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RESEARCH PAPER
1 Department of Botany and Plant Pathology, and 2 Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331, USA
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[Keywords: MicroRNA; RNA silencing; RNAi; virus-encoded suppressors; RISC]
Received March 8, 2004; revised version accepted March 31, 2004.
). These and other phenotypes are frequently associated with virus-encoded pathogenicity factors, many of which are suppressors of RNA silencing (Voinnet et al. 1999). RNA silencing functions as an adaptive immune response that restricts accumulation or spread of inducing viruses (Waterhouse et al. 2001). Suppressor proteins encoded by members of different virus families are distinct, suggesting that plant viruses evolved this counter-defensive mechanism independently on many occasions (Vaucheret et al. 2001; Tijsterman et al. 2002).
RNA silencing during virus infection is triggered by double-stranded RNA (dsRNA) generated during the course of virus replication or by the activity of a cellular RNA-dependent RNA polymerase (Ahlquist 2002). Processing of dsRNA by DICER or DICER-LIKE enzymes results in heterogeneous short interfering RNAs (siRNAs) of 21–25 nucleotides (Finnegan and Matzke 2003). siRNAs incorporate into RNA-induced silencing complexes (RISCs; Zamore et al. 2000; Elbashir et al. 2001b) and provide guide functions for sequence-specific ribonucleolytic activity. Protein components of RISC include ARGONAUTE family members, nucleases, and other factors (Hannon 2002).
microRNAs (miRNAs, 
21 nucleotides) are chemically similar to siRNAs, but they arise from processing of imperfect hairpin-forming RNA precursors transcribed from miRNA genes (Ambros et al. 2003). miRNA processing occurs by a multistep mechanism involving DICER (DICER-LIKE1 [DCL1] in Arabidopsis) activity to excise the miRNA from the hairpin stem (Bartel 2004). miRNAs function as negative regulators of target mRNAs through directing either site-specific cleavage by RISCs or translational repression (Bartel 2004). In plants, miRNAs target a wide range of mRNAs encoding transcription factors required for development (Park et al. 2002; Rhoades et al. 2002; Palatnik et al. 2003). These include factors required for meristem identity and maintenance, patterning, cell division, hormone signal ing, and developmental timing. In addition, plant miRNAs also target mRNAs encoding miRNA metabolic factors and factors of unknown function (Rhoades et al. 2002; Xie et al. 2003). Loss of miRNA biogenesis or activity in Arabidopsis results in pleiotropic defects during embryonic, vegetative, and reproductive development (Park et al. 2002; Schauer et al. 2002; Kasschau et al. 2003).
Despite differences in precursor structure for siRNAs (perfectly paired dsRNA) and miRNAs (imperfect hairpins), functional siRNA and miRNA molecules are incorporated into RISC through an asymmetric strand selection process. Precursor processing yields a duplex intermediate containing perfectly (siRNA) or imperfectly (miRNA) paired strands with two unpaired bases at each 3' end (Elbashir et al. 2001a). This duplex intermediate is unwound prior to, or during, incorporation of one strand into RISCs. Strand asymmetry depends on the strength of base-pair interactions at each end of the duplex, with the molecule containing the 5' end participating in the weakest interaction preferentially used (Khvorova et al. 2003; Schwarz et al. 2003). The nonselected strand (siRNA* or miRNA*) is rapidly degraded (Bartel 2004).
We previously showed that the Turnip mosaic virus (TuMV) silencing suppressor, P1/HC-Pro, interferes with miRNA-guided regulation of at least 10 target mRNAs in infected or transgenic Arabidopsis plants. The suppressor also caused multiple developmental defects, including some resembling those associated with dcl1 mutants (Kasschau et al. 2003). Here, we show that interference with miRNA-guided target cleavage/degradation and development in Arabidopsis is a general property of several, unrelated silencing suppressors encoded by evolutionarily distinct viruses. Through analysis of miRNA processing intermediates and suppressor-containing complexes in vivo, three of these suppressors were shown to inhibit the RISC assembly pathway (by two distinct mechanisms) after DCL1-catalyzed formation of miRNA/miRNA* duplexes.
