Plant Virus-Derived Small Interfering RNAs Origina
http://www.100md.com
病菌学杂志 2005年第12期
Agricultural Biotechnology Center, Plant Biology Institute, P. O. Box 411, H-2101 G?d?ll?, Hungary
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom
ABSTRACT
RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected with Cymbidium ringspot tombusvirus (CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show that Tobacco mosaic virus (TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation of PDS siRNAs than the corresponding antisense PDS sequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.
INTRODUCTION
Eukaryotic organisms have developed a highly adaptable and specific mechanism to protect their genomes against aberrant endogenous or exogenous RNA molecules. This phenomenon, referred to as RNA silencing, is an ancient defense mechanism induced by double-stranded RNAs (dsRNAs) that leads to homology-dependent degradation of target RNAs. RNA silencing is conserved across kingdoms and is manifested as quelling in fungi, RNA interference (RNAi) in animals, and cosuppression or posttranscriptional gene silencing (PTGS) in plants. The unifying feature of RNA silencing is the presence of 21- to 26-nucleotide (nt) small interfering RNAs (siRNAs) (17, 18, 40). In addition, biochemical and genetic analyses have shown that the core mechanisms of RNA silencing are shared among different eukaryotes (5, 20, 32, 40, 55, 60). RNA silencing is induced by dsRNAs or structured single-stranded RNAs (ssRNAs) that are processed into siRNAs by RNase III-like enzymes such as DICER (6, 37). siRNAs guide the sequence-specific degradation of target mRNAs by the RNA-induced silencing complex (RISC) (19). The RISC mediates the cleavage of a target mRNA when there is perfect or nearly perfect base pairing between the mRNA and a short guide RNA and mediates translation repression when there is partial complementarity (3, 10, 13, 23). siRNAs can also guide another effector complex, namely, the RNA-induced initiation of transcriptional gene silencing (RITS) complex, to direct the chromatin modification of homologous DNA sequences (53).
In addition to siRNAs, there are other short regulatory RNAs in the RNA silencing machinery called micro-RNAs (miRNAs), which are the products of endogenous noncoding genes. Mature miRNAs are derived by DICER-mediated cleavage from an inverted-repeat mRNA precursor harboring short dsRNA stem-loops (2, 7, 25, 26, 28, 35, 43). Importantly, miRNAs are encoded by genes distinct from those encoding the mRNAs whose expression they control, while siRNAs are generally derived from the same RNAs that are targeted by RNA silencing. A third class of small RNAs, the so-called trans-acting siRNAs, are distinct from miRNAs in that they are likely derived from long dsRNAs produced by an RNA-dependent RNA polymerase; they also appear to be different from exogenous siRNAs, as they primarily repress the expression of other genes rather than their own expression (39, 52).
Regardless of their origins, the occurrence of dsRNAs in the cytoplasm of plant cells induces PTGS. It has been demonstrated that RNA-dependent RNA polymerase is also involved in PTGS (11, 36), presumably by converting target ssRNAs into dsRNAs, which are then processed by DICER to generate siRNAs (58). Remarkably, PTGS can generate mobile silencing signals with sequence-specific information that spread from cell to cell through plasmodesmata (short-distance movement) and systemically through the vascular system (phloem-mediated long-distance movement) to different organs of the plant (21, 33).
An important characteristic of RNA silencing in plants is the involvement of two functionally distinct siRNA species that likely arise from separate DICER-like activities (17, 48, 50). The 21-nt siRNAs are sufficient for RISC-mediated cleavage of target transcripts and are believed to be involved in short-distance signaling, while the 25-nt siRNAs are associated with DNA methylation and the systemic spread of silencing (17, 21).
Plant viruses are known as strong inducers as well as targets of PTGS. During a virus infection, the accumulation of 21-nt double-stranded siRNAs is observed in local and systemic tissues (18, 49), indicating the activation of PTGS. Elevated siRNA levels are correlated with a reduction in viral titer, and in some cases, immunity or recovery in upper noninoculated leaves (41, 49). Thus, PTGS acts as an RNA-mediated defense response to protect plants against viral infection (34, 54, 57). Consequently, to counteract RNA silencing, many plant viruses have evolved proteins that suppress various steps of the silencing machinery (29, 47, 48, 56). It is not clear how most viral suppressor proteins counteract silencing at the molecular level. The tombusviral 19-kDa silencing suppressor protein p19 represents an exception. Indeed, recent advances in our understanding of the molecular mechanism underlying p19's suppressive activity revealed that p19 specifically binds 21-nt double-stranded siRNAs in vitro and in vivo, preventing siRNA incorporation into effector complexes such as the RISC (27, 48). The three-dimensional X-ray crystal structure of a p19-siRNA complex revealed that a p19 dimer acts as a caliper, binding the ends of the siRNA duplex while measuring its length (51, 59).
