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Journal of Virology, June 2005, p. 7410-7418, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7410-7418.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Adenosine Kinase Inhibition and Suppression of RNA Silencing by Geminivirus AL2 and L2 Proteins

Hui Wang,1 Kenneth J. Buckley,2 Xiaojuan Yang,1 R. Cody Buchmann,1 and David M. Bisaro1*

Department of Molecular Genetics, Plant Biotechnology Center,2 Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, Ohio 432101

Received 18 December 2004/ Accepted 13 February 2005


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ABSTRACT
 
Most plant viruses are initiators and targets of RNA silencing and encode proteins that suppress this adaptive host defense. The DNA-containing geminiviruses are no exception, and the AL2 protein (also known as AC2, C2, and transcriptional activator protein) encoded by members of the genus Begomovirus has been shown to act as a silencing suppressor. Here, a three-component, Agrobacterium-mediated transient assay is used to further examine the silencing suppression activity of AL2 from Tomato golden mosaic virus (TGMV, a begomovirus) and to determine if the related L2 protein of Beet curly top virus (BCTV, genus Curtovirus) also has suppression activity. We show that TGMV AL2, AL21-100 (lacking the transcriptional activation domain), and BCTV L2 can all suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene when silencing is induced by a construct expressing an inverted repeat GFP RNA (dsGFP). We previously found that these viral proteins interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance. Using the GFP-dsGFP system, we demonstrate here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins. In addition, AL2/L2 suppression phenotypes and nucleic acid binding properties are shown to be different from those of the RNA virus suppressors HC-Pro and p19. These findings provide strong evidence that ADK activity is required to support RNA silencing, and indicate that the geminivirus proteins suppress silencing by a novel mechanism that involves ADK inhibition. Further, since AL21-100 is as effective a suppressor as the full-length AL2 protein, activation and silencing suppression appear to be independent activities.


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INTRODUCTION
 
In eukaryotic cells, homology-dependent silencing that operates at the RNA level is involved in a number of fundamental processes, including cellular defense against viruses, control of transposon mobility, developmental gene regulation via micro-RNAs (miRNAs), de novo histone and DNA methylation, and the establishment of heterochromatin (8, 9, 60, 63). RNA silencing is triggered by double-stranded RNA (dsRNA), and a defining feature is the appearance of short interfering RNA (siRNA), 21- to 26-nucleotide (nt) dsRNA species homologous to the silenced gene (17, 68). These siRNAs are produced from inducing dsRNA by the action of RNase III-like enzymes called Dicer or Dicer-Like (12). In turn, the antisense strands of unwound siRNAs guide another RNase-containing complex, the RNA-induced silencing complex (RISC), to homologous single-stranded RNA (ssRNA) targets (usually mRNA) for degradation (19). Methylation of homologous nuclear DNA corresponding to transcribed regions also occurs, although the role of methylation in RNA silencing is unclear (3, 4, 26).

RNA silencing acts as an antiviral defense in both plant and animal (insect) cells, and viruses are both inducers and targets of the system and thus determine its specificity (31, 32, 55, 59, 65). To counter this adaptive defense, viruses from different families have elaborated a variety of apparently unrelated suppressor proteins that affect different, and possibly multiple, steps in the silencing pathway (33, 61). For example, P1/HC-Pro (HC-Pro) of Tobacco etch virus (TEV) and related potyviruses can reverse established silencing in plants and suppress local silencing of reporter genes in transient assays (2, 6, 27, 35). HC-Pro interacts with a cellular protein (rgs-CaM) that is itself a silencing suppressor, suggesting that the viral protein stimulates an endogenous regulatory pathway (1). It also at least partially inhibits dsRNA processing by Dicer (14, 37). In addition, HC-Pro appears to block unwinding of siRNA/siRNA* duplexes, thereby preventing the incorporation of targeting information into RISC (10). In contrast, the p19 protein of Cymbidium ringspot virus and related tombusviruses cannot reverse established silencing, although it blocks the production of a systemic silencing signal in plants and can suppress local silencing in transient assays. The activity of p19 is due to its ability to bind and sequester siRNAs, which could also prevent incorporation of siRNA into RISC (29, 46, 58, 67). Interestingly, HC-Pro and p19 impact both siRNA and miRNA metabolism, underscoring the similar and overlapping natures of these pathways (10, 14, 28). Further study of HC-Pro, p19, and other viral suppressor proteins will no doubt provide additional insight into the molecular mechanisms of RNA silencing and related processes.

