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Journal of Virology, June 2005, p. 7217-7226, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.7217-7226.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity{ddagger}

Zsuzsanna Mérai,1 Zoltán Kerényi,1 Attila Molnár,1,{dagger} Endre Barta,1 Anna Válóczi,1 György Bisztray,2 Zoltán Havelda,1 József Burgyán,1 and Dániel Silhavy1*

Agricultural Biotechnology Center, Gödöllö, Hungary,1 Department of Genetics and Horticultural Plant Breeding, Budapest University of Economic Sciences and Public Administration, Budapest, Hungary2

Received 1 September 2004/ Accepted 17 January 2005


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ABSTRACT
 
RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.


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INTRODUCTION
 
RNA silencing (also termed posttranscriptional gene silencing in plants and RNA interference in animals) is a conserved eukaryotic gene inactivation system that plays regulatory roles in many biological processes including development, maintenance of genome stability, and antiviral responses (2, 6, 12, 25, 54). RNA silencing is induced by accumulation of double-stranded RNAs (dsRNAs). dsRNAs are first processed by an RNase III-like nuclease called DICER (in plants termed DICER-LIKE, or DCL) into short (21 to 25 nucleotide [nt]) RNAs, and then these short RNAs incorporate and guide different silencing effector complexes to homologous nucleic acids for suppression (2, 6, 12, 16, 25, 54). In plants, RNA silencing acts at both single-cell (cell-autonomous silencing) and at whole-plant (systemic silencing) levels. Cell-autonomous silencing inactivates genes in the cells in which dsRNAs accumulated. Moreover, cell-autonomous silencing generates mobile silencing signals that confer suppression of homologous mRNAs in neighboring cells (short distance) and in distant tissues (long-distance systemic silencing) (29, 31, 32, 56).

DICERs can process dsRNAs into two functionally different small RNAs, micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are involved in the control of many endogenous protein-encoding mRNAs, while siRNAs mainly play a role in suppression of molecular parasites such as transposons, transgenes, and viruses (2, 6, 12, 16, 25). In Arabidopsis, matured miRNAs are 21- to 22-nt-long single-stranded RNAs (ssRNAs) that are excised from endogenous hairpin RNA precursors by DCL1 (60). siRNAs, which are generated from long dsRNAs, accumulate as short (21 to 22 nt) or long (23 to 25 nt) double-stranded molecules having 2-nt 3' overhangs.

miRNA- and short siRNA-mediated silencing pathways share components. Both types of small RNAs are incorporated into and guide a multicomponent nuclease (RNA-induced silencing complex, or RISC) to homologous mRNAs for suppression. RISC cleaves targeted mRNA in the case of (near) perfect base-pairing between mRNA and guide RNA. When the guide RNA is only partially complementary to the mRNA, RISC mediates translational repression (2, 6, 12). siRNAs also guide other silencing effector complexes. In addition to RISC, short siRNAs are supposed to provide sequence specificity for a host-encoded RNA-dependent RNA polymerase that transforms homologous mRNAs into dsRNAs, thus amplifying silencing. Moreover, short siRNAs could be involved in short-distance systemic silencing (15, 18). Long siRNAs would play a role in long-distance systemic silencing (15) and in transcriptional silencing by directing the histone/DNA methylation of homologous DNA (7, 15, 25, 51).

RNA silencing plays an antiviral role in plants, in insects, and perhaps in other eukaryotes (2, 13, 22, 37, 46, 59). DCL2 and perhaps other DCL enzymes generate viral siRNAs from double-stranded replicative intermediates of RNA viruses (61) or from hairpins of viral mRNAs (48). Viral siRNAs could target RISC to viral mRNAs for suppression. As RNA-dependent RNA polymerase mutant plants are more susceptible to certain viruses, it is likely that silencing amplification is also an important antiviral pathway against particular viruses (9, 11, 30, 61). Importantly, virus-induced silencing acts as a short-distance systemic defense system. Viral siRNAs might spread 10 to 15 cell layers and activate silencing in still noninvaded neighboring cells, thus limiting the extent of virus invasion (15, 17, 18, 43).

To counteract RNA silencing, most plant viruses express silencing suppressor proteins. Viral suppressors target different steps of the silencing response (22, 39, 46, 57, 59). Although many suppressors have been identified, the molecular basis of silencing inhibition and the evolution of suppressors are poorly understood.

Members of the Tombusviridae plant virus family have icosahedral particles and linear, small, single-stranded positive-sense RNA genomes (42). Different genera of Tombusviridae express distinct suppressors. The coat protein (CP) of Turnip crinkle virus (Tombusviridae, Carmovirus) has multiple functions; in addition to forming a capsid, it also suppresses silencing (36, 52, 64). By contrast, the 19-kDa suppressor protein (P19) of tombusviruses (Tombusviridae, Tombusvirus) is apparently required only for silencing inhibition, as it is dispensable for replication, movement, or virion formation (34, 35, 45, 57). P19 is a specific dsRNA binding protein, which binds dsRNAs size selectively (55, 62). P19 forms strong complexes with dsRNAs having 19-nt duplex regions, thus it binds siRNAs in vitro (45, 55, 62) and in vivo (8, 14, 21). Importantly, it binds shorter or longer dsRNAs with much weaker affinity. It is proposed that in tombusvirus-infected cells, P19 sequesters silencing-generated siRNAs, thereby suppressing antiviral silencing responses (21, 45). Indeed, in Cymbidium ringspot virus (CymRSV; Tombusviridae, Tombusvirus)-infected plants, viral siRNAs are present in complex with P19 (21). To better understand the evolution of silencing suppression within Tombusviridae, we wanted to identify and analyze the silencing suppressor of Pothos latent virus (PoLV), the type species of aureusviruses (Tombusviridae). The genome organization of aureusviruses is identical to that of tombusviruses, but the Aureusvirus genome is significantly smaller and the sequence similarity between the two genera is limited (26, 28, 41) (Fig. 1A). The PoLV open reading frame (ORF) 5-encoded 14-kDa protein (P14), like the tombusvirus ORF 5-encoded P19, increases the severity of viral symptoms (symptom determinant) (40). Infection of Nicotiana benthamiana plants with either PoLV{Delta}14, a mutant PoLV that fails to express P14 (40) (Fig. 1B), or a mutant CymRSV that is unable to express P19 (Cym19stop) leads to similar recovery phenotypes (48). As the recovery phenotype is supposed to be the manifestation of virus-induced systemic silencing (1, 17, 37, 48), it has been suggested that P14, like P19, operates as a silencing suppressor (38, 40). Interestingly, although the genomic positions of P14 and P19 are identical and both proteins are symptom determinant, no significant sequence homology has been detected between P19 and P14 (40).



