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Agricultural Biotechnology Center, Plant Biology Institute, P.O. Box 411, H-2101 Gödöllo, Hungary,1 Department of Genetics, Eötvös Lóránd University, Budapest, Hungary,2 University of East Anglia, Norwich, United Kingdom3
Received 5 June 2007/ Accepted 10 August 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In higher plants, AGO1 protein is the slicer component of RISC (43), and it recruits small silencing-related RNAs, such as miRNAs, trans-acting siRNAs (tasiRNAs), transgene-derived siRNAs (5), and virus-derived siRNAs (66). However, other members of the AGO family and different Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) proteins are also involved in diverse RNA-silencing pathways. In the nucleus, DCL3-, RDR2-, AGO4-, and NRPD1-dependent silencing is associated with DNA cytosine methylation, heterochromatin-associated modifications of histone H3 tails, and transcriptional silencing (19, 22, 29, 35, 60, 67). Posttranscriptional RNA-silencing-related pathways for endogenous gene regulation involve miRNAs (DCL1 dependent) or tasiRNAs (DCL4 and RDR6 dependent) (30). Virus-induced RNA silencing is triggered by dsRNA intermediates of cytoplasmically replicating viruses, RDR1- or RDR6-dependent formation of dsRNA, or structured regions of viral RNAs (34, 54). Virus-induced silencing leads to the sequence-specific degradation of viral RNA (37) and the generation of a mobile silencing signal that activates RNA silencing in uninfected cells (4, 54).
Systemic invasion of plants by viruses requires effective mechanisms to suppress RNA silencing. To counteract antiviral RNA silencing, most plant and many animal viruses have evolved silencing-suppressor proteins (45, 54). The large majority of plant virus suppressors of RNA silencing bind either to long dsRNA or to siRNA-miRNA duplexes (9, 25, 32, 46, 51). However, two exceptions have also been reported: the 2b protein of cucumoviruses (66), which binds to and inactivates the AGO1 effector protein of silencing, and the P0 RNA-silencing suppressor of the poleroviruses (40, 41), which does not have RNA binding activity (J. Burgyan, unpublished results).
The crucifer-infecting strain of Tobacco mosaic virus (cr-TMV) belongs to the genus Tobamovirus. Tobamoviruses are a group of rod-shaped plant viruses with undivided positive-sense RNA genomes encoding at least four proteins. The genomic RNA of cr-TMV directs the translation of a 122-kDa protein and its read-through product, a 178-kDa protein, which are involved in the replication of the viral RNA. The other two proteins, the 29-kDa movement protein and the 18-kDa coat protein, are translated from individual 3'-coterminal subgenomic RNAs (14). The p122 protein is composed of three domains: a methyltransferase domain of 42 to 466 amino acids, an intervening region (IR), and a helicase domain of 823 to 1,076 amino acids, whereas the 178-molecular-weight protein possesses an additional RDR motif. Little is known about the precise mechanism of action of p122 upon infection.
It has been demonstrated that the p126 protein of TMV strain OM (corresponding to p122 of cr-TMV) forms a heterodimer replicase complex with p183 and two or more host proteins (56). Only one specific interaction between p126 and p183 was identified, which was in the C-terminal half of the p126 IR and the N-terminal portion of the p183 helicase domain (17). The ratio between p126 and p183 is 1:1, although they are expressed in a 10:1 ratio during infection (56). The biological function of this excess amount of p126 remains to be determined. A substitution mutant of TMV strain L in which the amber stop codon of p126 was replaced by a tyrosine codon, thus expressing only the p183 read-through product, was shown to replicate in vivo in the absence of the p126 protein. The growth rate of this mutant virus was about 1/10 of the rate of the wild type (wt) (21). All these results imply that the open reading frame 1 product, besides being involved in replication complex actions, has other functions, as well. Indeed, the other analogous protein, p130 of tomato mosaic virus (ToMV), has been shown to have a silencing-suppressor function (24). It was also shown that p130 does not suppress the activity of the preexisting sequence-specific silencing machinery, suggesting that p130 blocks the utilization of small RNAs in the formation of new effector complexes (24); however, the molecular bases of the suppression remained to be determined.