The geminiviruses package ssDNA that replicates in the host cell nucleus through dsDNA intermediates that assemble into minichromosomes (15, 20, 41). Geminiviruses do not encode polymerases but specify multifunctional proteins that provide a cellular environment favorable to replication, initiate specific steps in replication and/or transcription, potentiate virus spread within and between hosts, and suppress host defenses. For example, the AL2 protein of Tomato golden mosaic virus (TGMV; genus Begomovirus) is a transcription factor required for the expression of late viral genes (49-51). The 15-kDa AL2 protein (also known as AC2, C2, or transcriptional activator protein) has a C-terminal activation domain that is functional in plant, yeast, and mammalian cells (22). AL2 is also a pathogenicity factor, and homologues from several begomoviruses have been shown to reverse RNA silencing in plants and to suppress local silencing in transient assays (56, 57, 61). In addition, TGMV AL2 and the related L2 protein of Beet curly top virus (BCTV; genus Curtovirus) condition a virus-nonspecific enhanced-susceptibility phenotype in transgenic plants which is attributable to their ability to inactivate SNF1-related kinase (21, 52). AL2 and L2 also interact with and inactivate adenosine kinase (ADK), which phosphorylates adenosine to produce 5'-AMP (64). Because AMP can stimulate SNF1 activity, the inactivation of SNF1 and ADK by AL2/L2 may represent a dual mechanism to counter SNF1-mediated antiviral responses.

ADK is generally considered to be a housekeeping enzyme involved in adenosine salvage. More recently, it has also been shown to play a key role in sustaining the methyl cycle and S-adenosylmethionine-dependent methyltransferase activity (30, 39, 45, 66). In yeast, methylation deficiency is the primary defect of ADK-null mutants. ADK deficiency also reduces methyltransferase activity in plants, and observational evidence suggests that this can compromise the maintenance of RNA silencing (39, 64).

In this report, a transient system is used to demonstrate that TGMV AL2 protein and the related L2 protein from the curtovirus BCTV can suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene. We also demonstrate that inhibiting cellular ADK activity causes similar silencing suppression, providing evidence that the TGMV AL2 and BCTV L2 proteins counter RNA silencing by reducing ADK activity. We further show that the AL2 and L2 proteins operate by mechanisms which differ from those of HC-Pro and p19.


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MATERIALS AND METHODS
 
Delivery of RNA silencing targets, inducers, and suppressors. Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana plants or N. benthamiana line 16c containing a GFP transgene (44) (provided by David Baulcombe, Sainsbury Laboratory) was based on the three-component system described by Johansen and Carrington (25). Equal volumes of A. tumefaciens cultures (optical density at 600 nm = 1) harboring plasmids designed to express the silencing target (GFP), the silencing inducer (an inverted repeat GFP RNA [dsGFP]), or a silencing suppressor or control were mixed and coinfiltrated to the underside of leaves using a 1-ml syringe. In some experiments, the ADK inhibitor A-134974 (38) (Sigma) was mixed with bacterial cultures to final concentrations of 0.01 to 100 µM just prior to infiltration. GFP fluorescence was observed using a 100-W, longwave UV lamp (Blak-Ray Model B 100YP; UV Products). Photographs were taken with UV and yellow filters and uniformly processed using Adobe Photoshop. Tissue was harvested from infiltration zones and used for RNA and protein isolation. RNA analysis is described below. ADK activity levels in protein extracts were measured as described previously (64).

Plasmids. In most cases, expression was driven from a pRTL2-derived cassette consisting of the enhanced Cauliflower mosaic virus 35S promoter, the TEV 5' nontranslated leader, and the 35S terminator (43). Constructs containing ß-glucuronidase (GUS), GFP, dsGFP, and HC-Pro were provided by James Carrington (Oregon State University) (25). Plasmids containing African cassava mosaic virus (ACMV) AC2, TGMV AL2 and AL21-100, and BCTV L2 were generated from earlier constructs (52) by inserting the genes into pRTL2 as NcoI-BglII fragments. Ti plasmid constructs were prepared by inserting the expression cassettes into HindIII-BamHI-digested pKJB5033 (a derivative of pBI121; Clontech; K. J. Buckley, unpublished). pBIN61-p19 was obtained from David Baulcombe (62).

The dsADK plasmid contained ~500 bp of Arabidopsis ADK2 (nt 306 to 814) (64) in the sense and antisense orientations separated by an intron containing GUS sequence and was expressed from the 35S promoter in pFGC1008 (47) (www.chromdb.org/plasmids/vectors2.html). ADK sequence was amplified by PCR from a cDNA library obtained from the Arabidopsis Biological Resource Center (Ohio State University). The 5' primer contained SpeI (italics) and AscI (underlined) restriction sites (5'-GAGACTAGTGGCGCGCCGGATGCTACAGCAGCTGG). The 3' primer contained BamHI (italics) and SwaI (underlined) restriction sites (5'-GAGGGATCCATTTAAATCCACTGGATCAGCGCCCTG). DNA fragments were digested with SpeI and BamHI or AscI and SwaI and sequentially inserted into pFGC1008. The dsSNF1 construct contained an SmaI-XbaI fragment from Arabidopsis SNF1 (AKIN 11; nt 1 to 1,028) (21) in the sense and antisense orientations separated by the GUS intron from pFGC1008 and was expressed from the 35S promoter in pBI121.