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  		FIG. 1. PoLV P14 is a symptom determinant. (A) Schematic representation of genome organization of a tombusvirus (CymRSV) and PoLV, the type species of aureusviruses. K, kilodalton. (B) PoLV P14 increases symptom severity. N. benthamiana plants were infected with PoLV and PoLV{Delta}14, a mutant virus that fails to express P14. PoLV{Delta}14 infection results in a recovery phenotype characterized by low virus titers and mild symptoms in the upper leaves. Plants were grown at 21°C.

Here we report that PoLV P14 is an efficient suppressor of both virus- and transgene-induced silencing. P14 is a dsRNA binding protein that binds dsRNA in vitro without obvious size selection. The potential mechanism of P14-mediated silencing suppression and the evolution of P14 and P19 suppressors will be discussed.


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MATERIALS AND METHODS
 
Plant materials and Agrobacterium tumefaciens infiltration. Transgenic N. benthamiana carrying the green fluorescent protein (GFP) ORF was described previously (4). The A. tumefaciens infiltration method was carried out as described previously (56). For coinfiltration, equal volumes of respective A. tumefaciens cultures (optical density at 600 nm, 0.25) were mixed before infiltration.

Silencing suppression assay and GFP imaging. The RNA silencing suppression assay was carried out as described previously (58). Visual detection of GFP fluorescence was performed using a 100-W handhold long-wave UV lamp (Black Ray model B 100AP; UV Products, Upland, CA).

Plasmid constructs. The infectious cDNA clones of PoLV, PoLV{Delta}14 (40), CymRSV (10), and Cym19stop (48) were described previously.

Silencing suppressors for agroinfiltration assays were cloned into pBIN61S (45). P19 and Sigma3 binary constructs were described previously (24, 45). P14 was PCR amplified with P14 5' and 3' primers corresponding to the first and last 20 nt of P14. The P14 5' primer carried an additional BamHI site, while the P14 3' primer contained an additional SalI site. The PCR product was cloned in reverse orientation into SmaI-cleaved pBluescript KS vector (KS-P14). To create the P14 binary construct, the BamHI-SalI fragment was cloned from KS-P14 into pBin61S. G-P14 was generated by cloning the BamHI fragment from KS-P14 into Gex-2T, and then the sense orientation was selected. PVX-P14 was generated by cloning a refilled BamHI-SalI fragment from KS-P14 into EcoRV-digested pP2C2S.

In vitro RNA transcription and plant inoculation. In vitro transcription from PoLV, PoLV{Delta}14 (40), CymRSV, Cym19stop, and PVX cDNA clones and inoculation of RNA transcripts onto plants were performed as described previously (10, 48).

Protoplast preparation and inoculation. Protoplasts were isolated from N. benthamiana and transfected with in vitro transcripts of PoLV or PoLV{Delta}14 (10, 20).

Protein separation and Western analysis. Proteins were separated in a 12% sodium dodecyl sulfate-polyacrylamide gel and than transferred onto a Hybond C Extra filter (Amersham Pharmacia Biotech). Rabbit P14 polyclonal antibodies (anti-P14) raised against the GST-P14 fusion protein were used for Western analysis.

RNA gel blot analysis. The same total RNA extract was used for high- and low-molecular-weight RNA gel blot analysis. RNA extraction and RNA gel blot analysis were carried out as described previously (48). PCR fragments labeled with the random priming method were used for Northern analyses of high-molecular-weight RNAs. Radioactively labeled in vitro transcripts corresponding to the positive strand of virus RNA and antisense strand of GFP were used as probes for Northern analyses of low-molecular-weight RNAs. Labeling was carried out as described previously (48).

Gel mobility shift assay. Synthetic siRNAs were labeled with T4 PNK. [{alpha}-32P]UTP-labeled in vitro RNA transcripts were used as long RNA probes. Transcripts were produced from a T7-T3 Bluescript PCR fragment by the T7 and T3 RNA polymerases, respectively (48).

To generate double-stranded siRNAs, 5'-phosphorylated complementary strand siRNAs in 5 times molar excesses were added to labeled single-stranded siRNAs, and then siRNAs were heated and annealed. To generate long dsRNAs, a 1:1 mixture of labeled T7 and T3 in vitro transcripts were heated and annealed. GST, G-P14, and G-P19 proteins were expressed and purified according to the manufacturer's protocols (Amersham Pharmacia Biotech).

To prepare protein extract, 0.25 g leaf tissue was grinded in 1 ml band shift buffer (83 mM Tris-HCl [pH 7.5], 0.8 mM MgCl2, 66 mM KCl, 100 mM NaCl, and 10 mM dithiothreitol), and then this crude extract was centrifuged twice for 15 min at 15,000 x g. The supernatant was frozen in aliquots at –70°C. In a binding reaction, labeled dsRNA (in a 1 nM concentration) was incubated with extract containing ~2 µg total protein. Binding reaction and mobility shift assays were carried out as described previously (48), except that 8 U RNasin was added to each 10-µl reaction mixture. For long dsRNA direct competition assays, 0.02% Tween 20 was added to the binding buffer.