Cymbidium ringspot virus (CymRSV) is a member of the Tombusvirus genus containing a positive single-stranded RNA genome with five open reading frames (ORFs) (44). It is widely assumed that positive-strand RNA viruses replicate their genomes via dsRNA intermediates that may activate the siRNA-generating machinery (1). However, the existence of long viral dsRNA intermediates and their accessibility for DICER cleavage have not been proven experimentally. In addition, our recent studies suggested that highly structured viral RNAs might also be processed into siRNAs in virus-infected plants (24, 49).
The work presented here addresses the origins and molecular nature of virus-derived siRNAs and reports the development of cloning and sequencing approaches to characterize viral siRNAs accumulating in infected plants. Based on our findings, we suggest that virus-derived siRNAs are predominantly produced by direct DICER cleavage of imperfect duplexes originating from highly base-paired structures from the positive-strand viral genomic RNA.
MATERIALS AND METHODS
Plasmid constructs, in vitro RNA transcription, and plant inoculation. The infectious cDNA clones of CymRSV, i.e., Cym19stop, TMV.hpPDS60, TMV.PDSas110, and PVX, were reported previously (9, 12, 24, 49). A full-length infectious clone of the tobacco mosaic virus (TMV) legume strain will be described elsewhere.
In vitro transcription of the previously mentioned viral constructs from linearized DNA templates and the inoculation of RNA transcripts onto Nicotiana benthamiana plants were performed as described earlier (49). TMV-derived plasmids were linearized with SmaI, and in vitro RNA transcripts were capped with a cap analogue (New England Biolabs, Hitchin, United Kingdom) as previously reported (24).
RNA extraction and analysis. The total RNA was extracted from 100 mg of leaf tissue (12). Briefly, the homogenized plant materials were resuspended in 600 μl of extraction buffer (0.1 M glycine-NaOH, pH 9.0, 100 mM NaCl, 10 mM EDTA, 2% sodium dodecyl sulfate, and 1% sodium lauroylsarcosine) and mixed with an equal volume of phenol. The aqueous phase was treated with equal volumes of phenol and chloroform, precipitated with ethanol, and resuspended in sterile water. RNA gel blot analysis of high-molecular-weight RNAs was performed as described previously (12).
Detection, isolation, labeling, and sequencing of virus-derived siRNAs. RNA gel blot analysis of siRNAs was performed as described previously (49). For preparations of labeled viral siRNAs, 15 to 20 μg of total RNA from virus-infected plants was subjected to electrophoresis through a 12% denaturing polyacrylamide gel, followed by staining with a 1x Tris-borate-EDTA, 0.5 μg/ml ethidium bromide solution for 20 min. ss- and dsRNAs were visualized with UV light and excised from the gel. The gel slices were crushed, covered with 2 volumes of elution buffer (80% formamide, 40 mM PIPES, pH 6.4, 1 mM EDTA, and 400 mM NaCl), and incubated overnight. The gel residues were pelleted by centrifugation, and the supernatants were ethanol precipitated. The purified siRNAs (approximately 1 μg) were dephosphorylated and subsequently labeled in 10-μl reaction mixtures in the presence of [-32P]ATP and RNasin with 8 U of T4 polynucleotide kinase. The labeled virus-specific siRNAs were used for hybridization (49).
Purified siRNAs were cloned according to the original siRNA cloning protocol as described previously (14), with some minor modifications. Briefly, denaturing gel-purified viral dephosphorylated small RNAs were ligated to 3'-terminal adapter oligonucleotides. The ligation products were 5' end labeled with [-32P]ATP and recovered after migration in a sequencing gel (8% polyacrylamide, 8 M urea). 5' adapters were then ligated to the phosphorylated intermediate products. These ligation products were then gel purified, excised, and eluted as previously described (14). After reverse transcription-PCR amplification, the oligonucleotide-linked cDNAs were cloned as concatemers into the pBluescript SK(+) vector. The recombinant clones were randomly selected and sequenced.
IP. For immunoprecipitation (IP), 2 g of CymRSV- or Cym19stop-infected N. benthamiana leaves showing systemic symptoms were collected at 6 days postinoculation and used to prepare extracts in IP buffer (30 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 66 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride), and the extracts were then centrifuged at 15,000 x g for 10 min. IPs were performed at 41°C for 3 to 5 h. Beads were centrifuged and washed in 1x IP buffer two times. Input extracts and IP eluates were used for Western blotting analysis and RNA isolation. RNA molecules were separated in a sequencing gel (12% polyacrylamide, 8 M urea), blotted, and hybridized with an appropriate probe.
RESULTS AND DISCUSSION
The majority of viral siRNAs are derived from viral positive-strand RNA. To evaluate the polarity of siRNAs in virus-infected plants, we extracted total RNAs from systemically infected leaves of CymRSV-infected N. benthamiana plants at 7 days postinoculation. The extracted RNA samples were separated by 8% denaturing polyacrylamide gel electrophoresis (PAGE), blotted, and hybridized with a coat protein (CP) probe (48) representing the central part of the virus genome. CP-specific siRNAs accumulated to a relatively high level and comigrated exclusively with a 21-mer RNA oligonucleotide (not shown). The purified siRNAs were cloned and sequenced to determine their exact size as well as the origin of CymRSV-specific siRNAs. Random sequencing of a total of 70 clones identified 228 siRNAs homologous to the sequence of the CymRSV genome (see Table S1 in the supplemental material). Eight siRNAs containing nonviral sequences were not analyzed further. The nucleotide sequences of the virus-derived siRNAs were derived from regions along the length (4,733 nt) of the viral genome (Fig. 1A). A size distribution analysis of the CymRSV siRNAs confirmed that these RNAs belonged to the short siRNA fraction, with an average size of 20 to 21 nucleotides (Fig. 1B).