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), Tomato bushy stunt virus p19 (Voinnet et al. 1999; Silhavy et al. 2002), Turnip crinkle virus coat protein (CP; Qu et al. 2003; Thomas et al. 2003), and Cucumber mosaic virus 2b (Brigneti et al. 1998; Guo and Ding 2002) silencing suppressors were analyzed and compared with P1/HC-Pro (Anandalakshmi et al. 1998; Brigneti et al. 1998; Kasschau and Carrington 1998). These suppressors belong to evolutionarily and structurally unrelated protein families (Dolja and Koonin 1991; Koonin et al. 1991; Reed et al. 2003; Vargason et al. 2003). To facilitate detection and experimental manipulation, constructs were expressed by using Cauliflower mosaic virus 35S promoter and terminator sequences and engineered such that an influenza hemagglutinin (HA) epitope tag was added to the C terminus of each protein (Fig. 1A). The HA-tagged proteins were tested for functionality as RNA silencing suppressors using Agrobacterium-mediated transient assays in Nicotiana benthamiana leaves (Johansen and Carrington 2001). This assay measures the ability of a suppressor to inhibit silencing of the GFP sequence triggered by an inverted-repeat hairpin construct. All five suppressors inhibited silencing in transient assays, although 2b was relatively weak (Supplementary Fig. S1A–C). Only CP inhibited siRNA accumulation (Supplementary Fig. S1D). Functionality of the HA-tagged proteins was also tested in side-by-side comparisons to nontagged forms of each suppressor. Inhibition of silencing by each HA-tagged protein was comparable to inhibition by the corresponding nontagged form (data not shown).
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To determine if induction of developmental phenotypes is a general property of silencing suppressors, the five HA-tagged constructs, as well as empty vector, were introduced as transgenes into Arabidopsis Col-0 plants (Fig. 1B). Growth and development parameters were analyzed by using a minimum of 58 primary transformants expressing each suppressor.
Plants expressing P1/HC-Pro, p21, p19, and CP exhibited moderate to severe defects in leaf and rosette development (Fig. 1C). Rosette leaves (growth stage 5.1 according to the scale described by Boyes et al. [2001]) were narrow, lobed or serrated, or curled (Fig. 1C; Supplementary Table S1). Rosette diameter and leaf area were reduced, as were the weight of total aerial tissue and the length of the primary bolt (Supplementary Fig. S2). Plants expressing CP displayed leaf and rosette phenotypes that were generally mild compared with those in plants expressing P1/HC-Pro, p21, and p19. Plants expressing 2b were indistinguishable from vector-transformed plants with respect to leaf morphology (Fig. 1C), although modest reductions in aerial tissue weight, leaf area, and rosette diameter were detected (Supplementary Fig. S2A).
Plants expressing P1/HC-Pro, p21, p19, and CP also had obvious flower phenotypes and were generally infertile (Fig. 1D; Supplementary Fig. S2B). They failed to release pollen and, in the case of P1/HC-Pro-expressing plants, had split or nonfused carpels (Supplementary Table S1; Supplementary Fig. S2B). P1/HC-Pro, p21, and CP expressing plants had narrow and unusually long sepals, whereas plants expressing p19 had short sepals (Fig. 1D; Supplementary Fig. S2B; Supplementary Table S1). In all four cases, however, organs in the internal whorls were exposed prior to opening (Fig. 1D). At a low frequency, plants expressing P1/HC-Pro, p19, or 2b contained additional trichomes on abaxial or adaxial sepals (Supplementary Table S1). Plants expressing 2b were fertile, although they contained fewer flowers per plant compared with control plants (Supplementary Fig. S2B).
Therefore, the correlation between strong RNA silencing suppressor activity (as measured in the hairpin dsRNA silencing assay) and strong developmental phenotypes in Arabidopsis is relatively high. The protein with weak silencing suppressor activity in the transient assay (2b) caused only mild developmental abnormalities in transgenic plants. The data also support the hypothesis that pathogenicity associated with these proteins involves, at least partly, interference with growth and development during virus infection.