Computer analysis. Multiple alignments of RNA and deduced protein sequences were carried out with ClustalX (53). Relationships among proteins were analyzed by the bootstrap parsimony (47) and maximum-likelihood methods (44).


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RESULTS
 
P14 is a silencing suppressor. Expression of a silencing suppressor from a heterologous virus intensifies viral symptoms (57). To test whether P14 is a silencing suppressor, we infected N. benthamiana and Nicotiana clevelandii plants with Potato virus X (PVX) that expressed P14 (PVXP14) and with PVX as a control. While PVX infection caused only mild symptoms on both hosts, PVXP14-infected plants showed strong symptoms including stunting and necrosis along the veins (Fig. 2A and data not shown). Findings that P14 increased PoLV and PVX symptoms suggest that P14 is a suppressor of virus-induced silencing.



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  		FIG. 2. P14 is an RNA silencing suppressor. (A) P14 increases the symptoms of PVX. N. benthamiana plants were infected with PVX or PVXP14, a modified PVX that expressed P14. (B) P14 suppresses sense transgene-induced RNA silencing. Leaves of N. benthamiana plants were infiltrated with an Agrobacterium (35SGFP) expressing GFP (–) or were coinfiltrated with 35SGFP and a second Agrobacterium expressing P14 (P14). Photos were taken at 6 d.p.i. (C) Effect of P14 on accumulation of GFP mRNAs and GFP-derived siRNAs (siRNS).

Viral silencing suppressors can be identified in sense transgene-induced silencing assays (58). Infiltration of the leaves of an N. benthamiana plant with an Agrobacterium carrying a plasmid that expresses GFP (35SGFP) leads to strong, transient GFP expression, but it also triggers GFP silencing (4, 58). Cell-autonomous GFP silencing is manifest as a weakening of green fluorescence, a decrease in the level of GFP mRNA, and an accumulation of both short (21 to 22 nt) and long (23 to 25 nt) GFP-specific siRNAs in the infiltrated patches (Fig. 2B and C). However, GFP silencing is partially or fully inhibited if 35SGFP is coinfiltrated with a second Agrobacterium expressing a silencing suppressor. To determine whether P14 suppresses sense transgene-induced silencing, N. benthamiana plants were coinfiltrated with 35SGFP and with a second Agrobacterium expressing P14 (P14). As the green fluorescence was much stronger and lasted longer in coinfiltrated patches than in leaves infiltrated with 35SGFP alone (Fig. 2B), we concluded that P14 inhibited sense transgene-induced cell-autonomous silencing.

The effect of silencing suppressors on accumulation of short and long siRNAs depends on the targeted step of a particular suppressor (15, 18). To investigate which step of silencing is targeted by P14, we studied the accumulation of GFP mRNA and the GFP-derived siRNAs in 35SGFP- and P14-coinfiltrated leaves. As Fig. 2C shows, in coinfiltrated leaves, GFP mRNAs accumulated to much higher levels than in leaves that were infiltrated with 35SGFP alone. Moreover, neither short nor long GFP-specific siRNAs could be detected in coinfiltrated leaves. These data indicate that P14 interferes with sense transgene-induced cell-autonomous silencing by preventing the accumulation of siRNAs. The effects of a silencing suppressor on transgene-induced short- and long-distance systemic silencing can be also studied in coinfiltration assays. If a GFP-transgenic N. benthamiana is infiltrated with 35SGFP, cell-autonomous GFP silencing generates mobile signals, which lead to systemic GFP silencing. Since P14 prevents the accumulation of either short or long siRNAs in coinfiltrated patches and siRNAs are supposed to play role in systemic silencing, we postulated that P14 also inhibits the development of systemic silencing. Indeed, coinfiltration of P14 prevented the development of both short- and long-distance systemic GFP silencing (data not shown).

Taken together, the findings that P14 increases viral symptoms and inhibits silencing in agroinfiltration assays indicate that P14 is an efficient suppressor of both virus and transgene-induced silencing.

P14 is a dsRNA binding protein. In a GFP coinfiltration (sense transgene-induced silencing) assay, P14 acts like the P19 suppressor of closely related tombusviruses (18, 34, 35, 45). Both proteins prevent the accumulation of GFP-specific short and long siRNAs in the coinfiltrated patches, thus inhibiting the development of cell-autonomous and systemic silencing. These data open up the possibility that P14 and P19 suppressors target an identical step in the silencing pathway. P19 inhibits silencing by sequestering double-stranded siRNAs. To test whether P14 could also suppress silencing by binding double-stranded siRNAs, we studied the RNA binding activity of P14 in gel mobility shift assays. P14 was expressed and purified as a GST fusion protein (G-P14), and then G-P14 was probed with labeled, synthetic single-stranded and double-stranded siRNAs. A GST fusion version of the previously characterized Carnation Italian ringspot tombusvirus (CIRV) P19 (G-P19) was used as a control (55). As expected, G-P19 did not shift ssRNAs, while it bound 21-nt double-stranded siRNAs. Importantly, G-P14 also failed to form complexes with ssRNAs but bound 21-nt double-stranded siRNA (Fig. 3A). These data suggest that G-P14, like G-P19, is a double-stranded siRNA binding protein. However, G-P14 forms complexes with double-stranded siRNAs with less efficiency than G-P19, since a similar shift required a much higher G-P14 concentration. The striking feature of P19-mediated dsRNA binding is its strong size selectivity. To test whether P14 is also a size-selective dsRNA binding protein, G-P14 was also probed with long (144 nt) dsRNAs. Interestingly, we found that unlike G-P19, G-P14 formed complexes with long dsRNAs (Fig. 3A). These data suggest that G-P14 is a dsRNA binding protein that lacks size specificity. Unfortunately, we failed to release functional P14 from G-P14 with thrombin cleavage; therefore, we cannot characterize the RNA binding activity of Escherichia coli-expressed native P14.