The most accepted virus-induced RNA silencing model (1, 54, 57) suggests that a DICER-like enzyme generates siRNAs from double-stranded replicative intermediates of RNA viruses, and thus the expected ratio between positive- and negative-strand-derived siRNAs should be 1:1. However, sequencing of the cloned siRNAs indicated that 80% of viral siRNAs were derived from the positive-strand viral RNA, reinforcing our previous observations of the nature of virus-derived siRNAs (49). In addition, the sequences of 228 siRNAs homologous to the CymRSV genomic RNA revealed that 194 virus-specific siRNA clones could be assigned to several clusters that likely represent hot spots for siRNA production (Fig. 1A). These results suggest that siRNAs in the CymRSV system are predominantly processed by DICER cleavage of imperfect duplexes, which can be formed from the positive-strand viral RNA. This process resembles the production of short-lived, imperfectly pairing, double-stranded intermediates during miRNA generation (4, 8). The preferential incorporation of positive-strand siRNAs into the RISC cannot explain this asymmetric accumulation because the sequence composition of siRNAs does not display a significantly reduced stability profile at the 5' end (see Table S1 in the supplemental material). This observation of asymmetry contrasts with other recent interpretations in which siRNAs were thought to arise from dsRNAs that were produced either by viral transcription events or by the action of host RNA-dependent RNA polymerase (1, 54, 57).
Virus-derived double-stranded siRNAs are imperfect duplexes. The finding that virus-derived siRNAs are asymmetric raised the question of whether these siRNAs are present in infected cells in a ss- or dsRNA form. To determine the structure of viral siRNAs, we examined the mobilities of these molecules under native nondenaturing conditions. RNA extracts of CymRSV-infected tissues were separated by 10% native PAGE along with both single-stranded and double-stranded 21-nt synthetic siRNAs. The results presented in Fig. 2A indicate that only one additional low-molecular-weight RNA fraction could be detected in virus-infected N. benthamiana plants compared to the noninfected plant extract. This low-molecular-weight RNA fraction comigrated with siRNA duplexes, strongly suggesting that it was composed of a double-stranded siRNA. Northern hybridization of the same samples confirmed that this double-stranded siRNA was derived from CymRSV RNA (27; data not shown).
If viral siRNAs are mostly derived from highly structured regions of the positive-strand viral RNA, then these double-stranded siRNAs (Fig. 2A) should contain non-base-paired nucleotides, which are nuclease sensitive. To confirm this hypothesis, we performed RNase A digestion to assess whether these molecules were perfect or imperfect duplexes. Perfect dsRNAs are resistant to RNase A in a high salt buffer (2x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate]), while imperfect dsRNAs with mismatches are rapidly degraded under the same conditions (45). The double-stranded siRNA fraction purified from the native gel (CymRSV siRNAnat) was sensitive to RNase A, indicating the presence of mismatches between the two viral siRNA strands (Fig. 2B, top panel). In contrast, control synthetic perfectly double-stranded siRNAs were completely resistant to RNase A, even at a high nuclease concentration and in high-salt buffer (Fig. 2B, bottom panel). In addition, labeled CymRSV-derived small RNAs isolated by native PAGE (CymRSV siRNAnat) hybridized more intensively to the negative-strand coat protein (CP) transcript than to the corresponding positive-strand transcript (Fig. 2C). The finding that virus-derived siRNAs are imperfect dsRNAs is consistent with the model that viral siRNAs are processed from highly base-paired structures. These observations also corroborate our previous results that artificially extended base-paired structures imbedded in viral ssRNAs give rise to correspondingly high levels of siRNAs. Together, these observations suggest that highly structured regions within viral ssRNAs are able to trigger RNA silencing (49).
p19-bound siRNAs have predominantly positive-strand polarity. We recently reported that the majority of virus-specific siRNAs are bound by p19 in tombusvirus-infected plants. These p19-bound siRNAs are double-stranded and can efficiently trigger the sequence-specific degradation of CymRSV viral RNA in a Drosophila melanogaster embryo extract (27). To test whether p19-bound double-stranded viral siRNAs also have an asymmetric composition with respect to strand specificity, we analyzed the ratio of positive to negative strands in p19-bound siRNAs. RNAs were extracted from anti-p19-immunoprecipitated p19-siRNA complexes derived from CymRSV-infected plants (27) and subsequently analyzed by Northern hybridization using strand-specific probes. As expected from the sequence analysis of siRNAs, the majority of p19-bound siRNAs originated from the positive strand of the viral RNA (Fig. 3), further supporting the model that CymRSV siRNAs originate from highly structured viral RNA regions. An alternative explanation for the specific accumulation of positive-strand siRNAs could be that the positive strand of a perfect double-stranded siRNA is preferentially incorporated into the RISC or other effector complexes instead of the negative strand, with the latter being rapidly eliminated by cellular nucleases. However, our previous observation that p19 sequesters siRNAs before their incorporation into the RISC, when they are still double-stranded siRNAs (27), strongly argues against this alternative model.