Here, we demonstrate that p122 has strong silencing-suppressor activity, and we explore in detail the molecular mechanism of silencing suppression by the p122 protein. Our findings demonstrate that p122 prevents si/miRNA assembly into RISCs, inhibiting the development of virus- or transgene-induced silencing activity. In contrast, p122 has no effect on the slicer activity of preassembled RISC, both in vitro and in vivo. In addition, we also demonstrate that p122 interferes with miRNA- and tasiRNA-mediated pathways. However, this interference is not a general effect; instead, it likely depends on the spatial and temporal coexpression of p122 and miRNAs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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p122 mutant virus clone was prepared by PCR mutagenesis, replacing the amber stop codon of p122 (TAG) with tyrosine (TAT). The suppressor p122 was PCR amplified using appropriate primer pairs, cloned into pBIN61, and used for agroinfiltration assays. The following constructs used for different transient assays were described previously: 35S-green fluorescent protein (GFP), 35S-GFP-IR, 35S-sigma 3 (28), 35S-His-HC-Pro (25), GFP-Cym, and GFP-Pothos latent virus (PoLV) (37). GFP-171.1 and GFP-171.2 were kindly provided by O. Voinnet and were described previously (38). Construct 35S-p122-His was prepared by amplifying the p122 open reading frame by PCR using a forward primer containing a start codon (italics) (5'-ATGGCACAATTTCAACAAACAATTGAC-3') and a reverse primer containing RGS His6 epitope codons (underlined) and a stop codon (italics) (5'-CTAGTGATGGTGATGGTGATGCGATCCTCTTTGTATCCCCGCTTCAACTCTATACATGTC-3'), and then this fragment was cloned into an SmaI-cleaved BIN61 vector. 35S-p122 was prepared like 35S-p122-His except that the His tag was omitted from the reverse primer. For the Pri-miR171c construct, we amplified the pri-miR171 sequence from cDNA of Arabidopsis thaliana with the forward (5'-TGAGCGCACTATCGGACATCAAATAC-3') and reverse (5'-TAAACGCGTGATATTGGCACGGCTC-3') primers and cloned it into the pBIN61-SmaI vector.
Agrobacterium tumefaciens infiltration. A. tumefaciens C58C1/pBIN61 harboring the appropriate plasmid (pBIN-GFP, pBIN-HC-Pro, pBIN-p122, or pBIN-sigma3) was infiltrated according to the method described previously (46). The GF-IR construction was made in the pHannibal vector (57) by amplifying GFP sequences with forward primer 5'-GAACTCGAGATGAGTAAAGGAGAAGAACTTTTCAC-3' or 5'-GAGGTACCCGTGTCTTGTAGTTCCCGTCATC-3' and reverse primer 5'-GAATCTAGAATGAGTAAAGGAGAAGAACTTTTCAC-3' or 5'-GAATCGATCGTGTCTTGTAGTTCCCGTCATC-3'. The GF-IR construction (bearing an intron between the GF and FG sequences) was transferred into the pBIN-61 vector and used in infiltration assays. The GFP-expressing Nicotiana benthamiana 16C line was coinfiltrated with 35S-GFP (optical density at 600 nm [OD600] = 0.3) or 35S-GF-IR (OD600 = 0.6) and suppressor protein constructs at an OD600 of 0.6. Total RNA was extracted from the infiltrated paths and analyzed by Northern and Western blotting.
Uninfected or Cym19stop-infected N. benthamiana plants were coinfiltrated with siRNA- or miRNA-sensor constructs (37, 38) at an OD600 of 0.15, and the suppressor protein constructs and the pri-miR171c construct were infiltrated at OD600s of 0.5 and 0.4, respectively.
RNA isolation and hybridization analyses.
In vitro transcription of the pUC19-cr-TMV or pUC19-cr-TMV-p122 viral construct from PmlI-linearized DNA templates and the inoculation of RNA transcripts onto N. benthamiana and A. thaliana Columbia-0 ecotype plants were performed as described previously (13). In vitro RNA transcripts were capped with a cap analogue (New England Biolabs, Hitchin, United Kingdom) as previously described (34). Total RNAs from mock- and cr-TMV-infected plants were isolated using Trizol reagent. RNA extraction was performed 4 days postinoculation (p.i.) for N. benthamiana and 14 days p.i. for A. thaliana. RNA gel blot analysis was performed as described previously (46).
Quantitative real-time RT-PCR analysis. Expression of the DCL1 gene in cr-TMV-infected and healthy control wt or mutant plants was estimated by quantitative real-time reverse transcription (RT)-PCR using the SYBR Green assay (Applied Biosystems) and the Rotor-Gene 3000 (Corbett Research) system. RT-PCR was performed with the DCL1 forward primer (5'-ACAACTGCTGCTTGGAAGGTTTTTCAACCTTTGC-3') and the DCL1 reverse primer (5'-GCATTGGAAGTGTCTCTGGTGTCACCATGG-3') (amplicon region, between bases 5501 and 5570 of the DCL1 mRNA). cDNA was synthesized in a 20-µl reaction volume using a QuantiTect Reverse Transcription Kit (QIAGEN). The quantitative real-time RT-PCR thermal profile was 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 60 s. The U6 snRNA was used as an internal standard in quantitative PCR analysis for each reaction. The primers for U6 were as follows: forward primer, 5'-GTCCCTTCGGGGACATCCGATAAAATTGGAACG-3', and reverse primer, 5'-AAAATTTGGACCATTTCTCGATTTATGCGTG-3'). The quantitative RT-PCR analysis was repeated five times, and the average was taken to determine the level of DCL1 mRNA.