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  		FIG. 3. P14 is a dsRNA binding protein. (A) RNA binding activity of P14 (G-P14) and P19 (G-P19) expressed as a GST fusion protein were studied in gel mobility shift assays. Shift assays with expressed GST or without additional protein (–) were used as negative controls. Synthetic 21-nt RNAs were labeled and used as ssRNA probes, while labeled 21-nt RNAs, which annealed to dsRNAs having a 19-nt duplex with 2-nt 3' overhangs (21 ds siRNA) were used as double-stranded siRNA probes; 144-nt-long, labeled, complementary in vitro transcripts were annealed and used as long dsRNA probes (144 dsRNA). Concentrations of G-P14 of 15 µM and 1.5 µM were used for the double-stranded siRNA and for the long dsRNA binding experiments, respectively. A 1.5 µM concentration of G-P19 was used for both double-stranded siRNA and long dsRNA binding tests. Free probes and protein-probe complexes are referred as F and C, respectively. (B) PoLV-expressed P14 efficiently binds double-stranded siRNAs. dsRNA binding activity of extracts prepared from PoLV- or PoLV{Delta}14-inoculated leaves were probed with labeled 21-nt double-stranded siRNAs. Extracts prepared from leaves inoculated with CymRSV and Cym19stop (C19stop), a mutant CymRSV that was unable to express P19, were used as controls. Extracts were isolated at 3 d.p.i. from N. benthamiana leaves inoculated with the corresponding viruses. (C) In planta-expressed P14 binds long dsRNA. Extracts prepared from mock (–)-, PoLV-, PoLV{Delta}14-, CymRSV-, and C19stop-inoculated N. benthamiana leaves (left panel) were probed with labeled 144-nt dsRNA. Extracts prepared from mock (–)-, 35SGFP (GFP)-, and P14-infiltrated N. benthamiana leaves (right panel) were also probed with labeled 144-nt dsRNA. Each extract was obtained at 3 d.p.i. An asterisk indicates a nonspecific binding activity that is not associated with P14 or P19 suppressors because it is present in all extracts prepared from PoLV{Delta}14, CymRSV, and C19stop virus-inoculated leaves. Similar nonspecific long dsRNA binding activity can be occasionally detected in extracts prepared from mock- or 35SGFP-infiltrated leaves. (D to G) Plant-expressed P14 binds double-stranded siRNAs without size selectivity. P14 and P19 extracts were obtained from P14- and P19-infiltrated N. benthamiana leaves at 3 d.p.i. Direct competition assays were carried out with labeled 21-nt (D and F) and 26-nt double-stranded siRNAs (E and G) and with cold competitors added in the indicated molar excesses. A 0 indicates that the shift assay was conducted in the absence of competitor.

Although in vitro G-P14 fusion protein inefficiently forms complexes with double-stranded siRNAs, it is possible that native P14 efficiently binds double-stranded siRNAs in PoLV-infected cells. Moreover, full double-stranded siRNA binding activity of P14 might require plant-specific posttranslational modification and/or the presence of host/viral factors. Therefore we wanted to directly analyze the double-stranded siRNA binding activity of PoLV-expressed P14. To study whether PoLV-expressed P14 binds double-stranded siRNAs, we compared the double-stranded siRNA binding activity of crude extracts prepared from PoLV (PoLV extract)- and PoLV{Delta}14 (PoLV{Delta}14 extract)-inoculated N. benthamiana leaves. Importantly, PoLV extract efficiently bound 21-nt double-stranded siRNAs, while PoLV{Delta}14 extract failed to form complexes with 21-nt double-stranded siRNAs (Fig. 3B). As P14 protein was expressed only in PoLV-infected leaves (see Materials S1 and Fig. S1A in the supplemental material), it is likely that P14 provided the double-stranded siRNA binding capacity for the PoLV extract. Moreover, PoLV extract causes almost as strong a shift on 21-nt double-stranded siRNAs as the extract that was prepared from CymRSV tombusvirus-inoculated leaves (CymRSV extract) (Fig. 3B). This result suggests that PoLV-expressed P14 effectively forms complexes with double-stranded siRNA.

To investigate whether P14 can bind double-stranded siRNAs in the absence of viral factors, we analyzed the double-stranded siRNA binding activity of crude extract prepared from P14-agroinfiltrated N. benthamiana leaves (P14 extract). Extract prepared from 35SGFP-infiltrated leaves was used as a negative control (GFP extract). We could not detect double-stranded siRNA binding activity in the GFP extract (data not shown), while P14 extract caused a strong shift on 21-nt double-stranded siRNAs (Fig. 3D, second lane). Moreover, as both GFP and P14 extracts weakly and identically bound single-stranded siRNAs (data not shown), it is likely that P14 does not bind ssRNAs. Therefore, we conclude that in planta expressed P14 is a double-stranded siRNA binding protein which does not require viral factors for dsRNA binding.