The preferential accumulation of positive-strand siRNAs is a general characteristic of positive-strand RNA viruses. To investigate whether the characteristics of CymRSV siRNAs reflect the general rule for virus-derived siRNA generation in virus-infected plants, we also included two other nonrelated positive-strand RNA viruses, Potato virus X (PVX) (9) and TMV (46), in our analysis. As with CymRSV, infections of N. benthamiana plants with PVX and TMV resulted in the appearance of single siRNA fractions of 21 to 22 nt (Fig. 4A). To analyze whether PVX- and TMV-derived siRNAs are also asymmetric, we induced the transcription of the CP-encoding regions of the two viruses with both positive and negative polarities. These transcripts were then subjected to Northern hybridization and probed with 5'-labeled siRNAs isolated from either PVX- or TMV-infected plants. In both cases, the majority of the labeled virus-specific siRNAs hybridized more strongly to the negative transcript, indicating that similar to the case with CymRSV, PVX and TMV siRNAs are derived mainly from viral RNAs with a positive polarity (Fig. 4B).
It was recently reported that the expression of a host gene fragment as a short inverted repeat from TMV leads to more efficient silencing of the cognate mRNA than the expression of a similar sequence in just the sense or antisense orientation (24). To better understand this observation at the molecular level, we compared the siRNA accumulation in plants infected with a viral vector carrying a hairpin structure to that in plants infected with an antisense sequence. To this end, viral transcripts carrying 60-bp inverted repeats (TMV.hpPDS60) or the corresponding antisense sequence (TMV.PDSas110) homologous to the endogenous phytoene desaturase (PDS) gene (24) were used to inoculate N. benthamiana plants. RNAs were extracted from TMV-infected leaves showing the white coloration characteristic of the photobleaching phenotype observed for PDS-silenced plants and then analyzed for both viral RNA accumulation and virus-derived siRNA composition (Fig. 5). The photobleaching phenotype of PDS-silenced plants was more pronounced with the TMV.hpPDS60 hairpin construct than in plants infected with the TMV.PDSas110 antisense construct (Fig. 5A), in spite of similar viral RNA accumulation in virus-infected plants (Fig. 5B, top panel). More importantly, the siRNA accumulation originating from the hairpin sequence was significantly higher for both the plus (12 times) and minus (5 times) polarities than that from the antisense sequence containing the 60-nt sequence of the corresponding hairpin construct (Fig. 5B, bottom panels).
Taken together, these results strongly suggest that one or more of the plant-encoded DICER-like enzymes are able to recognize highly structured regions within viral ssRNAs and process them into siRNAs. This property resembles the DICER-like activity involved in the generation of miRNAs from short-hairpin precursors (16, 22, 30, 38, 42, 58), although recent data indicate that siRNAs and miRNAs require different DICER-like activities in plants (15, 50, 58). The discrete size (21 nt) of virus-derived siRNAs, however, suggests that one of the still unidentified plant DICERs has evolved specifically to process highly structured viral RNAs and is likely a key player in the PTGS-mediated antiviral defense. A recent report demonstrated that an Arabidopsis dcl2 mutant transiently accumulated lower levels of Turnip crinkle virus-derived siRNAs than did wild-type plants (58). However, there is likely to be some degree of redundancy because dcl1, dcl2, and dcl3 mutants were not defective in siRNA production when they were infected with other viruses.
The fact that plant DICER-like proteins generate predominantly positive-strand siRNAs makes the siRNA-based antiviral surveillance system more effective since the siRNAs potentially target the negative viral strand for RISC-mediated cleavage, which is required for replication and is much less abundant than the positive-strand viral RNA. In further support of this model, it was reported that transgenic plants expressing a positive-strand viral sequence specifically target the negative-strand RNA of TMV. This suggests that the transgenically expressed sense viral sequence can be processed into positive-strand siRNAs, which in turn specifically target the negative-strand viral RNA replicative intermediate for degradation (31). Since the sense siRNAs derived from folded RNA are partially complementary to the positive-strand viral RNA, positive siRNAs could also guide the translational repression of viral RNA in a manner analogous to miRNA-mediated translational control, which relies on only partial complementarity between the miRNA and the target. This type of gene silencing is common in animal systems (2, 5) but also seems to operate in plants (3, 10). Further experiments are required to address whether this additional level of control occurs.
ACKNOWLEDGMENTS
We thank Daniel Silhavy, Gy?rgy Szittya, Gábor Giczey, and Alan Herr for critical readings of and helpful comments on the manuscript.
L.L. is a recipient of a Bolyai János Fellowship. This research was supported by grants from the Hungarian Scientific Research Fund (OTKA; T046728), the "RIBOREG" EU project (LSHG-CT-2003503022), and the Scientia Amabilis Foundation.