Interference with miRNA-guided mRNA cleavage by three silencing suppressors
Developmental abnormalities in Arabidopsis plants expressing P1/HC-Pro correlate with inhibition of miRNA-guided target regulation (Kasschau et al. 2003). To determine if this is a general property of strong RNA silencing suppressors, the levels of three mRNAs (ARF8, ARF10, and SCL6-IV) that are normally under negative regulation by miRNAs (miR167, miR160, and miR171, respectively) were measured in transgenic plants expressing each of the suppressors and compared relative to the levels in vector-transformed plants. The ARF8 and ARF10 mRNAs accumulated to higher levels in plants expressing P1/HC-Pro, p21, and p19 (Fig. 2A,B, lanes 3–8). Also, the ratio of SCL6-IV full-length to 3' cleavage product detected by blot assay was higher in plants expressing P1/HC-Pro, p21, and p19 (Fig. 2C, lanes 3–8). In plants expressing CP, only slight increases in ARF8 and ARF10 mRNA levels, and no change in the ratio of SCL6-IV RNAs, were measured (Fig. 2, lanes 9,10). Plants expressing 2b showed no increases in the levels or ratios of miRNA targets (Fig. 2, lanes 11,12).
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; Rhoades et al. 2002; Kasschau et al. 2003). Increased accumulation of these target mRNAs in the presence of silencing suppressors could conceivably be the result of lower levels of miRNAs, although this would be inconsistent with previous studies (Mallory et al. 2002; Kasschau et al. 2003). Indeed, there was no consistent decrease among the three miRNAs caused by any of the suppressors (Fig. 3).
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The P1/HC-Pro, p21, and p19 silencing suppressors interfered with siRNA-guided and miRNA-guided target cleavage but not with siRNA or miRNA formation, suggesting that inhibition is likely associated with RISC assembly or RISC activity (Figs. 2, 3; Supplementary Fig. S1B–D). Assembly of RISCs containing miRNA requires unwinding of the miRNA/miRNA* duplex intermediate, followed by (or concurrent with) incorporation of miRNA into RISC and degradation of miRNA* (Khvorova et al. 2003; Schwarz et al. 2003). If the strong suppressors inhibit RISC assembly at the point of unwinding miRNA/miRNA*, we predicted that miRNA* species would accumulate specifically in plants expressing these proteins. The miR167b*, miR160c*, and miR171* levels were below the detection limit in vector-transformed plants (Fig. 3, lanes 1,2). However, they were each detected in plants expressing P1/HC-Pro, p21, and p19 (Fig. 3, lanes 3–8), with the highest levels of each accumulating in P1/HC-Pro-expressing plants. In contrast, each miRNA* accumulated to relatively low or nondetectable levels in plants expressing CP or 2b proteins (Fig. 3, lanes 9–12). Therefore, strong suppression of miRNA-guided target cleavage correlated with accumulation of normally labile miRNA* species, suggesting that the strong suppressors interfered with unwinding of miRNA/miRNA* duplexes.
Interaction of silencing suppressors and miRNA/miRNA* in vivo and in vitro
Tombusvirus p19 dimers bind duplex siRNAs (Silhavy et al. 2002; Vargason et al. 2003; Ye et al. 2003). This led to a competitive inhibition model in which p19 interferes with RISC assembly by sequestering siRNA intermediates (Lakatos et al. 2004). We tested the hypothesis that each of the three strong suppressors interacted with miRNA/miRNA* complexes in vivo by coimmunoprecipitation (co-IP) assays using anti-HA monoclonal antibody. Precipitated complexes from inflorescence (P1/HC-Pro and p21) and total aerial (P1/HC-Pro, p21, and p19) tissues were analyzed for suppressor protein, miRNAs, and miRNAs*. As controls, IP assays were done by using extracts from vector-transformed plants and by using a heterologous monoclonal antibody mixture specific for NIa and NIb proteins of Tobacco etch virus.