In vitro, P19 binds dsRNAs size selectively, while G-P14 binds dsRNAs without size specificity. To test whether in planta-expressed P14 and P19 proteins also differ in dsRNA preference, we defined the relative affinity of plant-produced P14 and P19 for 21-nt and 26-nt double-stranded siRNAs. To aim this, direct competition assays were carried out with P14 and P19 extracts prepared from agroinfiltrated leaves. Labeled 21-nt double-stranded siRNAs were incubated with plant extracts and with increasing molar concentrations of cold 21-nt and 26-nt double-stranded siRNA competitors (Fig. 3D and F). Competition experiments were repeated with labeled 26-nt double-stranded siRNAs (Fig. 3E and G). In line with results obtained with P19 expressed in E. coli, the P19 extract showed a much higher affinity for 21-nt double-stranded siRNAs than for 26-nt double-stranded siRNAs (55, 62). A large molar excess of 26-nt double-stranded siRNAs (320x) was required for detectable competition when 21-nt double-stranded siRNA was labeled (Fig. 3F), while 21-nt double-stranded siRNAs outcompeted 26-nt double-stranded siRNAs even at a low (20x) molar excess (Fig. 3G). By contrast, the P14 extract had no obvious size specificity because approximately the same molar excess of cold 21-nt double-stranded siRNAs and 26-nt double-stranded siRNAs was required for outcompeting either labeled 21- or 26-nt double-stranded siRNAs (Fig. 3D and E). These results suggest that both suppressors bind double-stranded siRNAs efficiently but with different selectivities. P19 might be more specific for small double-stranded siRNAs, whereas P14 could bind short and long double-stranded siRNAs with comparable affinity.

In vitro G-P14 fusion protein binds long dsRNAs. To test whether in planta-expressed P14 also binds long dsRNAs, labeled 144-nt dsRNA was incubated with PoLV and with P14 extract. As negative controls, PoLV{Delta}14 and GFP extract were used, respectively. As Fig. 3C shows, a long dsRNA binding activity could be detected in both PoLV and P14 extracts that was lacking in either the PoLV{Delta}14 or GFP extract. Therefore, it is very likely that P14 provided the long dsRNA binding activity for PoLV and for P14 extracts. Findings that long dsRNA binding of P14 extract could be outcompeted with cold dsRNA but not with ssRNA further support the notion that in planta-expressed P14 is a dsRNA binding protein (see Materials S2 and Fig. S1B in the supplemental material). Moreover, in line with results obtained with G-P19 fusion protein, labeled long dsRNA was not bound by CymRSV-expressed P19 (Fig. 3C).

Collectively, mobility shift assays revealed that P14 and P19 suppressors are different dsRNA binding proteins: P19 is a strict size-specific dsRNA binding protein, while P14 binds dsRNAs without strong size preference.

P14 and P19 act differently in hairpin-induced agroinfiltration assays. As dsRNAs play a key role in silencing and P14 is a dsRNA binding protein, we postulated that P14-mediated silencing is based on sequestering a dsRNA component of the silencing machinery. P14 might sequester long dsRNAs, the inducers of silencing machinery, or double-stranded siRNAs, the specificity determinants of the silencing system. To distinguish between these two possibilities, we studied the suppressor activity of P14 in hairpin transcript-induced silencing assays.

Agroinfiltration with an inverted repeat GFP construct (GFP IR) leads to expression of hairpin GFP RNAs (GFP-ir). As hairpin transcripts are rapidly processed into siRNAs by the silencing machinery, GFP-ir transcripts are barely detectable, while GFP-ir-derived siRNAs accumulate to high levels in the infiltrated leaves (Fig. 4C). Moreover, coinfiltration of GFP IR with 35SGFP (GFP IR plus 35SGFP) prevents transient GFP activity (Fig. 4A and B) because GFP-ir-derived siRNAs direct early degradation of GFP mRNAs (19). However, coinfiltration of GFP IR plus 35SGFP with dsRNA binding proteins such as reovirus Sigma3 (24) or P19 (50) result in strong green fluorescence and accumulation of GFP mRNAs (Fig. 4A and B). Sigma3 and P19 suppress hairpin-induced silencing at different steps. Sigma3 is a strong dsRNA binding protein that forms complexes only with dsRNAs longer than ~30 nt (63). Sigma3 is proposed to suppress silencing by sequestering hairpin transcripts (24) because, in coinfiltrated leaves, GFP-ir transcripts accumulate to high levels, while siRNAs could not be detected (Fig. 4B). By contrast, in P19-coinfiltrated leaves GFP-ir transcripts could not be detected, while siRNAs are easily detected (50) (Fig. 4B). These data are interpreted to mean that the siRNA-specific dsRNA binding protein P19 inhibits hairpin-induced silencing by sequestering double-stranded siRNAs (50).



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  		FIG. 4. P14 suppresses hairpin-induced RNA silencing. (A) N. benthamiana leaves were infiltrated with 35SGFP and a second Agrobacterium (GFP IR) expressing hairpin GFP transcripts (–) or coinfiltrated with GFP IR plus 35SGFP and P14, P19, and Sigma3 suppressors, respectively. Photos were taken at 3 d.p.i. (B) Effect of P14 on accumulation of hairpin transcripts, GFP mRNAs, and siRNAs. GFP-ir indicates hairpin transcripts derived from GFP IR, while GFP refers to GFP mRNA transcribed from 35SGFP (upper panel). RNA samples were isolated at 3 d.p.i. from GFP IR plus 35SGFP-infiltrated (–) or from GFP IR plus 35SGFP and dsRNA binding protein-coinfiltrated leaves. Note that the probe we used for siRNA hybridization (bottom panel) detected both hairpin transcript and GFP mRNA-derived siRNAs (GFP siRNA). (C) P14 prevents the generation of siRNAs from hairpin transcripts. RNA samples were isolated at 3 d.p.i. from GFP IR-infiltrated (–) or from GFP IR plus dsRNA binding protein-coinfiltrated leaves.