RNA analysis. RNA was obtained using TRIzol reagent (Invitrogen). For mRNA detection, 15 µg was fractionated on formaldehyde-agarose gels and transferred to nitrocellulose. For siRNA detection, 45 µg was separated by electrophoresis in 15% polyacrylamide gels containing 7 M urea in Tris-borate-EDTA buffer (pH 8.0), transferred to Hybond-NX membrane (Amersham), and UV cross-linked (1,200 µJ; Stratalinker; Stratagene). Probes were prepared by in vitro transcription using the Strip-EZ RNA kit (Ambion) in the presence of [{alpha}-32P]UTP. Antisense GFP probes were used to detect GFP mRNA, and probes of sense and antisense polarities were used separately or together for analysis of GFP-derived siRNA.

The Superscript One-Step RT-PCR kit (Invitrogen) was used both to clone and to detect SNF1 and ADK mRNA sequences from total cellular RNA obtained from N. benthamiana tissue. First, N. benthamiana ADK and SNF1 cDNA fragments were generated using a primer set designed from Arabidopsis ADK2 sequence (nt 223 to 860; 5' primer, 5'-ATGGGATCCATTGGAAAGGAC; 3' primer, 5'-TCACCTGCACCGTTGGTG) or from Arabidopsis SNF1 sequence (AKIN11, nt 598 to 1299; 5' primer, 5'-GATGTATGGAGTTGCGG; 3' primer, 5'-CCATCGACATTTCATGTT). The N. benthamiana ADK cDNA (GenBank accession number AY741533) and SNF1 cDNA (GenBank accession number AY919676) fragments were cloned into a T-vector (pKJB5048; Buckley, unpublished) and the sequences used to design primers for reverse transcription (RT)-PCR detection of ADK or SNF1 mRNA in RNA preparations from infiltration zones. Primers for either ADK (5' primer, 5'-GGGAGAAAATGAAGAACAATGC; 3'-GGCAACGGTATTACAGGGAAC) or SNF1 (5' primer, 5'-GCTCTTCTCTGTGGCACCCTTC; 3'-CACACATTTAGTTCTTGCAGAGC) and 18S rRNA (internal control) were used in each reaction. The rRNA primer set was derived from Arabidopsis 18S rRNA sequence (nt 3 to 1293; 5' primer, 5'-CCTGGTTGATCCTGCCAGTAG; 3' primer, 5'-ACCAACTAAGAACGGCCATGC). Equivalent samples, normalized by the amount of 18S rRNA product, were loaded on agarose gels and subjected to DNA gel blot hybridization analysis using N. benthamiana ADK or SNF1 sequences as probes. Probes were prepared using the Rediprime II random primer labeling system (Amersham) with [{alpha}-32P]dCTP.

Mobility shift analysis. Expression and purification of a glutathione S-transferase-AL2 fusion protein (GST-AL2) from Escherichia coli has been described previously (22). A GST-p19 expression construct was provided by Herman Scholthof (Texas A&M University), and GST-p19 protein and GFP siRNA were gifts from Yijun Qi, Xuehua Zhong, and Biao Ding (Ohio State University). The dsRNA starting material used to prepare the siRNA was generated by annealing in vitro transcription products synthesized from sense and antisense GFP strands in the presence of [{alpha}-32P]UTP. The internally labeled dsRNA was incubated in wheat germ extract essentially as previously described (53). Small RNA products were analyzed by electrophoresis in 15% polyacrylamide gels containing 8 M urea, and siRNA bands were eluted. The resulting siRNA probe was incubated with 1 µg GST, GST-p19, or GST-AL2 in 10 mM Tris-HCl (pH 7.5)-5 mM MgCl2-50 mM NaCl-66 mM KCl-5 mM dithiothreitol for 20 min at room temperature.

A DNA fragment (~500 bp) from pUC18 was end labeled using [{gamma}-32P]ATP and T4 DNA kinase. Labeled DNA was separated from unincorporated nucleotides by Sephadex G-50 chromatography, boiled for 5 min, and quick cooled on ice. The resulting ssDNA probe was incubated with 1 µg GST or GST-AL2 in 10 mM Tris-HCl (pH 7.5)-1 mM MgCl2-50 mM NaCl-0.5 mM EDTA-0.5 mM dithiothreitol-40 µg/ml poly(dI-dC) for 20 min at room temperature. Protein-nucleic acid complexes were resolved by electrophoresis in nondenaturing 5% polyacrylamide gels in Tris-borate-EDTA buffer at 4°C and visualized using a phosphorimager (Bio-Rad Molecular Imager FX).