Each suppressor protein was detected in total extracts (IP inputs) and in immunoprecipitated material using HA antibody, but not in IP fractions using NIa/NIb antibody (Fig. 4A,B, top). The input extracts from the suppressor-expressing plants contained each of the miRNAs and miRNAs* (miR167, miR171, miR167b*, miR171*, and miR160c*) analyzed in the respective tissues (Fig. 4A [lanes 4,7], B [lanes 4,7,10]). No miRNAs or miRNAs* were detected in any IP fractions from plants expressing P1/HC-Pro (Fig. 4A,B, lanes 5,6). In contrast, each miRNA and miRNA*, but no 5S rRNA, coimmunoprecipitated with p21 in both tissue types (Fig. 4A,B, lane 9). Similarly, miR167, miR167b*, miR171, and miR160c* each specifically coimmunoprecipitated with p19 in aerial tissue extracts (Fig. 4B, lane 12), although the proportion of small RNA in the IP fractions relative to the input extract was less using p19 expressing plants compared to p21 expressing plants. In vector-transformed plant extracts, miR167, miR171, and 5S rRNA were detected (Fig. 4A,B, lane 1), but none were present in the IP fractions (Fig. 4, lanes 2,3). Faster migrating forms of some miRNAs and miRNAs* were detected in total extracts or IP fractions in the presence of P1/HC-Pro or p19 (Fig. 4, lanes 4,9,12). Truncated miRNAs in the presence of p19 were also observed previously by others (Papp et al. 2003).
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21 nucleotides) coimmunoprecipitated with p19, as expected from the well-characterized binding properties (Fig. 4C, lane 9; Silhavy et al. 2002; Vargason et al. 2003; Ye et al. 2003). No siRNA was detected in IP fractions from tissue expressing GFP siRNA and P1/HC-Pro, although the efficiency of IP of P1/HC-Pro after transient expression was relatively low (data not shown). We tested the hypothesis that p21 binds small RNA duplexes directly by electrophoretic mobility shift assays by using purified recombinant p21 and synthetic miR171, miR171*, miR171/miR171* duplex, or an siRNA duplex. The miR171/miR171* duplex contained two mismatched positions and two G:U base-pairs. In the absence of p21 protein, each single-stranded and duplex RNA migrated to near the bottom of the gel (Fig. 4D, lanes 1,3,5,7). In the presence of p21, slower-migrating complexes were detected by using the siRNA duplex and miR171/miR171* duplex (Fig. 4D, lanes 2,8). No p21 complexes were detected using single-stranded miR171 or miR171* (Fig. 4D, lanes 4,6). These data indicate that p21 interacts directly with small RNA duplexes, regardless of whether or not the duplex contains perfectly complementary (siRNA) or mismatched (miRNA) strands. By using DNA oligonucleotides corresponding to a duplex siRNA, low levels of p21 complex were detected (<10% of the level detected using the RNA duplexes; data not shown).
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). Cytoplasmic localization of p21 is in accord with this hypothesis (Reed et al. 2003).
Although P1/HC-Pro was the most effective miRNA pathway suppressor tested, we obtained no evidence that it interacts with miRNAs or miRNAs* in vivo. However, it clearly inhibited miRNA* turnover, again suggesting that miRNA/miRNA* unwinding and RISC assembly was suppressed. One possibility is that the interaction between P1/HC-Pro and miRNA/miRNA* is too weak to be detected by using an IP assay. However, one would not expect the suppressor causing the strongest effects on inhibition of miRNA-guided target cleavage, promotion of miRNA* accumulation, and development to interact most weakly with small RNAs if direct binding was the mode of action. We suggest an alternative model in which P1/HC-Pro interferes with a protein (Fig. 5, protein X) or complex associated with miRNA/miRNA* duplexes (as well as siRNA duplexes). P1/HC-Pro might also inhibit the miRNA/miRNA* complex indirectly by suppressing a factor required for production of one or more components associated with the complex. In either case, interaction between P1/HC-Pro and a pathway component would prevent unwinding and assembly of active RISC. Among the known RNAi factors from animals, the dsRNA-binding protein R2D2 plays a role in transfer of siRNAs between DICERs and RISCs (Liu et al. 2003). A functionally equivalent protein in plants, however, has yet to be identified.
