNA was silencing (cis-silencing).39,40 In order to analyze if a transgene can extracted from a pool of agroinfiltrated leaves and subjected to mimick an endogene with introns and thus, become less prone to Northern blot analysis. Our data revealed that primary GFP RNA silencing, we constructed an intron-containing endogene- siRNAs, originating from the agroinfiltrated hpF construct, resembling GFP transgene (GFPendo). We have previously shown targeted GFPendo and GFPcDNA transcripts for degradation (Fig. 3, that upon agroinfiltration, GFPendo was poorly processed into probe F). In addition to the production of primary siRNAs, siRNAs.41 In this study, we generated transgenic N. benthamiana transitivity-derived secondary siRNAs may also be generated GFPendo plants (Nb-GFPendo) and we explored the response of by RDR6/DCLs processing of abRNA. However, in samples GFPendo against RNA silencing. In summary, our data indicated of locally silenced tissue, no secondary 5′ and 3′ siRNAs were that, similar to most endogenes, GFPendo was no target for RdDM detectable under the applied conditions (Fig. 3, probes G and P). and systemic silencing. Secondary siRNAs production may be slow23 and may take longer
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than 8 dpi. However, progressive physiological degeneration of agroinfiltrated tissue 8 dpi did not allow analysis at later stages. Analysis of RdDM in locally silenced leaves PTGS is tightly related to RdDM. In the case of transgenes, silencing of mRNA is generally associated with RdDM of its coding region, which in turn, may contribute to maintain PTGS.46 Endogenes and transgenes are efficiently targeted for trans-PTGS. However, why only transgenes are prone to RdDM46 is still elusive. In order to investigate if the endogene-resembling GFPendo differs from GFPcDNA in view of being a target for RdDM, the methylation status of the transgene constructs was analyzed by bisulfite sequencing using pooled samples Figure 2. GFP monitoring of hpF-agroinfiltrated GFPendo and GFPcDNAN. benthamiana plants. The develfrom locally silenced leaf tissue (8 opment of RNA silencing was monitored at local levels (4 and 8 dpi) and systemic levels (21 and 46 dpi). dpi) (Fig. 2). We found that GFPcDNA was heavily methylated, not only in Analysis of RdDM in systemically silenced leaves the region targeted by the hpF silencing trigger, but also in the In grafting experiments employing transgenic plants, silenced 3′ downstream sequence (Figs. 4 and S2). This finding was in agreement with another study reporting on RdDM spreading.47 scions induced systemic PTGS and RdDM in the rootstock.30,32,50 Notably, the observed 3′ spreading of RdDM was not correlated In a reverse experiment using a silenced rootstock, the scion became with the accumulation of detectable amounts of 3′ siRNAs systemically silenced, but de novo methylation of the transgene (Fig. 3, probe P). In contrast to the GFPcDNA, GFPendo was poorly was not detectable suggesting that the signals for induction of methylated in locally silenced tissue (Figs. 4 and S3). This PTGS and RdDM are not identical.33,51,52 In contrast to grafting finding was striking, since the silencing triggers of Nb-GFPendo experiments, we induced local silencing in leaves of individual and Nb-GFPcDNA were identical (hpF). In addition, abundant Nb-GFPcDNA and Nb-GFPendo plants. DNA was extracted from a primary hpF-derived siRNAs were detectable in Nb-GFPendo and pool of newly emerged Nb-GFPcDNA (almost fully silenced) and Nb-GFPcDNA samples (Fig. 3, probe F). Thus, one would have Nb-GFPendo (non-silenced) leaves 21 dpi, respectively (Fig. 2). expected RdDM of GFPendo, at least, in the region targeted by The DNA samples were subjected to bisulfite sequencing. The hpF. These findings suggest that the mere presence of siRNAs sequencing data showed that, as expected, the GFPendo was not was not sufficient to