Protein analysis. Infiltrated leaf tissues were homogenized 3 days p.i. in extraction buffer (150 mM Tris-HCl, pH 7.5, 6 M urea, 2% sodium dodecyl sulfate, and 5% ß-mercaptoethanol). Samples were boiled, and cell debris was removed by centrifugation at 18,000 x g at 4°C for 10 min. The supernatants were resolved on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blot analysis. The proteins were visualized using anti-GFP, anti-His, and anti-hemagglutinin (HA) antibodies by chemiluminescence (ECL kit; Amersham) according to the manufacturer's instructions. Commercially available antibodies were used for detection of endoplasmic reticulum-targeted GFP and His6-tagged proteins. Ponceau red staining was used to check the global protein contents of the samples.
In vitro RNA silencing. Drosophila lysate preparation, target RNA labeling, and siRNA annealing were described previously (18). In a 10-ml reaction mixture, 2 ml of lysate and siRNA in 5 nM final concentrations were used in 1x lysis buffer containing 10% (vol/vol) glycerol. GFP target RNA was in vitro transcribed with T7 polymerase in the presence of [32P]UTP and used in 0.5 nM final concentration. In direct-competition assays, the reaction mixtures were incubated for 1 h. In active RISC assays, siRNA and the extract were incubated for 30 min to allow RISC assembly, and then the target RNA and suppressor proteins were added to the reaction. Samples were deproteinized, and the RNA was analyzed on an 8% denaturing gel.
Native gel electrophoresis. Native gel electrophoresis for separation of silencing complexes was essentially as described previously (42) with some modifications. In direct-competition assays, in vitro reaction mixtures that were used for target cleavage assays were incubated for 30 min with 5 nM 32P-labeled siRNA and suppressor protein and diluted with 10 µl of loading buffer (1x lysis buffer, 6% Ficoll 400), and part of the mixture was analyzed on a 3.9% (39:1 acrylamide-bisacrylamide) native acrylamide gel. The gels were dried and exposed to a storage phosphor screen, and bands were quantified with a Genius Image Analyzer (Syngene).
Detection of 2' and 3' OH on the last nucleotide of miRNAs. Periodate treatment and ß-elimination were performed as previously described (2).
Gel mobility shift assay.
For RNA binding reactions, labeled single-stranded RNA or dsRNA (0.5 nM) was incubated with agrobacterium-infiltrated N. benthamiana leaf extract containing 
1 µg total protein or the relevant dilutions. Binding reactions and mobility shift assays were carried out as described previously (32).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To explore the molecular bases of the silencing suppression of p122, 35S-p122, and 35S-p122-His were coexpressed with 35S-GFP or/and 35S-GF-IR in N. benthamiana leaves. The well-characterized HC-Pro of Tobacco etch virus, which specifically binds to 21-nt siRNAs, and reovirus sigma 3, which exclusively binds to long dsRNA (28), were used as controls in the agroinfiltration assay (25). The transiently expressed p122 suppressed GFP silencing with the same efficiency as 35S-HC-Pro, based on the bright fluorescence in the coinfiltrated areas (Fig. 1F). The suppression of GFP silencing by p122 was also very effective in the presence of 35S-GF-IR expression (Fig. 1F). To confirm the visual observations, we also checked the GFP mRNA and GFP-specific siRNA accumulations. Total RNA samples were extracted from the infiltrated area 60 h after infiltration and analyzed by Northern blotting (Fig. 1G). In the presence of p122 or HC-Pro, the GFP mRNA accumulated to a high level, even when 35S-GF-IR was simultaneously coexpressed, while the level of GFP mRNA was strongly reduced when GFP was coexpressed with GF-IR or expressed alone in the absence of suppressors (Fig. 1G). An RNA band corresponding to the GF-IR transcript was observed only in the presence of sigma 3 protein, which inhibits DICER activity by dsRNA binding (25), but not in the presence of HC-Pro or p122 (Fig. 1G, top, compare lanes 5, 6, and 7 with 8), indicating that p122 does not interfere with DICER activity, similar to HC-Pro, which was demonstrated previously (25). As we expected, GFP siRNA accumulated in the leaf infiltrated only with GFP (Fig. 1G, bottom, hybridized with GFP probe), while the presence of