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), but not DCL1 required for miRNA processing. Nevertheless, CP had small but measurable effects on miRNA-directed target cleavage, which may partly explain the developmental consequences of CP expression in Arabidopsis. The CMV 2b protein had very little effect on miRNA-guided functions and development in Arabidopsis, and may function by mechanisms that are quite distinct from the others analyzed (Brigneti et al. 1998; Guo and Ding 2002). Finally, is there a physiologic advantage to viruses that interfere with miRNA-guided gene regulation? If miRNAs are required for defense responses or for expression of genes required for susceptibility to a broad range of viruses, then evolution of virus-encoded miRNA inhibition functions can be easily rationalized. However, in view of the functions of known miRNA target genes in plants, and the lack of effects of dcl1 (miRNA-deficient) mutations on virus susceptibility (Z. Xie and J.C. Carrington, unpubl.), no evidence for such requirements is available. It seems more likely that interference with the miRNA pathway by suppressors such as P1/HC-Pro, p21, and p19 is a consequence of inhibition of shared steps in the silencing pathways involving siRNAs for antiviral defense and miRNAs for development.
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Materials and methods |
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Suppressors were derived from the following viruses: P1/HC-Pro, TuMV; p21, Beet yellows virus; p19, Tomato bushy stunt virus; CP, Turnip crinkle virus; and 2b, Cucumber mosaic virus. Coding sequences for each suppressor were amplified by PCR using a 3' primer that added a C-terminal HA epitope (primer sequences available upon request). Resulting DNA fragments were cloned into a modified pCB-302 plant transformation vector (Peng and Dolja 2000) using NcoI and XbaI sites. Suppressor constructs contained a Cauliflower mosaic virus 35S promoter anda5' nontranslated leader sequence from Tobacco etch virus (Carrington and Freed 1990). Resulting constructs were introduced into Agrobacterium tumefaciens strains GV2260 and GV3101.
Transgenic plants
Arabidopsis thaliana Col-0 plants were transformed by the vacuum-infiltration method (Clough and Bent 1998) by using A. tumefaciens GV3101 carrying constructs for expression of epitope-tagged P1/HC-Pro, p21, p19, CP, 2b, or the empty expression vector. Seed from primary transformants was grown under selection for phosphoinothricin resistance in a standard greenhouse.
Protein and RNA blot analysis
Protein extracts were prepared and normalized for SDS-PAGE by using the Bradford assay (Bio-Rad). Immunoblot analysis of total protein samples (10 µg) was done using anti-HA-peroxidase conjugate (Roche). Total RNA was extracted from independent pools of leaf or inflorescence tissues by using Trizol reagent (Johansen and Carrington 2001). Low-molecular-weight RNA was isolated with RNA/DNA Midi Kits (Qiagen). Blot hybridization of normalized total or low-molecular-weight RNA (5 µg) was done as described (Llave et al. 2002a), and hybridization intensities were quantified by using a PhosphorImager or Scanning Densitometer (Molecular Dynamics). 32P-radiolabeled probes for mRNAs were synthesized by random-priming of cloned genomic sequences (Feinberg and Vogelstein 1983). 32P-Radiolabeled miRNA probes were produced by end-labeling of complementary oligonucleotides. Sequences for miRNA* probes were predicted from miRNA precursor structures (Llave et al. 2002a; Reinhart et al. 2002). Accumulation of miRNA-targeted mRNAs was normalized to levels of mRNA from the control gene TyrAT (At 2 g 20610). Accumulation of SCL6-IV full-length mRNA ("a" form) relative to the 3' cleavage product ("b" form) was represented as a ratio as described (Llave et al. 2002b).