efficiently trigger RdDM. methylated (Figs. 4 and S3). In contrast, the coding region of Systemic PTGS analysis the GFPcDNA construct was heavily methylated (Figs. 4 and S2). Endogenes and transgenes are efficiently targeted for local Importantly, RdDM patterns were not restricted to the siRNAPTGS, but only transgenes appeared to be prone to systemic mapping 3′ region but were established along the entire coding PTGS.25,37,48 Thus, we were interested to investigate if locally region (Fig. S2). Bisulfite sequencing data were validated by silenced Nb-GFPendo plants also become systemically silenced. For Southern blot analysis using the methylation sensitive restriction this reason, 12 Nb-GFPcDNA and Nb-GFPendo plants displaying endonucleases, NcoI, DdeI and PvuII (Fig. S4). In summary, our local silencing post hpF-agroinfiltration were monitored up to data suggested that in a non-grafting system, systemic PTGS 46 dpi for systemic silencing. All Nb-GFPcDNA plants exhibited of GFPcDNA is associated with efficient RdDM of the silenced systemic PTGS already 21 dpi (Fig. 2). Importantly, and in transgene. agreement with previous observations, only 3′ GFP siRNAs were detectable in systemically silenced leaves by Northern blot analysis (Fig. 3, probe P).33,49 In contrast, 21 dpi and even 46 dpi Discussion no systemic GFP silencing was observed in Nb-GFPendo plants. At 46 dpi Nb-GFPcDNA plants were fully silenced (Fig. 2). We The RdDM machinery is considered to be guided by thus conclude that, similar to most endogenes, GFPendo failed to 24-nt siRNAs.53-55 In our experiments, primary GFP siRNAs induce the production of and/or failed to respond to systemic were detected in samples of agroinfiltrated Nb-GFPendo and silencing signals. Nb-GFPcDNA leaves. However, GFPendo was inefficiently
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Figure 3. Northern blot analysis of RNA samples of hpF-agroinfiltrated GFPendo and GFPcDNA N. benthamiana plants. Large and small RNA analysis was performed at local levels (4 and 8 dpi) and systemic levels (21 dpi). The regions corresponding to hybridization probes are indicated in Figure 1. Ethidium bromide staining and miR159 accumulation served as loading controls. ‘Mock’ corresponds to 10 mM MgCl2 infiltration.
targeted for RdDM. Thus, our data suggests that either the mere presence of siRNAs is not sufficient to trigger RdDM, or molecules, other than siRNAs may target genomic DNA for de novo methylation.56,57 Establishment of RdDM is not only based on the presence of RdDM trigger molecules, but also on the transcription of RdDM targets (DNA and/or nascent transcripts). Typically, DNA-DEPENDENT RNA POLYMERASES IV and V (POLIV and POLV, respectively) are thought to transcribe RdDM targets, thus producing scaffold transcripts for the binding of trigger molecules.13,58,59 In addition, RDR2 and RDR6 transcription of target transcripts appeared to be essential for the establishment and/ or amplification of RdDM.43,47 Differential POLIV/POLV/ RDR2/RDR6 processing of GFPendo and GFPcDNA may also account for their differential methylation patterns we observed. Production of systemic silencing signals does not require DCL1, DCL2, DCL3, DCL4, RDR2, RDR6 and POLIV,29 indicating that systemic silencing is not triggered by a typical siRNA. However, upon reception, the response to systemic signals depends on RDR6, RDR2, DCL3 and POLIV.29 Our previous data showed that, when agroinfiltrated into Nb-GFPcDNA leaves, GFPendo induced diminished local and delayed systemic silencing of GFPcDNA.41 Here, we found that upon agroinfiltration of hpF, GFPendo did not respond to systemic silencing signals. Since systemic silencing requires RDR6, one may speculate that GFPendo does not produce RNA molecules that serve as a substrates for RDR6. Thus, similar to typical endogenes,36 transgenes resembling endogenes seem not to respond to systemic silencing signals.