To test whether P14 can suppress hairpin-induced silencing, we coinfiltrated leaves with GFP IR plus 35SGFP and with P14. We found that GFP mRNAs accumulated to high levels and green fluorescence was strong in P14 coinfiltrated leaves, indicating that P14 suppressed hairpin-induced silencing efficiently (Fig. 4A). Surprisingly, we failed to detect either hairpin transcripts or siRNAs in P14-coinfiltrated leaves (Fig. 4B). To prove that P14 directly affects on hairpin-derived siRNA accumulation, we infiltrated leaves with GFP IR or coinfiltrated leaves with GFP IR and P14. As expected, in GFP IR-infiltrated samples, siRNA accumulated to high levels, while hairpin transcripts could not be detected. By contrast, in GFP IR- and P14-coinfiltrated leaves, neither hairpin transcripts nor siRNAs could be found (Fig. 4C).

Collectively, GFP IR coinfiltration studies revealed that P14-mediated suppression of hairpin-induced silencing is mechanistically different than either Sigma3- or P19-mediated silencing inhibition. We suggest that these differences are the consequences of the different dsRNA binding preferences of the Sigma3, P19, and P14 proteins (see Discussion).

P14 fails to prevent accumulation of viral siRNAs. The observation in agroinfiltration assays that P14 prevents the accumulation of hairpin transcript-derived siRNAs suggests that P14 suppresses virus-induced silencing by preventing the accumulation of viral siRNAs. To test this model, we monitored the accumulation of viral RNAs and siRNAs in PoLV- and PoLV{Delta}14-inoculated N. benthamiana leaves. By 1 day postinoculation (d.p.i.), PoLV and PoLV{Delta}14 viral RNAs accumulated to detectable levels, while by 2 d.p.i., PoLV and PoLV{Delta}14 RNAs were abundant in the inoculated leaves (Fig. 5A). Surprisingly, virus-specific siRNAs could be identified in both PoLV- and PoLV{Delta}14-inoculated leaves (Fig. 5A). As the P14 protein could already be detected at 1 d.p.i. in PoLV-infected leaves (Fig. 5A bottom panel), we concluded that P14 fails to prevent the accumulation of viral siRNAs. However, viral siRNA/viral genomic RNA ratios were higher in PoLV{Delta}14-inoculated leaves than in PoLV-inoculated ones (Fig. 5A). These data indicate that virus-induced silencing operated less efficiently in PoLV-inoculated leaves than in PoLV{Delta}14-infected tissues, confirming that P14 acts as an efficient suppressor of aureusvirus-induced silencing.



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  		FIG. 5. P14 does not prevent accumulation of PoLV-derived siRNAs. (A) Accumulation of high- and low-molecular-weight viral RNAs (viral siRNA) in the inoculated leaves of PoLV- and PoLV{Delta}14-infected N. benthamiana plants. G indicates genomic virus RNAs, while Sg1 and Sg2 refer to subgenomic 1 and subgenomic 2 RNAs, respectively. Note that P14 was already detectable at 1 d.p.i. and that the level of PoLV Sg2 declines. Numbers below the bottom panel refer to the ratios of viral siRNA/viral genomic RNAs. The sample loaded in the first lane was taken as 100% (1), and others were normalized to it. {alpha}-P14 refers to polyclonal antibody raised against P14. (B) Virus-induced cell-autonomous silencing fails to control accumulation of suppressorless PoLV. RNA samples were isolated from N. benthamiana protoplasts transfected with in vitro transcripts of PoLV or PoLV{Delta}14. A Western blot assay with the available polyclonal antibody failed to detect P14 protein in PoLV-transfected cells, therefore conclusions about the role of P14 in suppression of virus induced cell-autonomous silencing cannot be drawn (see Materials S3 in the supplemental material).

Suppressors of aureusviruses and tombusviruses derive from a common ancestor protein. Findings that both P14 and P19 proteins bind double-stranded siRNAs and suppress silencing suggest that these proteins evolved from a common ancestor. However, the nonrelated P21 suppressor of Beet yellows virus (Closteroviridae, Closterovirus) also binds double-stranded siRNAs (8), indicating that dsRNA binding silencing suppressors evolved more than once. Therefore, it is also possible that P14 and P19 suppressors evolved independently.

To clarify whether P14 and P19 proteins evolved independently or have a common ancestor, multiple-sequence alignments were carried out with many tombusvirus and aureusvirus sequences. In both genera, ORF 5 is completely nested within ORF 4 but it is translated in the third frame related to ORF 4 (Fig. 1A). Therefore, we first defined the biologically meaningful gaps that optimized both the alignment of ORF 4-encoded movement proteins (MP) and the alignment of suppressor proteins (for details regarding defining gaps and creating protein alignments, see Materials S4 and Fig. S2 in the supplemental material).

Multiple-sequence alignments of proteins deduced from tombusvirus and aureusvirus ORF 4 (see Materials S4 and Fig. S3 in the supplemental material) and ORF 5 (Fig. 6) RNA sequences revealed that similarity was strong for both movement and suppressor proteins within a genus but weak between aureusviruses and tombusviruses (see Fig. S4 in the supplemental material). Amino acids that were identical or similar between aureusviruses and tombusviruses could be found all along the MPs except in the C-terminal region (see Materials S4 and Fig. S3 in the supplemental material). By contrast, conserved amino acid positions were limited to short regions in the suppressor alignment (Fig. 6). Interestingly, the regions of similarity in the suppressor sequences coincide with previously identified secondary structural elements (ß1, ß2, ß3, ß4, and the last {alpha}-helix) that play key roles in double-stranded siRNA binding by P19. ß4 and the last {alpha}-helix contribute to the P19 homodimer formation, while the four ß-strands form a concave ß sheet that makes contact with the sugar-phosphate backbone of double-stranded siRNA (3, 55, 62). No similarity was found between aureusvirus and tombusvirus suppressors in the region corresponding to the P19 {alpha}2 helix (reading head) that interacts with the end of siRNAs (3, 55, 62) (Fig. 6).