Immunoprecipitation
Aerial or inflorescence tissue from transgenic Arabidopsis plants (5 wk old), and leaf tissue from Agrobacterium-infiltrated N. benthamiana, were ground under liquid nitrogen and homogenized in 5 mL/g lysis buffer (50 mM Tris-HCl at pH 7.4, 100 mM KCl, 2.5 mM MgCl2, 0.1% NP-40, and 2x complete protease inhibitor cocktail; Roche). Cell debris was pelleted by centrifugation for 15 min at 9500 x g. The clarified lysate was precleared for 20 min at 4°C with 10 µL bed volume protein A-agarose (30 µg protein A) per milliliter. Precleared lysates were reacted with 4 µg anti-HA (Roche) or anti-NIa/NIb (Slade et al. 1989) per milliliter for 1 h at 4°C, then with 50 µL bed volume protein A-agarose (150 µg protein A) per milliliter for 3 h at 4°C. Precipitates were washed three times in lysis buffer and divided for protein and RNA analysis. Nucleic acid was recovered by treatment with 3 v proteinase K solution (100 mM Tris-HCl at pH 7.4, 10 mM EDTA, 150 mM NaCl, 2% SDS, and 0.2 µg/µL proteinase K) for 15 min at 65°C, extraction with saturated phenol and phenol:chloroform, and ethanol precipitation. For miRNA and miRNA* blot assays, 5 µg of RNA recovered from the input extract, or RNA from IP fractions representing 150 mg tissue, was used. Five micrograms of RNA recovered from input extracts corresponded to the equivalent of 5 mg tissue. As a control, blots were stripped and rehybridized with an oligonucleotide probe specific to 5S rRNA.
For IP assays using Agrobacterium-infiltrated leaves, GFP silencing was induced as described (Johansen and Carrington 2001). A. tumefaciens cultures were injected at the following concentrations: 35S:dsGFP-FAD2, O.D.600 = 0.1; 35S:vector,35S:P1/HC-Pro, 35S:p21, or 35S:p19, O.D.600 = 0.9. Infiltrated tissue was harvested and processed 72 h postinjection. 32P-Radiolabeled probes for siRNAs were synthesized by random-priming of cloned smGFP sequence (Johansen and Carrington 2001).
Electrophoretic mobility shift assays
The p21 coding sequence was amplified by PCR using a 5' primer that added an N-terminal hexahistidine tag (primer sequences available upon request). Resulting DNA fragments were cloned into pET16b (Novagen) by using NcoI and BamHI sites. The resulting construct was introduced into Escherichia coli strain BL21(DE3) (Novagen), and protein was expressed and purified under native conditions by using Ni-NTA resin (Qiagen) following the recommendations of the manufacturer.
Probes were synthesized by end-labeling RNA oligonucleotides miR171 (5'-UGAUUGAGCCGCGCCAAUAUC-3'), miR171* (5'-UAUUGGCCUGGUUCACUCAGA-3'), siRNA (5'-CGUAC GCGGAAUACUUCGAUU-3'), or siRNA* (5'-UCGAAGUAUUC CGCGUACGUG-3'; Dharmacon) using [32P]ATP. Duplexes were formed during annealing reactions similar to those described by others (Silhavy et al. 2002). Formation of duplexes was confirmed by electrophoresis mobility assays. p21 complex formation reactions contained 1 µM p21 and 0.1 µM oligonucleotide in 10 µL binding buffer (0.1 M KCL, 25 mM HEPES, and 10 mM DTT at pH 7.6; Ye et al. 2003) and were done for 15 min at 23°C.
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Article published online ahead of print. Article and publication date are at http://www.genesdev.org/cgi/doi/10.1101/gad.1201204.
3 Present address: Biochemistry & Biophysics, Texas A&M University, College Station, TX 77843, USA. 
E-MAIL carrington{at}cgrb.oregonstate.edu; FAX (541) 737-3045.