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The diverse response of GFPendo and GFPcDNA to systemic silencing and RdDM indicated that the availability of RDR6 substrates, probably abRNA, is a critical step in these processes. RDR6 seems to prefer abRNAs that lack either 5′ cap and/or 3′ polyadenylation tail.17,18 Most likely, both GFPendo and GFPcDNA produce abRNAs. In the case of GFPcDNA, abRNAs may refer to truncated and/or read-through transcripts, lacking 5′ cap and/or 3′ polyadenylation tail, thus serving as optimal RDR6 substrates.17,18 In the case of GFPendo, abRNAs may in addition, include intron-containing mispliced and/or unspliced transcripts that were ‘overlooked’ by the spliceosome. We suggest that, while GFPcDNA abRNAs are predominantly copied by RDR6 and induce RNA silencing, GFPendo abRNAs are not processed by RDR6 but are instead eliminated via exonucleolytic RNA decay mechanism.17,60,61 GFPendo differed in several aspects from GFPcDNA (promoter, UTRs, introns, terminator, integration site). In support of our suggestion, while the presence of UTRs and introns is positively correlated with RNA decay, it is negatively correlated with RNA silencing.23,60,62-64 Spliceosome association likely excludes RDR6 recruitment.16 Thus, endogenous siRNAs matching the transcripts of intron-containing genes are underrepresented in sRNA libraries of Arabidopsis, while siRNAs derived from intronless gene transcripts are highly abundant.63 Nevertheless, a couple of endogenous transcripts are targeted by RDR6 leading to the production of endogenous siRNAs.34,65 In conclusion, we have shown that an endogene-resembling transgene is resistant to systemic silencing and DNA methylation, most probably due to its poor RDR6-mediated processing. The presence of each endogenous element (promoter, 5′ UTR,
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introns, 3′ UTR, terminator) may have its own contribution to this phenomenon, but we find of particular importance the presence of introns. Yet, investigating the role of each parameter was beyond the scope of our analysis and would have required the production of an enormous amount of independent transgene constructs comprising all free combinations of the individual RuBisCO gene elements. Moreover, such work would not include the introduction of the introns at different GFP coding region sides or the analysis of an alternative reporter gene. Our data suggest that endogene-resembling transgenes may point toward a new generation of transgenes, where high transcription rates would not be compromised by concomitant susceptibility to RNA silencing.
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Materials and Methods Generation of transgenic plants The generation of pG104-GFPendo construct has been previously described.41 Agrobacterium tumefaciens (strain ATHV) containing pG104-GFPendo was used to transform N. benthamiana via leaf disc transformation, as previously described.66 Since pG104GFPendo did not contain a selection marker, co-transformation with A. tumefaciens (strain GV3101) containing the binary vector pPCV702SM-Δ67 was conducted for kanamycin selection. Independent T1 lines were characterized by Southern and northern blot analysis for copy number and expression levels of the transgene (Fig. S5). At least two independent GFPendo lines (18 and 25) containing multiple transgene copies, displayed GFP expression levels and GFP fluorescence similar that of GFPcDNA line 16C. Segregants of the GFPendo line 25, containing 2–5 transgene copies (Fig. S5) were used for subsequent analysis. RNA extraction and northern blot analysis RNA was isolated with NucleoSpin miRNA kit (MachereyNagel, www.mn-net.com) according to the manufacturer‘s instructions. Large and small RNA fractions were separately isolated and used for mRNA and siRNA analysis. For mRNA analysis, 5 µg of large RNA fraction was analyzed in 1.2% agarose/formaldehyde gels, and blotted