HC-Pro, p122, or sigma 3 protein abolished the accumulation of GFP siRNAs. In contrast, when GFP was coinfiltrated with GF-IR, a very large amount of GFP siRNA accumulated in either the presence or the absence of p122 and Hc-Pro (Fig. 1G, bottom, lanes 5, 6, and 7). The accumulation of siRNA was inhibited only in the presence of sigma 3, which compromises DICER activity (25). This result demonstrated that p122 inhibits the accumulation of siRNA when the RNA silencing is triggered by sense GFP transcript. However, p122 did not inhibit the processing of GF-IR dsRNA into siRNAs but interfered with the silencing machinery downstream of siRNA generation. We also tested the effect of p122 on the accumulation of RDR6-dependent secondary siRNAs (33). We have shown that p122 was able to inhibit the accumulation of secondary siRNAs detected by P-specific probe (Fig. 1G, compare lane 1 to lane 3). The inhibition of secondary siRNA (P-specific siRNA) accumulation by p122 was very efficient regardless of whether the GFP silencing was triggered by GFP alone or GFP plus GF-IR expression (Fig. 1G, compare lanes 1 to 3 and 5 to 7). It is worth noting that the majority of primary siRNAs were 24 nt long and only one-third of the siRNAs were 21/22 nt long when the silencing was triggered by GF-IR (Fig. 1G, middle, lanes 5, 6, and 7, GF-specific siRNAs). In contrast, the majority of secondary siRNAs (P specific) were 21/22 nt long, suggesting that they were the products of DCL4 and DCL2, respectively. We also analyzed the accumulation of virus-derived siRNA in cr-TMV-infected plants. Total RNA was extracted from N. benthamiana at 4 days p.i. and from A. thaliana at 14 days p.i. A large amount of viral siRNA accumulated in the virus-infected, but not in the mock-inoculated, plants (Fig. 1E), suggesting that p122 does not compromise the generation of siRNAs.
p122 inhibits siRNA-directed RNA cleavage and the assembly of silencing-related complexes in Drosophila embryo extracts in a dose-dependent manner. The Drosophila embryo extract-based RNA-silencing system has been used successfully to characterize silencing-suppressor proteins (25). This system allows the analysis of RNA-silencing complex formation and the cleavage activity of programmed RISC (42, 49). To better understand how the p122 silencing protein works, we tested the siRNA-programmed RISC activity in the presence of p122 protein. Repeated attempts to express p122 in bacteria were not successful; therefore, p122 was expressed in plants using a binary expression vector (32). We set up two sets of reactions for the RISC cleavage assay. In the direct-competition assay, the inducer siRNA, the labeled target RNA containing the sequence complementary to the inducer siRNA, the Drosophila embryo extract, and the p122-containing plant extract made from 35S-p122 agroinfiltrated N. benthamiana leaves at 3 days p.i. were added simultaneously. RISC activity was measured by quantifying the amount of 5' cleavage product of the target RNA (75 nt) over a dilution series of the suppressor protein extract (Fig. 2, lanes 3 to 7). For the control reaction, we used empty-vector-infiltrated plant extract at the highest concentration used for p122 (Fig. 2, lane 1) and we performed a reaction without embryo extract as a negative control (lane 2). p122 was able to inhibit the target cleavage (Fig. 2, lanes 3 to 5), likely preventing the assembly of RISC. To analyze the effect of p122 on preassembled-RISC activity (indirect competition), we preincubated the Drosophila embryo extract with inducer siRNA for 20 min, and then the target and the suppressor-containing plant extract or mock-infiltrated plant extract were added (Fig. 2, lines 8 to 12). p122 proved to be a potent inhibitor of RISC cleavage at higher concentrations (50% inhibitory concentration at 10-fold dilution) in the direct-competition assay but had no effect on preassembled-RISC activity. The activity of programmed RISC was refractory to the p122 suppressor protein regardless of the concentration. These results suggested that the siRNA sequestration model may also explain the mechanism of p122-mediated suppression, as was demonstrated for p19, HC-Pro, and p21 (10, 25, 46, 51, 62).
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p122 binds double-stranded siRNAs in a size-specific manner.