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  		FIG. 6. P14 and P19 suppressors appear to be evolutionarily related. Multiple alignments of amino acid sequences deduced from many available aureusvirus and tombusvirus ORF5 sequences. Defined gaps (see Materials S4 in the supplemental material) were incorporated into the deduced protein sequences. PoLV (indicated as PLV), Cucumber leaf spot virus (CLSV), and Johnsongrass chlorotic stripe mosaic virus (JCSMV) are aureusviruses, and other viruses included in the alignments are tombusviruses. CIRV refers to the P19 protein of CIRV, whereas CRSV shows CymRSV P19. List of viruses, which were not used in this study but were included in the comparison are available in the supplemental material (see Materials S4 and Table S1 in the supplemental material). Different colors show different groups of amino acids. Asterisks indicate amino acids that are perfectly conserved in each aligned protein, while colons and periods refer to conservative and semiconservative substitutions, respectively. Secondary structural elements defined for CIRV P19 are shown at the top.

As tombusvirus and aureusvirus suppressors show similarity in conserved regions that are important for P19-mediated suppression, we suggest that these proteins have evolved from an ancient suppressor, which was nested in the common ancestor of the MPs of tombusviruses and aureusvirures. Tombusvirus and aureusvirus MPs belong to the 30-kDa MP superfamily (27). To explore the evolution of tombusvirus-aureusvirus suppressors, we sought to determine whether related MPs also encoded a nested suppressor. The closest relatives to tombusvirus and aureusvirus MPs are the umbro, tobra (27), and trichovirus MPs (E. Barta, unpublished data). Importantly, none of these MP encodes a nested protein (data not shown).


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DISCUSSION
 
Previously P14 was identified as a symptom determinant (40). Here we demonstrate that P14 is a dsRNA binding protein that inhibits virus- and transgene-induced silencing.

P14 is a dsRNA binding protein. P14 is a dsRNA binding protein which forms complexes with 21- or 26-nt double-stranded siRNAs and with long dsRNAs (Fig. 3). It was an unexpected finding because the related P19 preferentially bound 21-nt double-stranded siRNAs (55, 62). Sequence alignments might explain the molecular basis of different double-stranded siRNA binding of these two suppressors. Regions that play roles in forming the dsRNA binding surface are conserved between aureusviruses and tombusviruses, while the reading head, which is involved in size-specific binding of P19, cannot be identified in aureusviruses (Fig. 6). Therefore, it is possible that P14 and P19 suppressors bind double-stranded siRNAs with similar structure, except that P14 does not interact with the 5' ends of RNAs.

P14-mediated silencing suppression. Previous results have shown that dsRNA binding proteins could act as silencing suppressors. P19 and P21 suppressors bind double-stranded siRNAs in vivo (8, 14, 21), indicating that these proteins inhibit silencing by sequestering double-stranded siRNAs. It is proposed that the influenza Ns1 protein also inactivates silencing in plant and insect cells by binding double-stranded siRNAs (5, 23). Moreover, as Sigma3 suppresses transgene-induced silencing efficiently, it is likely that binding to long dsRNAs could also inhibit certain silencing pathways (24). Although we cannot exclude that P14-mediated silencing suppression depends on its interaction with a host protein, the simplest explanation is that P14 targets silencing by binding long dsRNAs and/or double-stranded siRNAs. We suggest that P14 inhibits hairpin-induced silencing by binding long dsRNAs, while it suppresses virus-induced silencing by sequestering double-stranded siRNAs or by delaying the generation of viral siRNAs.

Coinfiltration of P14 with GFP IR plus 35SGFP prevents the accumulation of both hairpin transcript- and hairpin-derived siRNA (Fig. 4). These findings can be explained if P14 does not interfere with the generation of siRNAs, instead it accelerates the degradation of them. However, viral siRNAs accumulate to high levels in PoLV-infected cells (Fig. 5). Therefore, we prefer the alternative explanation that P14 binds hairpin transcripts (long dsRNAs); thus, it inhibits DCL-mediated processing of hairpin transcripts but allows their degradation by alternative decay systems. Sigma3 might bind long dsRNAs more strongly than P14; therefore, it protects hairpin transcripts. P19, which does not bind long dsRNAs, fails to prevent the generation of siRNAs from hairpin transcripts, instead it inhibits silencing by sequestering hairpin-derived double-stranded siRNAs.

Findings that P14 increases PVX and PoLV symptoms and that virus-induced silencing is more intense in PoLV{Delta}14-inoculated leaves than in PoLV-inoculated ones strongly indicate that P14 acts as an efficient suppressor of virus-induced silencing. Viral siRNAs can be easily detected in PoLV-infected leaves, suggesting that P14 inhibits virus-induced silencing by targeting a step downstream of siRNA generation. As in vitro P14 binds double-stranded siRNAs, we postulate that in virus-infected cells P14, like P19, suppresses silencing by sequestering double-stranded siRNAs. However, we failed to coimmunoprecipitate viral siRNAs from PoLV-infected leaves with P14 polyclonal antibody (Z. Merai, unpublished data). It could be due to technical difficulties (for instance, the P14 double-stranded siRNA complex is weak and dissociates during manipulation or the P14 antibody is not suitable for coimmunoprecipitation), and we cannot exclude that in vivo P14 does not bind double-stranded siRNAs. An alternative model for P14-mediated suppression of virus-induced silencing could be that P14 binds the precursors of viral double-stranded siRNAs; thus, it slightly delays the accumulation of siRNAs. Importantly, observation that viral siRNAs are relatively more abundant in PoLV{Delta}14-infected leaves than in PoLV-infected ones is consistent with both suppression models.