onto positively charged nylon membranes BioBond Plus (Sigma-Aldrich, www. sigmaaldrich.com) and UV312nm-cross-linked (300 mJ/cm2). The PerfectHyb Plus 1x (Sigma-Aldrich) was used for overnight hybridization at 64 °C. Membranes were washed at 64 °C with buffer 1 (2x SSC, 0.1% SDS (w/v)) for 30 min and with buffer 2 (0.5x SSC, 0.1% SDS (w/v)) for 15 min. For siRNA analysis, 5 µg of small RNA fraction was analyzed in 15% TBE-Urea gels and blotted onto positively charged nylon membranes Bright Star (Life Technologies, www.lifetechnologies.com) by electroblotting at 300mA for 1 h and UV312nm-cross-linked (300 mJ/ cm2). The PerfectHyb Plus 1x (Sigma-Aldrich) was used for overnight hybridization at 42 °C and membranes were washed twice with buffer 1 (2x SSC, 0.1% SDS (w/v)) at 42 °C for 30 min. For the generation of probes, templates obtained by PCR on GFP sequence were used in a random labeling reaction with DecaLabel DNA labeling kit (ThermoScientific, www. thermoscientific.com) with a-32P dATP. For probe G (291 bp,
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Figure 4. Histogram representing bisulfite sequencing data from GFPcDNA and GFPendo. Non-agroinfiltrated, 8 dpi local and 21 dpi systemic tissue was used for analysis. 10–15 clones were sequenced and data were analyzed with CyMate software.68 Histogram represents the data presented in Fig. S2 and S3. In non-agroinfiltrated GFPcDNA, transgenerationally maintained CG methylation was detected, in agreement with previous observations.41,70
62% A/T context), the primers 5′-ATG AAG ACT AAT CTT TTT CT-3′ and 5′-ATC TGG GTA TCT TGA AAA GC-3′ were used. For probe F (144 bp, 47% A/T context), the primers 5′-CAT ATG AAG CGG CAC GAC TT-3′ and 5′-CTC GAT CCT GTT GAC GAG GG-3′ were used. For probe P (336 bp, 53% A/T context), the primers 5′-CTT AAG GGA ATC GAT TTC AA-3′ and 5′-TAG TTC ATC CAT GCC ATG TG-3′ were used. Probes G, F and P are continuous and cover the full GFP sequence. For the detection of miR159, the oligonucleotide 5′-AAG AGC TCC CTT CAA TCC AAA-3′ was used in an end-labeling reaction using T4 polynucleotide kinase (New England Biolabs, www.neb.com) and γ-32P ATP. DNA extraction and Southern blot analysis To 1 g of grinded fresh leaf material, 4 ml extraction buffer (2% (w/v) CTAB, 20 mM EDTA, 1.4 M NaCl, 100 mM TRISHCl, pH = 8), 40 µl proteinase K solution and 8 µl RNase A were added, the mixture was incubated at 65 °C for 1 h then briefly cooled on ice. Four ml of chloroform:octanol (24:1) was added and the sample was centrifuged (6000 rpm, 15 min, 4 °C) on Rotanda 360R. To the upper phase, 400 µl CTAB/NaCl solution (10% (w/v) CTAB, 0.7 M NaCl) and 4 ml chloroform:octanol (24:1) were added and the sample was centrifuged (6000 rpm, 15 min, 4 °C). To the upper phase 4 ml precipitation buffer (1% (w/v) CTAB, 10 mM EDTA, 50 mM TRIS-HCl, pH = 8) were added and the mixture was kept overnight at 37 °C. The DNA was precipitated (6000 rpm, 15 min, 4 °C) and the pellet was resuspended in 1 ml high salt TE solution (1 M NaCl, 0.1 mM EDTA, 10 mM TRIS-HCl, pH = 8) at 65 °C for 30 min. The DNA was re-precipitated (6000 rpm, 15 min, 4 °C) with 1 ml isoporopanol and washed twice (centrifugation at 6000 rpm, 15 min, 4 °C) with 1 ml ethanol 70% (v/v). Finally, the DNA pellet was air-dried for 5 min and resuspended in water. For Southern blot analysis, 10 µg DNA were digested with the corresponding restriction endonucleases, separated onto 1% agarose gels and treated for 30 min with denaturation buffer (1 M NaCl, 0.4 N