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References |
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Ambros, V., Bartel, B., Bartel, D.P., Burge, C.B., Carrington, J.C., Chen, X., Dreyfuss, G., Eddy, S.R., Griffiths-Jones, S., Marshall, M., et al. 2003. A uniform system for microRNA annotation. RNA 9: 277–279.
Anandalakshmi, R., Pruss, G.J., Ge, X., Marathe, R., Smith, T.H., and Vance, V.B. 1998. A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. 95: 13079–13084.
Bartel, D. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116: 281–297.[CrossRef][Medline]
Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman, N.E., Davis, K.R., and Görlach, J. 2001. Growth stage-based phenotypic analysis of Arabidopsis: A model for high throughput functional genomics in plants. Plant Cell 13: 1499–1510.
Brigneti, G., Voinnet, O., Wan-Xiang, L., Ding, S.W., and Baulcombe, D.C. 1998. Viral pathogenicity determinants are suppressors of transgene silencing. EMBO J. 17: 6739–6746.[CrossRef][Medline]
Carrington, J.C. and Freed, D.D. 1990. Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region. J. Virol. 64: 1590–1597.
Clough, S.J. and Bent, A.F. 1998. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743.[CrossRef][Medline]
Dolja, V.V. and Koonin, E.V. 1991. Phylogeny of capsid proteins of small icosahedral RNA plant viruses. J. Gen. Virol. 72: 1481–1486.
Elbashir, S.M., Lendeckel, W., and Tuschl, T. 2001a. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes & Dev. 15: 188–200.
Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. 2001b. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20: 6877–6888.[CrossRef][Medline]
Feinberg, A.P. and Vogelstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 257: 8569–8572.
Finnegan, E.J. and Matzke, M.A. 2003. The small RNA world. J. Cell. Sci. 116: 4689–4693.
Guo, H.S. and Ding, S.W. 2002. A viral protein inhibits the long range signaling activity of the gene silencing signal. EMBO J. 21: 398–407.[CrossRef][Medline]
Hannon, G.J. 2002. RNA interference. Nature 418: 244–251.[CrossRef][Medline]
Hull, R. 2001. Matthews' plant virology, 4th ed. Academic Press, San Diego, CA.
Johansen, L.K. and Carrington, J.C. 2001. Silencing on the spot: Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol. 126: 930–938.
Kasschau, K.D. and Carrington, J.C. 1998. A counter-defensive strategy of plant viruses: Suppression of posttranscriptional gene silencing. Cell 95: 461–470.[CrossRef][Medline]
Kasschau, K.D., Xie, Z., Allen, E., Llave, C., Chapman, E.J., Krizan, K.A., and Carrington, J.C. 2003. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev. Cell 4: 205–217.[CrossRef][Medline]
Khvorova, A., Reynolds, A., and Jayasena, S.D. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 115: 209–216.[CrossRef][Medline]
Koonin, E.V., Choi, G.H., Nuss, D.L., Shapira, R., and Carrington, J.C. 1991. Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses. Proc. Natl. Acad. Sci. 88: 10647–10651.
Lakatos, L., Szittya, G., Silhavy, D., and Burgyán, J. 2004. Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J. 23: 876–884.[CrossRef][Medline]
Liu, Q., Rand, T., Kalidas, S., Du, F., Kim, H.E., Smith, D.P., and Wang, X. 2003. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301: 1921–1925.
Llave, C., Kasschau, K.D., Rector, M.A., and Carrington, J.C. 2002a. Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605–1619.
Llave, C., Xie, Z., Kasschau, K.D., and Carrington, J.C. 2002b. Cleavage of Scarecrow-like mRNA targets is directed by a class of Arabidopsis miRNA. Science 297: 2053–2056.
Mallory, A.C., Reinhart, B.J., Bartel, D., Vance, V.B., and Bowman, L.H. 2002. A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco. Proc. Natl. Acad. Sci. 99: 15228–15233.