Antiviral silencing operates as a cell-autonomous and systemic response. Because viral RNAs accumulate to high levels in PoLV{Delta}14-transfected protoplasts (40) (Fig. 5B) even though viral siRNAs are present (Fig. 5B), we conclude that cell-autonomous antiviral silencing is unable to limit the accumulation of the rapidly replicating virus. By contrast, infection of PoLV{Delta}14 leads to a recovery phenotype (40) (Fig. 1A), indicating that colonization of the host plant requires the suppression of systemic silencing (1, 17, 18, 46, 49). We suggest that, in wild-type infection, P14 inhibits the development of systemic silencing by sequestering 21-nt double-stranded siRNAs and/or by delaying the generation of siRNAs; thus, PoLV spreads more quickly than the silencing signal and colonizes the plant. These models, in which P14 suppresses virus-induced systemic silencing by binding double-stranded siRNAs and/or precursor dsRNAs, predict that it inhibits systemic silencing in a dose-dependent manner. Indeed, at a high temperature (27°C) where siRNA generation is efficient (49), even PoLV infection leads to a recovery phenotype, indicating that at 27°C P14 fails to completely inhibit systemic silencing (Merai, unpublished).

Evolution of dsRNA binding silencing suppressors of aureusviruses and tombusviruses. Both MP and suppressor proteins of aureusviruses are homologous to the corresponding tombusvirus proteins, indicating that the common ancestor of these two genera already carried an ancestral silencing suppressor nested in the MP. Because nested protein could not be found in any related MP, we suggest that this ancestral suppressor has evolved within the common ancestor of aureusvirus-tombusvirus MP after it diverged from other 30-kDa MPs but before the branching of Aureusvirus and Tombusvirus genera. Moreover, as P19 and P14 suppressors are dsRNA binding proteins and as the conserved suppressor regions are important in forming the dsRNA binding structure of P19, it is likely that the ancestral suppressor was a dsRNA binding protein. P19 is a unique dsRNA binding protein because it binds dsRNAs size selectively, while all other characterized dsRNA binding proteins binds dsRNAs without strict size specificity. Therefore, we speculate that the common ancestor of the aureusvirus-tombusvirus suppressor was a size-independent dsRNA binding protein.

Suppressors have evolved to target antiviral responses, but they have also been selected for causing as little damage as possible to the host. It is proposed that, as size-specific double-stranded siRNA binding is a result of such a dual selection, P19 can efficiently bind siRNAs that play a role in antiviral response, while it might not interfere with long double-stranded siRNA programmed silencing pathways such as RNA-mediated epigenetic gene regulation (3, 33, 55). It is conceivable that expression of a size-independent dsRNA binding protein would cause additional damages, for instance, P14 might interfere with RNA-directed epigenetic regulation or bind structured host mRNAs. Interestingly, the level of Sg2 RNA, from which P14 is translated, declines after 2 to 3 d.p.i. in PoLV-infected plants (40) (Fig. 5A), while CymRSV Sg2 is abundant even at 10 d.p.i (48). It has been suggested that decreased expression of PoLV Sg2 is a viral control measure to reduce the toxicity of P14 (40). Indeed, infection with a PoLV mutant that constitutively expresses Sg2 RNA results in rapid plant death (40). It is appealing to speculate that the common ancestor suppressor might have evolved by two ways to reduce the damage to the host. In tombusviruses, it has evolved into a size-specific double-stranded siRNA binding suppressor, while in PoLV, it could have evolved into a temporally/spatially controlled suppressor.

Is dsRNA binding a frequent suppressor strategy? P14 suppressed hairpin-induced silencing by preventing the accumulation of hairpin transcript and siRNAs. Interestingly, transgenic expression of peanut clump virus P15, potato virus X P25, and turnip crinkle virus CP silencing suppressors in an Arabidopsis line that expressed hairpin transcripts of chalcone synthase also lead to similar inhibition of hairpin-induced silencing (14). In all three cases, hairpin-derived siRNA levels were dramatically reduced even though hairpin transcripts were not protected (14). These findings suggest that (some of) these suppressors target silencing like P14. They might be size-independent dsRNA binding proteins, which inhibit hairpin-induced silencing by preventing DCL-mediated processing. If these proteins bind dsRNAs like P14, they also form complexes with double-stranded siRNAs; hence, they could suppress silencing by sequestering double-stranded siRNAs. Indeed, P15 and CP suppressed siRNA-mediated silencing efficiently in HeLa cells (14). Since these proteins are nonrelated (8), we speculate that dsRNA binding is a frequent suppression strategy, which has evolved independently many times. In this study, we described a rapid mobility shift assay using crude extracts prepared from either virus-infected or agroinfiltrated leaves that is suitable for recognition and characterization of dsRNA binding proteins. We think that this convenient method could facilitate the identification of other dsRNA binding viral suppressors.


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ACKNOWLEDGMENTS
 
We are grateful to M. Russo for kindly providing full-length cDNA clones of PoLV and PoLV{Delta}14 and to D. Baulcombe for the GFP plant and 35SGFP constructs. We are especially grateful to J. Vargason and T. Tanaka Hall for providing siRNAs and for help with conducting and analyzing competition assays. We thank T. Tanaka Hall, G. Szittya, and L. Lakatos for useful comments on the manuscript, G. Takacs for help with figure preparations, and Edina Kapuszta for excellent technical assistance.

This research was supported by grants from the Hungarian Scientific Research Fund (OTKA) (T15042787). D.S. was financed by the Bolyai scholarship.


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FOOTNOTES
 
* Corresponding author. Mailing address: Agricultural Biotechnology Center, Plant Science Institute, P.O. Box 411, H-2101 Gödöllö, Hungary. Phone: 36 28 526 194. Fax: 36 28 526 145. E-mail: silhavy{at}abc.hu. Back

{ddagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back

{dagger} Present address: The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom. Back


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Journal of Virology, June 2005, p. 7217-7226, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.7217-7226.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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