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NaOH). DNA was capillary transferred overnight onto positively charged nylon membranes (BioBond Plus, Sigma-Aldrich, www. sigmaaldrich.com) with denaturation buffer. After transfer, the membranes were treated with equilibration buffer (1 M NaCl, 1 M TRIS-HCl, pH = 7), air-dried and UV312nm-cross-linked (100 mJ/cm2). For the generation of probe 35S, templates obtained by PCR (primers 5′-CCG GAA ACC TCC TCG GAT TCC3′ and 5′-GAT GAT CCC CCT CTC CAA ATG-3′) on 35S promoter sequence were used in a random labeling reaction with DecaLabel DNA labeling kit (ThermoScientific, www. thermoscientific.com) and a-32P dATP. The PerfectHyb Plus 1x (Sigma-Aldrich, www.sigmaaldrich.com) was used for overnight hybridization at 64 °C. Membranes were washed at 64 °C with buffer 1 (2x SSC, 0.1% (w/v) SDS) for 30 min and with buffer 2 (1x SSC, 0.1% (w/v) SDS) for 15 min. Membranes were exposed to FujiFilm Imaging Plates (Fuji, www.fujifilm.com) for 2 d and scanned using PharosFX Plus PhosphorImager (BioRad, www. bio-rad.com). Western blot analysis Protein was extracted from Nb-GFPendo and Nb-GFPcDNA and subjected to western blot analysis with GFP antibody as previously described.67 Bisulfite sequencing For bisulfite sequencing, 500 ng DNA were pre-digested with DraI and subjected to bisulfite treatment using the EZ DNA Methylation-Lightning Kit (ZYMO Research, www. zymoresearch.com) according to the manufacturer‘s instructions. The recovered material was amplified by PCR with Zymo Taq DNA Polymerase (ZYMO Research, www.zymoresearch.com) with the primer pair 5′-TTT GTT YYA AAA AAA AAA GAA GAA GAA G-3′ and 5′-RRA RTT RTA RTT RTA TTC CAA References 1. Martínez de Alba AE, Elvira-Matelot E, Vaucheret H. Gene silencing in plants: a diversity of pathways. Biochim Biophys Acta 2013; 1829:1300-8; PMID:24185199; http://dx.doi.org/10.1016/j. bbagrm.2013.10.005 2. Pumplin N, Voinnet O. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nat Rev Microbiol 2013; 11:745-60; PMID:24129510; http://dx.doi. org/10.1038/nrmicro3120 3. Voinnet O. Use, tolerance and avoidance of amplified RNA silencing by plants. Trends Plant Sci 2008; 13:317-28; PMID:18565786; http://dx.doi. org/10.1016/j.tplants.2008.05.004 4. Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell 2009; 136:669-87; PMID:19239888; http://dx.doi.org/10.1016/j. cell.2009.01.046 5. Fusaro AF, Matthew L, Smith NA, Curtin SJ, Dedic-Hagan J, Ellacott GA, Watson JM, Wang MB, Brosnan C, Carroll BJ, et al. RNA interferenceinducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep 2006; 7:116875; PMID:17039251; http://dx.doi.org/10.1038/ sj.embor.7400837 6. Yang Z, Ebright YW, Yu B, Chen X. HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2′ OH of the 3′ terminal nucleotide. Nucleic Acids Res 2006; 34:667-75; PMID:16449203; http://dx.doi.org/10.1093/nar/ gkj474
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CTT-3′ for GFPendo and the primer pair 5′-CTT TRA TRC CRT TCT TTT RCT TRT C-3′ and 5′-TTY AAG GAY GAY GGG AAY TAY AAG A-3′ for GFPcDNA at the following conditions: 10 min at 95 °C, 44 amplification cycles (95 °C for 30 s, gradient 45–47–51 °C for 30 s and 72 °C for 30 s), and 72 °C for 10 min. Gradient amplicons were gel excised with Qiagen gel extraction kit (Qiagen, www.qiagen.com), pooled and ligated into pGEM T Easy (Promega, www.promega.com). 10–15 clones were sequenced for each experiment. For interpretation of bisulfite sequencing data the CyMate software was used.68 Agroinfiltration assay Agrobacterium tumefaciens (strain GV3101) containing the binary vector pPCV702SM-GpG (hpF) 67 was used for N. benthamiana leaf agroinfiltration at an optical density (OD) of 1.0, as previously described.69 GFP monitoring Pharos FX molecular scanning (Biorad, www.bio-rad-com) was used for scanning of GFP fluorescence, as recommended by the manufacturer‘s instructions. Leaves were scanned by CY3 and FITC filter channels and the merged picture depicted GFP fluorescence. Acknowledgments
This project was supported by the German Research Foundation (DFG) (grants: Wa1019/8–1). We thank Dr. Mirko Moser and Nora Schwind for their contribution in the production of transgenic plants. Supplemental Materials
Supplemental materials may be found here: www.landesbioscience.com/journals/rnabiology/article/29623/
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