Palatnik, J.F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J.C., and Weigel, D. 2003. Control of leaf morphogenesis by microRNAs. Nature 425: 257–263.[CrossRef][Medline]
Papp, I., Mette, M.F., Aufsatz, W., Daxinger, L., Schauer, S.E., Ray, A., van der Winden, J., Matzke, M., and Matzke, A.J. 2003. Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiol. 132: 1382–1390.
Park, W., Li, J., Song, R., Messing, J., and Chen, X. 2002. CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12: 1484–1495.[CrossRef][Medline]
Peng, C.W. and Dolja, V.V. 2000. Leader proteinase of the beet yellows closterovirus: Mutation analysis of the function in genome amplification. J. Virol. 74: 9766–9770.
Qu, F., Ren, T., and Morris, T.J. 2003. The coat protein of Turnip crinkle virus suppresses posttranscriptional gene silencing at an early step. J. Virol. 77: 511–522.
Reed, J.C., Kasschau, K.D., Prokhnevsky, A.I., Gopinath, K., Pogue, G.P., Carrington, J.C., and Dolja, V.V. 2003. Suppressor of RNA silencing encoded by Beet yellows virus. Virology 306: 203–209.[CrossRef][Medline]
Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., and Bartel, D.P. 2002. MicroRNAs in plants. Genes & Dev. 16: 1616–1626.
Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B., and Bartel, D.P. 2002. Prediction of plant microRNA targets. Cell 110: 513–520.[CrossRef][Medline]
Schauer, S.E., Jacobsen, S.E., Meinke, D.W., and Ray, A. 2002. DICER-LIKE1: Blind men and elephants in Arabidopsis development. Trends Plant Sci. 7: 487–491.[CrossRef][Medline]
Schwarz, D.S., Hutvágner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P.D. 2003. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115: 199–208.[CrossRef][Medline]
Silhavy, D., Molnár, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M., and Burgyán, J. 2002. A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J. 21: 3070–3080.[CrossRef][Medline]
Slade, D.E., Johnston, R.E., and Dougherty, W.G. 1989. Generation and characterization of monoclonal antibodies reactive with the 49-kDa proteinase of tobacco etch virus. Virology 173: 499–508.[CrossRef][Medline]
Thomas, C.L., Leh, V., Lederer, C., and Maule, A.J. 2003. Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306: 33–41.[CrossRef][Medline]
Tijsterman, M., Ketting, R.F., and Plasterk, R.H. 2002. The genetics of RNA silencing. Annu. Rev. Genet. 36: 489–519.[CrossRef][Medline]
Vargason, J.M., Szittya, G., Burgyán, J., and Tanaka Hall, T.M. 2003. Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115: 799–811.[CrossRef][Medline]
Vaucheret, H., Béclin, C., and Fagard, M. 2001. Post-transcriptional gene silencing in plants. J. Cell. Sci. 114: 3083–3091.
Voinnet, O., Pinto, V.M., and Baulcombe, D.C. 1999. Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants. Proc. Natl. Acad. Sci. 96: 14147–14152.
Waterhouse, P.M., Wang, M.B., and Lough, T. 2001. Gene silencing as an adaptive defence against viruses. Nature 411: 834–842.[CrossRef][Medline]
Xie, Z., Kasschau, K.D., and Carrington, J.C. 2003. Negative feedback regulation of Dicer-like1 (DCL1) in Arabidopsis by microRNA-guided mRNA degradation. Curr. Biol. 13: 784–789.[CrossRef][Medline]
Xie, Z., Johansen, L.K., Gustafson, A.M., Kasschau, K.D., Lellis, A.D., Zilberman, D., Jacobsen, S.E., and Carrington, J.C. 2004. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2: E104 (Epub ahead of print).[CrossRef][Medline]
Ye, K., Malinina, L., and Patel, D.J. 2003. Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426: 874–878.[CrossRef][Medline]
Zamore, P.D. 2004. Plant RNAi: How a viral silencing suppressor inactivates siRNA. Curr. Biol. 14: R198–R200.[CrossRef][Medline]
Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. 2000. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33.[CrossRef][Medline]
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