Supplemental material for this article may be found at http://jvi.asm.org/.
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Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom
ABSTRACT
RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected with Cymbidium ringspot tombusvirus (CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show that Tobacco mosaic virus (TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation of PDS siRNAs than the corresponding antisense PDS sequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.
INTRODUCTION
Eukaryotic organisms have developed a highly adaptable and specific mechanism to protect their genomes against aberrant endogenous or exogenous RNA molecules. This phenomenon, referred to as RNA silencing, is an ancient defense mechanism induced by double-stranded RNAs (dsRNAs) that leads to homology-dependent degradation of target RNAs. RNA silencing is conserved across kingdoms and is manifested as quelling in fungi, RNA interference (RNAi) in animals, and cosuppression or posttranscriptional gene silencing (PTGS) in plants. The unifying feature of RNA silencing is the presence of 21- to 26-nucleotide (nt) small interfering RNAs (siRNAs) (17, 18, 40). In addition, biochemical and genetic analyses have shown that the core mechanisms of RNA silencing are shared among different eukaryotes (5, 20, 32, 40, 55, 60). RNA silencing is induced by dsRNAs or structured single-stranded RNAs (ssRNAs) that are processed into siRNAs by RNase III-like enzymes such as DICER (6, 37). siRNAs guide the sequence-specific degradation of target mRNAs by the RNA-induced silencing complex (RISC) (19). The RISC mediates the cleavage of a target mRNA when there is perfect or nearly perfect base pairing between the mRNA and a short guide RNA and mediates translation repression when there is partial complementarity (3, 10, 13, 23). siRNAs can also guide another effector complex, namely, the RNA-induced initiation of transcriptional gene silencing (RITS) complex, to direct the chromatin modification of homologous DNA sequences (53).
In addition to siRNAs, there are other short regulatory RNAs in the RNA silencing machinery called micro-RNAs (miRNAs), which are the products of endogenous noncoding genes. Mature miRNAs are derived by DICER-mediated cleavage from an inverted-repeat mRNA precursor harboring short dsRNA stem-loops (2, 7, 25, 26, 28, 35, 43). Importantly, miRNAs are encoded by genes distinct from those encoding the mRNAs whose expression they control, while siRNAs are generally derived from the same RNAs that are targeted by RNA silencing. A third class of small RNAs, the so-called trans-acting siRNAs, are distinct from miRNAs in that they are likely derived from long dsRNAs produced by an RNA-dependent RNA polymerase; they also appear to be different from exogenous siRNAs, as they primarily repress the expression of other genes rather than their own expression (39, 52).
Regardless of their origins, the occurrence of dsRNAs in the cytoplasm of plant cells induces PTGS. It has been demonstrated that RNA-dependent RNA polymerase is also involved in PTGS (11, 36), presumably by converting target ssRNAs into dsRNAs, which are then processed by DICER to generate siRNAs (58). Remarkably, PTGS can generate mobile silencing signals with sequence-specific information that spread from cell to cell through plasmodesmata (short-distance movement) and systemically through the vascular system (phloem-mediated long-distance movement) to different organs of the plant (21, 33).
An important characteristic of RNA silencing in plants is the involvement of two functionally distinct siRNA species that likely arise from separate DICER-like activities (17, 48, 50). The 21-nt siRNAs are sufficient for RISC-mediated cleavage of target transcripts and are believed to be involved in short-distance signaling, while the 25-nt siRNAs are associated with DNA methylation and the systemic spread of silencing (17, 21).
Plant viruses are known as strong inducers as well as targets of PTGS. During a virus infection, the accumulation of 21-nt double-stranded siRNAs is observed in local and systemic tissues (18, 49), indicating the activation of PTGS. Elevated siRNA levels are correlated with a reduction in viral titer, and in some cases, immunity or recovery in upper noninoculated leaves (41, 49). Thus, PTGS acts as an RNA-mediated defense response to protect plants against viral infection (34, 54, 57). Consequently, to counteract RNA silencing, many plant viruses have evolved proteins that suppress various steps of the silencing machinery (29, 47, 48, 56). It is not clear how most viral suppressor proteins counteract silencing at the molecular level. The tombusviral 19-kDa silencing suppressor protein p19 represents an exception. Indeed, recent advances in our understanding of the molecular mechanism underlying p19's suppressive activity revealed that p19 specifically binds 21-nt double-stranded siRNAs in vitro and in vivo, preventing siRNA incorporation into effector complexes such as the RISC (27, 48). The three-dimensional X-ray crystal structure of a p19-siRNA complex revealed that a p19 dimer acts as a caliper, binding the ends of the siRNA duplex while measuring its length (51, 59).
Cymbidium ringspot virus (CymRSV) is a member of the Tombusvirus genus containing a positive single-stranded RNA genome with five open reading frames (ORFs) (44). It is widely assumed that positive-strand RNA viruses replicate their genomes via dsRNA intermediates that may activate the siRNA-generating machinery (1). However, the existence of long viral dsRNA intermediates and their accessibility for DICER cleavage have not been proven experimentally. In addition, our recent studies suggested that highly structured viral RNAs might also be processed into siRNAs in virus-infected plants (24, 49).