To explore the affinity of p122 for siRNAs, we carried out a more detailed analysis of the RNA binding affinity of this protein. Electrophoretic mobility shift assays were performed using labeled synthetic single- or double-stranded RNA oligonucleotides in different sizes and diluted plant protein extract of 35S-p122-infiltrated N. benthamiana leaf. In these experiments, the well-characterized 35S-p19 of Carnation Italian ringspot virus (25)-infiltrated N. benthamiana leaf extract was used as a control (data not shown). p122 did not show any single-stranded RNA binding activity irrespective of the length of the RNA (data not shown). The relative dissociation constant (Kr) of p122 for double-stranded siRNA was in the same range as p19-infiltrated extract (not shown). p122 bound to 21-nt RNA duplexes with 2 nt of 3' overhang with the highest affinity (Fig. 5A and C). The size and the 3' overhang of the siRNA proved to be important, because the binding affinity was slightly reduced when 19-nt blunt (Kr/19) (Fig. 5A and C) or 21-nt blunt (Kr/21) (Fig. 5C) duplex RNAs were used (Kr/19 = 2 and Kr/21 = 6; the Kr of p122 for 21-nt-long siRNA is considered to be 1). The affinity for 24-nt siRNA species (Kr/24) (Fig. 5C) was much lower: Kr/24 = 20. p122 did not bind the other RNAs tested: 19-nt RNA with a 2-nt 3' overhang; 26-nt RNA with a 2-nt 3'overhang; 49-nt duplex RNA (Fig. 5C); and 19-, 21-, and 49-nt single-stranded RNAs (data not shown). Since viral siRNAs are predominantly 21 to 22 nt long, these results suggest a specific adaptation of the virus to counteract the antiviral silencing machinery. To analyze the natural function of p122 during the course of virus infection, we tested the siRNA binding affinity of plant extract derived from wt cr-TMV-infected plants. Figure 5E shows that the plant extract from wt virus-infected plants had the same binding activity as transiently expressed p122: it bound 21-nt siRNAs but did not bind 49-nt siRNAs. The mobility of the p122-siRNA complex was the same as that of the wt virus protein-siRNA complex, suggesting that p122 is the only viral protein that binds siRNA and that the p178 viral replicase does not have siRNA binding activity. We were not able to directly test p178 for RNA binding because a 35S-p178-HA construct failed to express a detectable amount of p178-HA. However, indirect evidence suggesting that p178 did not bind siRNAs was obtained. We created a mutant cr-TMV in which the amber stop codon of p122 was replaced by a tyrosine codon. The mutant virus (cr-TMV-122) was able to replicate, although at a lower rate than the wt (not shown), and the protein extract from cr-TMV-122-infected plants did not show siRNA binding activity (Fig. 5F). This result suggests that the p178 read-through product of p122 does not have siRNA binding activity.
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To explore the possible effect of p122 on miRNA pathways, A. thaliana Columbia-0 plants were infected with cr-TMV. After 14 days, we observed severe mottling of systemically infected leaves, and the edges of the leaves became serrated (Fig. 1A), similar to those of plants in which the miRNA pathways were compromised by mutation (36) or silencing-suppressor proteins were expressed (15). This observation suggested that p122 may also interfere with miRNA pathways. We hypothesized that if p122 could bind miRNA-miRNA* duplexes in vivo, the accumulation of miRNA* would increase because the miRNA duplexes would be stabilized by p122 binding, similarly to the previously reported silencing suppressors, which bind miRNA duplexes (10, 25, 26). Indeed, upon cr-TMV infection, both mature and star strands of all tested endogenous miRNAs (miR160c, miR162a, miR171c, miR168a, and miR172) accumulated to a higher level than in the mock-infected control plants (Fig. 6A). These observations are in line with our in vitro findings and strengthen the idea that p122 can interfere with miRNA pathways by sequestering miRNA duplexes and stabilizing them. However, virus infection itself without silencing-suppressor protein can also increase the accumulation of miRNAs (Burgyan, unpublished). Interestingly the accumulation of miR168 was particularly high in cr-TMV-infected plants. This result prompted us to analyze the expression of AGO1, which is controlled by miR168, since it was recently reported that the expression of miR168 and its target, AGO1, are coregulated as a consequence of AGO1 homeostasis (52). Indeed, the expression of AGO1 in cr-TMV-infected plants was also elevated similarly to miR168, and the high level of AGO1 was neither influenced by the lack of methylation in a hen1-1 plant (11) nor in an hst-15 plant (59) in which the miRNA nuclear export was affected (39) (Fig. 6B). It is worth noting that although miR162 (Fig. 6A), which regulates DCL1, accumulated at higher levels in virus-infected plants, we did not observe significant downregulation of DCL1 mRNA using quantitative RT-PCR analysis (data not shown). The finding that miR162* also accumulated at higher levels in virus-infected plants (Fig. 6A) suggests that p122 binds miR162/miR162* duplexes and thus prevents the miR162-mediated downregulation of DCL1.
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We have also shown that p122 recognized the 3' 2-nt overhangs of siRNA duplexes, predicting that p122 may interfere with 3'-end methylation of endogenous small RNAs and viral siRNAs. Plant short RNAs are likely methylated by HEN1 methyltransferase at their 3'-terminal nucleotides at the 2'-hydroxyl group (7, 65). The methylation appears to protect them from oligouridilation and subsequent degradation (27), and it is present in all species of the small-RNA family (siRNA, miRNA, tasiRNA, and sense- and hairpin transgene-derived and transposon- and repeat-derived siRNAs). The methylation statuses of small RNAs can be assessed by treatment of the RNAs with sodium periodate, followed by ß-elimination. The free 2'-OH groups are sensitive to the chemical modification, which results in the elimination of the last nucleotide from the RNA. The resulting molecules migrate faster in gel electrophoresis.