The work presented here addresses the origins and molecular nature of virus-derived siRNAs and reports the development of cloning and sequencing approaches to characterize viral siRNAs accumulating in infected plants. Based on our findings, we suggest that virus-derived siRNAs are predominantly produced by direct DICER cleavage of imperfect duplexes originating from highly base-paired structures from the positive-strand viral genomic RNA.
MATERIALS AND METHODS
Plasmid constructs, in vitro RNA transcription, and plant inoculation. The infectious cDNA clones of CymRSV, i.e., Cym19stop, TMV.hpPDS60, TMV.PDSas110, and PVX, were reported previously (9, 12, 24, 49). A full-length infectious clone of the tobacco mosaic virus (TMV) legume strain will be described elsewhere.
In vitro transcription of the previously mentioned viral constructs from linearized DNA templates and the inoculation of RNA transcripts onto Nicotiana benthamiana plants were performed as described earlier (49). TMV-derived plasmids were linearized with SmaI, and in vitro RNA transcripts were capped with a cap analogue (New England Biolabs, Hitchin, United Kingdom) as previously reported (24).
RNA extraction and analysis. The total RNA was extracted from 100 mg of leaf tissue (12). Briefly, the homogenized plant materials were resuspended in 600 μl of extraction buffer (0.1 M glycine-NaOH, pH 9.0, 100 mM NaCl, 10 mM EDTA, 2% sodium dodecyl sulfate, and 1% sodium lauroylsarcosine) and mixed with an equal volume of phenol. The aqueous phase was treated with equal volumes of phenol and chloroform, precipitated with ethanol, and resuspended in sterile water. RNA gel blot analysis of high-molecular-weight RNAs was performed as described previously (12).
Detection, isolation, labeling, and sequencing of virus-derived siRNAs. RNA gel blot analysis of siRNAs was performed as described previously (49). For preparations of labeled viral siRNAs, 15 to 20 μg of total RNA from virus-infected plants was subjected to electrophoresis through a 12% denaturing polyacrylamide gel, followed by staining with a 1x Tris-borate-EDTA, 0.5 μg/ml ethidium bromide solution for 20 min. ss- and dsRNAs were visualized with UV light and excised from the gel. The gel slices were crushed, covered with 2 volumes of elution buffer (80% formamide, 40 mM PIPES, pH 6.4, 1 mM EDTA, and 400 mM NaCl), and incubated overnight. The gel residues were pelleted by centrifugation, and the supernatants were ethanol precipitated. The purified siRNAs (approximately 1 μg) were dephosphorylated and subsequently labeled in 10-μl reaction mixtures in the presence of [-32P]ATP and RNasin with 8 U of T4 polynucleotide kinase. The labeled virus-specific siRNAs were used for hybridization (49).
Purified siRNAs were cloned according to the original siRNA cloning protocol as described previously (14), with some minor modifications. Briefly, denaturing gel-purified viral dephosphorylated small RNAs were ligated to 3'-terminal adapter oligonucleotides. The ligation products were 5' end labeled with [-32P]ATP and recovered after migration in a sequencing gel (8% polyacrylamide, 8 M urea). 5' adapters were then ligated to the phosphorylated intermediate products. These ligation products were then gel purified, excised, and eluted as previously described (14). After reverse transcription-PCR amplification, the oligonucleotide-linked cDNAs were cloned as concatemers into the pBluescript SK(+) vector. The recombinant clones were randomly selected and sequenced.
IP. For immunoprecipitation (IP), 2 g of CymRSV- or Cym19stop-infected N. benthamiana leaves showing systemic symptoms were collected at 6 days postinoculation and used to prepare extracts in IP buffer (30 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 66 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride), and the extracts were then centrifuged at 15,000 x g for 10 min. IPs were performed at 41°C for 3 to 5 h. Beads were centrifuged and washed in 1x IP buffer two times. Input extracts and IP eluates were used for Western blotting analysis and RNA isolation. RNA molecules were separated in a sequencing gel (12% polyacrylamide, 8 M urea), blotted, and hybridized with an appropriate probe.
RESULTS AND DISCUSSION
The majority of viral siRNAs are derived from viral positive-strand RNA. To evaluate the polarity of siRNAs in virus-infected plants, we extracted total RNAs from systemically infected leaves of CymRSV-infected N. benthamiana plants at 7 days postinoculation. The extracted RNA samples were separated by 8% denaturing polyacrylamide gel electrophoresis (PAGE), blotted, and hybridized with a coat protein (CP) probe (48) representing the central part of the virus genome. CP-specific siRNAs accumulated to a relatively high level and comigrated exclusively with a 21-mer RNA oligonucleotide (not shown). The purified siRNAs were cloned and sequenced to determine their exact size as well as the origin of CymRSV-specific siRNAs. Random sequencing of a total of 70 clones identified 228 siRNAs homologous to the sequence of the CymRSV genome (see Table S1 in the supplemental material). Eight siRNAs containing nonviral sequences were not analyzed further. The nucleotide sequences of the virus-derived siRNAs were derived from regions along the length (4,733 nt) of the viral genome (Fig. 1A). A size distribution analysis of the CymRSV siRNAs confirmed that these RNAs belonged to the short siRNA fraction, with an average size of 20 to 21 nucleotides (Fig. 1B).