To test whether p122 is able to interfere with the methylation of small RNAs, we checked the methylation statuses of several miRNAs, tasiRNAs, and viral siRNAs generated upon virus infection or in agroinfiltration transient assays. In virus-infected A. thaliana leaf RNA extracts, the tested miRNAs and tasiRNAs showed a partial inhibition in the 3'-methylation level compared to mock-inoculated plants (Fig. 6A). All miRNAs tested were fully methylated in mock-inoculated plants, while miRNAs, both mature and star strands, derived from cr-TMV-infected plants were partially nonmethylated, which was indicated the by the faster migration of miRNAs that underwent ß-elimination (Fig. 6A). Interestingly, the methylation of miR160c, miR171c, and miR168a* was only slightly affected in the presence of cr-TMV. Moreover, the mature strands, as well as the star strands, of mir172a migrated in three different size classes in virus-infected plants in contrast to mock-inoculated plants, in which only the 21-nt miR172a accumulated. The appearance of the 22- and 23-nt species was not DCL2 or DCL3 dependent (data not shown). Similarly to miR172a, the 22- and 23-nt-long species of miR168a, miR159a, miR160c, miR162a, and miR171c or shorter forms of miR171c were also observed (data not shown). The partial inhibition of miRNA and viral-siRNA methylation in the virus-infected plants was somewhat surprising, since HEN1 has been suggested to be localized in the nucleus while cr-TMV replicates exclusively in the cytoplasm (20). Furthermore, we can rule out the existence of other, unidentified methylases that may operate in the cytoplasm, since none of the miRNAs or siRNAs were methylated in the in hen1-1 plants. These findings suggested that HEN1 is active either in the nucleus or in the cytoplasm. Moreover, the ability of virus to interfere with miRNA methylation may suggest that miRNAs exported from the nucleus to the cytoplasm are in both methylated and nonmethylated forms. To better understand the effect of virus infection on the methylation of miRNAs and siRNAs, hst-15 plants, in which miRNA nuclear export is affected (39), were also infected with cr-TMV. The virus infection resulted in elevated accumulation of all tested miRNAs, including both mature and star strands. More importantly, these miRNAs were completely methylated, suggesting that the virus infection could not inhibit the methylation of miRNAs (Fig. 6A). This finding could be a consequence of the fact that the nuclear export of miRNAs to the cytoplasm is compromised. In contrast to miRNAs, the methylation of virus siRNAs was partially inhibited, similarly to the wt, suggesting that the inactivated HST has no role in virus siRNA biogenesis. The methylation status of tasiRNA (siR255) was similar to those of miRNAs in wt plants. siR255 was fully methylated in the mock-inoculated plants and partially methylated in the virus-infected plants (we were not able to analyze TAS3D7+ RNA because it accumulated at a very low level in virus-infected plants). However, siR255 behaved differently in virus-infected hst-15 plants, in which siR255 methylation was partially inhibited, suggesting that siR255 duplexes were available for p122, since they were generated in the cytoplasm, similarly to viral siRNAs. These results further support HEN1 activity in the cytoplasm.
We have shown that p122 efficiently binds double-stranded siRNAs and miRNA intermediates, which strongly suggests that the small-RNA binding activity of p122 is responsible for the inhibition of small-RNA methylation. To further support our hypothesis, p122 was coexpressed with 35S-pri-miRNA171c hairpin (which generates miRNA171c duplexes) using agroinfiltration. As a control, 35S-pri-miRNA171c was also coinfiltrated with 35S-HC-Pro or empty vector. When 35S-pri-miRNA171c was coinfiltrated with the empty vector, the generated miR171c mature and miR171c* RNAs were resistant to the ß-elimination treatment. It is worth noting that the amount of miRNA in the infiltrated patch was in high excess compared to the natural miRNA levels, but even in this case, the methylation was 100% (Fig. 7A, lanes 3 and 4), suggesting that this step in miRNA biogenesis is very efficient. Importantly, the ß-elimination reaction was complete, as demonstrated by the synthetic oligonucleotide controls (Fig. 7A, lanes 9 to 12). The coexpressed p122 partially inhibited miR171c methylation (Fig. 7A, lanes 7 and 8), similarly to virus-infected plants (Fig. 6) and to HC-Pro (Fig. 7, lanes 5 and 6). Interestingly, the miR171c* strand was fully methylated in p122-infiltrated plants, suggesting that one of the two termini of miR171c-miR171c* is not protected by p122 and is accessible for HEN1-mediated methylation (Fig. 7A, lanes 7 and 8).
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DISCUSSION |
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Indeed, our finding demonstrated that the p122 protein of cr-TMV (the protein corresponding to p126) also functions as a silencing-suppressor protein, apart from its function in virus replication. The combination of replicase and silencing-suppression functions in the same protein is likely to be advantageous for the virus, since the replicating RNA is probably the form of the viral genome most exposed to silencing-mediated RNA degradation.
In this work, we presented an extensive characterization of the silencing function of the cr-TMV p122 protein.
The p122 protein of cr-TMV efficiently suppresses RNA silencing by siRNA sequestration. The analysis of the effect of the p122 silencing suppressor in agroinfiltration assays indicated that p122 inhibits RNA silencing triggered by both sense RNAs and dsRNAs. We propose that the molecular basis of inhibition in both cases relies on the ability of p122 to sequester siRNA duplexes. Indeed, when GFP RNA was coexpressed with GF-IR and p122, Dicer activity was clearly not inhibited, although p122 efficiently blocked GFP RNA targeting, probably by inhibiting siRNA incorporation in RISCs via siRNA binding. Moreover, we also showed in this experiment that p122 inhibited the accumulation of secondary siRNAs (Fig. 1G, P-specific siRNAs), which requires the action of RDR6 (33). We also suggest that p122 inhibits the action of RDR6 in the case of GFP sense RNA silencing by binding the minute amount of primary siRNA, which is essential for RDR6-mediated amplification of RNA silencing through the production of secondary siRNAs. In agreement with our observation, it was shown very recently that several silencing-suppressor proteins expressed transgenically inhibited RDR6-dependent steps in the RNA-silencing pathways (33). In line with our siRNA sequestration model, we also showed that p122 inhibited RNA silencing in a quantitative manner, which can explain our observation that the silencing of GFP expression in Amp plants was complete, while in Amp x GFP plants it was only partially suppressed. In the case of Amp x GFP, the silencing is much more robust and generates a much higher level of GFP-specific siRNAs (12), and it is very likely that the large amount of siRNA cannot be sequestered completely by p122. This may suggest that in the veins of Amp x GFP plants, the level of GFP siRNAs is lower (as low as in Amp plants) than in the interveinal tissue.
Our in vivo and in vitro studies further demonstrated that p122 acts upstream of the RISC-programming step, since the loaded siRISC and miRISC cannot be inhibited by the presence of the p122 protein. This feature resembles the ToMV suppressor p130, which has been suggested to suppress GFP silencing but cannot interfere with the preprogrammed RNA-silencing machinery (24). Indeed, our results demonstrated that the molecular basis of silencing suppression by p122 is the sequestration of siRNAs, thus preventing the assembly of siRNA-containing effector complexes in the antiviral silencing response. We have shown that p122 selects for bona fide siRNA for binding, similarly to p19 of tombusviruses and HC-Pro of Tobacco etch virus (25, 32). This finding further supports the model of siRNA-RISC-mediated targeting of viral RNA (37, 66), in contrast to the theoretical possibility that Dicer activity alone could control efficient viral invasion. Since p122 is part of the replicase complex, it is interesting that p122 does not bind 49-nt-long double-stranded or single-stranded RNA molecules, although it is possible that in the replicase complex p122 has different RNA binding activity or that another component of the viral replicase complex binds the replicating long viral RNAs.
cr-TMV infection enhances the accumulation of miRNAs and downregulates tasiRNAs. p122 efficiently binds double-stranded miRNA intermediates in vitro, and the simultaneous accumulation of miRNA-miRNA* in cr-TMV-infected plants suggests that p122 also binds miRNA duplexes in planta. The symptoms of cr-TMV-infected plants may indicate that the miRNA pathways are compromised. The in vitro band shift assay demonstrated that the miRNA mismatches did not significantly alter the binding affinity of the p122 protein for miRNA duplexes, which is in line with the finding that the accumulation of tested miRNAs and miRNA*s was increased in virus-infected plants. It is not clear whether this is due to stabilization of the miRNA duplexes in the miRNA-p122 complex or whether the large amounts of viral siRNAs alter the synthesis-degradation equilibrium of miRNA biogenesis. The miRNAs are produced by the DCL1 protein, which is regulated through a feedback loop by miR162 RNA (61). An elevated level of DCL1 mRNA was detected in dcl1 and hen1-1 mutant plants and in plants expressing a viral suppressor, TuMV P1/HC-Pro, which inhibits miRNA-guided degradation of DCL1 mRNA (61). In cr-TMV-infected leaves, both miR162a and miR162a*, similarly to other miRNA-miRNA* pairs, accumulated at a higher level. Their methylation was partially blocked, suggesting that p122 binds miR162a-miR162a* duplexes in vivo, which can lead to stabilization and thus the increased accumulation of these miRNAs. Alternatively or additionally, the elevated level of miRNAs might also be the consequence of enhanced DCL1 enzyme activity upon viral infection and not simply the result of p122 expression. However, p122 may mask the effect of enhanced miRNA levels by sequestering many of the miRNA duplexes, including miR162, thus blocking the downregulation of DCL1 mRNA.