The most accepted virus-induced RNA silencing model (1, 54, 57) suggests that a DICER-like enzyme generates siRNAs from double-stranded replicative intermediates of RNA viruses, and thus the expected ratio between positive- and negative-strand-derived siRNAs should be 1:1. However, sequencing of the cloned siRNAs indicated that 80% of viral siRNAs were derived from the positive-strand viral RNA, reinforcing our previous observations of the nature of virus-derived siRNAs (49). In addition, the sequences of 228 siRNAs homologous to the CymRSV genomic RNA revealed that 194 virus-specific siRNA clones could be assigned to several clusters that likely represent hot spots for siRNA production (Fig. 1A). These results suggest that siRNAs in the CymRSV system are predominantly processed by DICER cleavage of imperfect duplexes, which can be formed from the positive-strand viral RNA. This process resembles the production of short-lived, imperfectly pairing, double-stranded intermediates during miRNA generation (4, 8). The preferential incorporation of positive-strand siRNAs into the RISC cannot explain this asymmetric accumulation because the sequence composition of siRNAs does not display a significantly reduced stability profile at the 5' end (see Table S1 in the supplemental material). This observation of asymmetry contrasts with other recent interpretations in which siRNAs were thought to arise from dsRNAs that were produced either by viral transcription events or by the action of host RNA-dependent RNA polymerase (1, 54, 57).
Virus-derived double-stranded siRNAs are imperfect duplexes. The finding that virus-derived siRNAs are asymmetric raised the question of whether these siRNAs are present in infected cells in a ss- or dsRNA form. To determine the structure of viral siRNAs, we examined the mobilities of these molecules under native nondenaturing conditions. RNA extracts of CymRSV-infected tissues were separated by 10% native PAGE along with both single-stranded and double-stranded 21-nt synthetic siRNAs. The results presented in Fig. 2A indicate that only one additional low-molecular-weight RNA fraction could be detected in virus-infected N. benthamiana plants compared to the noninfected plant extract. This low-molecular-weight RNA fraction comigrated with siRNA duplexes, strongly suggesting that it was composed of a double-stranded siRNA. Northern hybridization of the same samples confirmed that this double-stranded siRNA was derived from CymRSV RNA (27; data not shown).
If viral siRNAs are mostly derived from highly structured regions of the positive-strand viral RNA, then these double-stranded siRNAs (Fig. 2A) should contain non-base-paired nucleotides, which are nuclease sensitive. To confirm this hypothesis, we performed RNase A digestion to assess whether these molecules were perfect or imperfect duplexes. Perfect dsRNAs are resistant to RNase A in a high salt buffer (2x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate]), while imperfect dsRNAs with mismatches are rapidly degraded under the same conditions (45). The double-stranded siRNA fraction purified from the native gel (CymRSV siRNAnat) was sensitive to RNase A, indicating the presence of mismatches between the two viral siRNA strands (Fig. 2B, top panel). In contrast, control synthetic perfectly double-stranded siRNAs were completely resistant to RNase A, even at a high nuclease concentration and in high-salt buffer (Fig. 2B, bottom panel). In addition, labeled CymRSV-derived small RNAs isolated by native PAGE (CymRSV siRNAnat) hybridized more intensively to the negative-strand coat protein (CP) transcript than to the corresponding positive-strand transcript (Fig. 2C). The finding that virus-derived siRNAs are imperfect dsRNAs is consistent with the model that viral siRNAs are processed from highly base-paired structures. These observations also corroborate our previous results that artificially extended base-paired structures imbedded in viral ssRNAs give rise to correspondingly high levels of siRNAs. Together, these observations suggest that highly structured regions within viral ssRNAs are able to trigger RNA silencing (49).
p19-bound siRNAs have predominantly positive-strand polarity. We recently reported that the majority of virus-specific siRNAs are bound by p19 in tombusvirus-infected plants. These p19-bound siRNAs are double-stranded and can efficiently trigger the sequence-specific degradation of CymRSV viral RNA in a Drosophila melanogaster embryo extract (27). To test whether p19-bound double-stranded viral siRNAs also have an asymmetric composition with respect to strand specificity, we analyzed the ratio of positive to negative strands in p19-bound siRNAs. RNAs were extracted from anti-p19-immunoprecipitated p19-siRNA complexes derived from CymRSV-infected plants (27) and subsequently analyzed by Northern hybridization using strand-specific probes. As expected from the sequence analysis of siRNAs, the majority of p19-bound siRNAs originated from the positive strand of the viral RNA (Fig. 3), further supporting the model that CymRSV siRNAs originate from highly structured viral RNA regions. An alternative explanation for the specific accumulation of positive-strand siRNAs could be that the positive strand of a perfect double-stranded siRNA is preferentially incorporated into the RISC or other effector complexes instead of the negative strand, with the latter being rapidly eliminated by cellular nucleases. However, our previous observation that p19 sequesters siRNAs before their incorporation into the RISC, when they are still double-stranded siRNAs (27), strongly argues against this alternative model.
The preferential accumulation of positive-strand siRNAs is a general characteristic of positive-strand RNA viruses. To investigate whether the characteristics of CymRSV siRNAs reflect the general rule for virus-derived siRNA generation in virus-infected plants, we also included two other nonrelated positive-strand RNA viruses, Potato virus X (PVX) (9) and TMV (46), in our analysis. As with CymRSV, infections of N. benthamiana plants with PVX and TMV resulted in the appearance of single siRNA fractions of 21 to 22 nt (Fig. 4A). To analyze whether PVX- and TMV-derived siRNAs are also asymmetric, we induced the transcription of the CP-encoding regions of the two viruses with both positive and negative polarities. These transcripts were then subjected to Northern hybridization and probed with 5'-labeled siRNAs isolated from either PVX- or TMV-infected plants. In both cases, the majority of the labeled virus-specific siRNAs hybridized more strongly to the negative transcript, indicating that similar to the case with CymRSV, PVX and TMV siRNAs are derived mainly from viral RNAs with a positive polarity (Fig. 4B).
It was recently reported that the expression of a host gene fragment as a short inverted repeat from TMV leads to more efficient silencing of the cognate mRNA than the expression of a similar sequence in just the sense or antisense orientation (24). To better understand this observation at the molecular level, we compared the siRNA accumulation in plants infected with a viral vector carrying a hairpin structure to that in plants infected with an antisense sequence. To this end, viral transcripts carrying 60-bp inverted repeats (TMV.hpPDS60) or the corresponding antisense sequence (TMV.PDSas110) homologous to the endogenous phytoene desaturase (PDS) gene (24) were used to inoculate N. benthamiana plants. RNAs were extracted from TMV-infected leaves showing the white coloration characteristic of the photobleaching phenotype observed for PDS-silenced plants and then analyzed for both viral RNA accumulation and virus-derived siRNA composition (Fig. 5). The photobleaching phenotype of PDS-silenced plants was more pronounced with the TMV.hpPDS60 hairpin construct than in plants infected with the TMV.PDSas110 antisense construct (Fig. 5A), in spite of similar viral RNA accumulation in virus-infected plants (Fig. 5B, top panel). More importantly, the siRNA accumulation originating from the hairpin sequence was significantly higher for both the plus (12 times) and minus (5 times) polarities than that from the antisense sequence containing the 60-nt sequence of the corresponding hairpin construct (Fig. 5B, bottom panels).
Taken together, these results strongly suggest that one or more of the plant-encoded DICER-like enzymes are able to recognize highly structured regions within viral ssRNAs and process them into siRNAs. This property resembles the DICER-like activity involved in the generation of miRNAs from short-hairpin precursors (16, 22, 30, 38, 42, 58), although recent data indicate that siRNAs and miRNAs require different DICER-like activities in plants (15, 50, 58). The discrete size (21 nt) of virus-derived siRNAs, however, suggests that one of the still unidentified plant DICERs has evolved specifically to process highly structured viral RNAs and is likely a key player in the PTGS-mediated antiviral defense. A recent report demonstrated that an Arabidopsis dcl2 mutant transiently accumulated lower levels of Turnip crinkle virus-derived siRNAs than did wild-type plants (58). However, there is likely to be some degree of redundancy because dcl1, dcl2, and dcl3 mutants were not defective in siRNA production when they were infected with other viruses.
The fact that plant DICER-like proteins generate predominantly positive-strand siRNAs makes the siRNA-based antiviral surveillance system more effective since the siRNAs potentially target the negative viral strand for RISC-mediated cleavage, which is required for replication and is much less abundant than the positive-strand viral RNA. In further support of this model, it was reported that transgenic plants expressing a positive-strand viral sequence specifically target the negative-strand RNA of TMV. This suggests that the transgenically expressed sense viral sequence can be processed into positive-strand siRNAs, which in turn specifically target the negative-strand viral RNA replicative intermediate for degradation (31). Since the sense siRNAs derived from folded RNA are partially complementary to the positive-strand viral RNA, positive siRNAs could also guide the translational repression of viral RNA in a manner analogous to miRNA-mediated translational control, which relies on only partial complementarity between the miRNA and the target. This type of gene silencing is common in animal systems (2, 5) but also seems to operate in plants (3, 10). Further experiments are required to address whether this additional level of control occurs.
ACKNOWLEDGMENTS
We thank Daniel Silhavy, Gy?rgy Szittya, Gábor Giczey, and Alan Herr for critical readings of and helpful comments on the manuscript.
L.L. is a recipient of a Bolyai János Fellowship. This research was supported by grants from the Hungarian Scientific Research Fund (OTKA; T046728), the "RIBOREG" EU project (LSHG-CT-2003503022), and the Scientia Amabilis Foundation.
Supplemental material for this article may be found at http://jvi.asm.org/.
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