Mir168a and its corresponding target, AGO1, accumulated at particularly high levels during the development of virus infection, suggesting that AGO1 homeostasis (52) keeps the balance between miR168 and AGO1 expression.
In contrast to the enhanced accumulation of miRNAs, the accumulation of the tested tasiRNAs was reduced in cr-TMV-infected plants. Although the levels of miRNAs, including miR173 and miR390, were higher in virus-infected plants, their biological activities were likely inhibited by p122 sequestration, as suggested by the low level of tasiRNA accumulation, which requires the cleavage of TAS1 and TAS3 RNAs mediated by miR173 and miR390, respectively. In addition to inhibition of miR173 and miR390 actions, p122 may interfere with tasiRNA biogenesis at an additional stage. Indeed, we showed that the tested tasiRNAs were only partially methylated in cr-TMV-infected plants, demonstrating that the virus infection interferes with tasiRNA-mediated pathways at different steps. Our findings are in line with recent observations that tasiRNAs (siR255) are downregulated and partially unmethylated in plants infected by another tobamovirus, Oilseed rape mosaic virus (6).
p122 inhibits the 3'methylation of small RNAs. Previous results demonstrated that transgenically expressed silencing-suppressor proteins, which are able to bind siRNAs, inhibit 3'-terminal methylation (64). In addition, inhibition of HEN1-mediated methylation of small RNAs has been observed in Oilseed rape mosaic virus-infected Arabidopsis plants (1, 6).
We have shown in this study that the methylation of viral siRNAs, transgene-derived siRNAs, and miRNAs is inhibited by p122, likely through the binding of miRNA-siRNA duplexes. Our results also demonstrated that HEN1 operates in both the nucleus and cytoplasm, since cr-TMV, like many other positive-strand RNA viruses, replicates in the cytoplasm and the virus-derived siRNAs were partially methylated (Fig. 6). Importantly, we can exclude the activity of another, unidentified methylase that may operate in the cytoplasm, since all miRNAs and siRNAs were not methylated in hen1-1 plants. The ability of the replicating virus to interfere with miRNA methylation suggests that miRNAs are exported from the nucleus to the cytoplasm in both methylated and nonmethylated forms. The infection of hst-15 plants, in which miRNA nuclear export might be compromised, resulted in the elevated accumulation of all tested miRNAs, and these miRNAs were completely methylated. This demonstrated that the virus infection could not inhibit the methylation of miRNAs, likely because they were separated in different compartments, while the methylation of virus siRNAs was partially inhibited (as in wt plants), since they accumulated in the same compartment where the virus replicated. Indeed, the activity of HEN1 in the cytoplasm was supported by a recent report, which demonstrated that HEN1 is present in both the nucleus and the cytoplasm (16).
Another puzzling result was the asymmetrical methylation of the miRNAs upon virus infection. In some cases, the mature strand was protected by the methyl group and the star strand was not (miR159-mir159*) (not shown), while in other cases we observed the opposite situation, in which miR171c methylation was partially blocked but that of miR171* was not (Fig. 7). The asymmetrical methylation could be a consequence of two-step kinetics of the HEN1 enzyme. In this scenario, the suppressor binds the miRNA after the first step, in which one of the strands is methylated. It is also possible that the p122-miRNA complex is not symmetrical, and therefore, HEN1 and/or p122 recognizes the ends of miRNA duplexes asymmetrically. The difference in the suppression of the methylation of different species could be due to a spatial and temporal compartmentalization of the different miRNAs and the p122 protein.
We also observed that the transgene-derived 21-nt siRNAs were sensitive to the ß-elimination reaction in the p122-infiltrated tissue, indicating that they were not methylated, while the 24-nt siRNAs were fully methylated. These results are likely the consequence of the preferential binding of 21-nt siRNAs versus 24-nt siRNAs by the p122 suppressor protein, further supporting our finding that p122 binds siRNA duplexes in a size-specific manner.
In conclusion, the multifunctional p122 protein of cr-TMV is a very potent silencing-suppressor protein that blocks the intermediate steps of antiviral and endogenous silencing pathways, preventing the assembly of DCL enzyme-generated si/miRNA duplexes into effector molecules, such as RDR6, HEN1, and RISC.
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ACKNOWLEDGMENTS |
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T.C. was the recipient of a grant from the Ministry of Education and Culture for Hungarians Abroad (A/3-2004008). J.B. and T.D. received an International Joint Project grant (2006/3) from the Royal Society and Hungarian Academy of Science. This research was supported by grants from the Hungarian Scientific Research Fund (OTKA T046728 and OTKA NK60352) and the SIROCCO EU project LSHG-CT-2006-037900.
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FOOTNOTES |
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Published ahead of print on 22 August 2007.
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