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RESULTS
 
RNA silencing is suppressed by TGMV AL2 and BCTV L2 proteins. We set out to confirm that TGMV AL2, like its counterparts from begomoviruses such as ACMV, can suppress RNA silencing and to determine whether the AL2 transcriptional activation domain is necessary for this activity. We also asked whether the related BCTV (curtovirus) L2 protein, which unlike AL2 is not required for late viral gene expression, can also suppress RNA silencing (24, 48). This study employed a transient system that involves coinfiltration of N. benthamiana leaves with A. tumefaciens cultures harboring Ti plasmids that express GFP, an inverted repeat GFP RNA as a strong silencing inducer (dsGFP), and a test construct or control (25). Constructs expressing HC-Pro, p19, and ACMV AC2 (AL2) were used as positive controls, and a construct expressing GUS was a negative control. GFP expression in infiltration zones could be visualized under UV light as green or yellow fluorescence against a red background of chlorophyll fluorescence. The accumulation of GFP mRNA and silencing-derived, GFP-specific siRNAs in infiltrated tissues was assessed by RNA gel blot hybridization.

We found that AL2 and L2 can suppress GFP-directed silencing in the transient system, as judged by the appearance of green or yellow fluorescence (Fig. 1A) and by the increased accumulation of GFP mRNA in infiltration zones relative to the GUS negative control (Fig. 1B). However, GFP fluorescence was less intense with the geminivirus proteins than with HC-Pro and p19 (Fig. 1A). AL21-100, which lacks the transcriptional activation domain, suppressed silencing to about the same degree as full-length AL2. Consistent with visual inspection, the greatest GFP mRNA accumulation was observed with p19, while HC-Pro also supported robust but somewhat lower accumulation of this transcript. The geminivirus proteins behaved as weaker suppressors by comparison (Fig. 1B). However, they proved about as effective as p19 in reducing the accumulation of GFP-specific siRNAs over the 5-day course of the experiments (Fig. 1C). That these species are dsRNA was indicated by their ability to hybridize with probes of sense and antisense polarities (data not shown). In contrast, GFP-derived siRNA accumulated to relatively large amounts after 5 days in the HC-Pro and GUS treatments, although GUS did not support GFP mRNA accumulation. The same results were obtained in four independent experiments using RNA preparations from infiltrated wild-type N. benthamiana or N. benthamiana line 16-C plants, which express a GFP transgene (44). However, it should be noted that relatively weak activity coupled with inherent experimental variability precluded a precise ordering of the relative abilities of geminivirus proteins to suppress silencing. Our results with the positive control proteins HC-Pro, p19, and AC2 were similar to those obtained previously by others, although some differences in siRNA accumulation have been observed in different studies (see Discussion).



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  		FIG. 1. Silencing suppression by AL2 and L2 proteins and treatments that reduce ADK activity. N. benthamiana leaf tissues were coinfiltrated with Agrobacterium cultures delivering Ti plasmids expressing GFP, dsGFP and the indicated proteins, or inverted repeat RNA constructs. One treatment included the adenosine inhibitor A-134974 as the third component. (A) Representative leaves were photographed under UV light 5 days postinfiltration. Each half-leaf was infiltrated, and under these conditions GFP fluorescence appears green or yellow. The GFP control (GFP only) was infiltrated with a mixture of Agrobacterium cultures containing the GFP-expressing construct and empty vector. (B and C) Analysis of GFP mRNA and GFP-derived siRNAs, respectively. Total RNA was isolated from infiltration zones 3 and 5 days postinfiltration (dpi) and subjected to RNA gel blot hybridization analysis. In panel B, agarose gel-fractionated RNA (15 µg) was hybridized with a 32P-labeled antisense GFP riboprobe. In panel C, RNA (45 µg) was fractionated on a 15% polyacrylamide gel containing 7 M urea and hybridized with mixed-sense and antisense GFP riboprobes. The positions of 21- and 26-nt markers are indicated. rRNAs are shown as loading controls.

We concluded that TGMV AL2 and the related BCTV L2 protein similarly suppress silencing directed against a reporter gene, although their activity can be characterized as weak in comparison to p19 and HC-Pro. Further, since AL21-100 is as effective a suppressor as full-length AL2 protein, the activation and silencing suppression activities of AL2 appear to be independent.

RNA silencing is suppressed by an ADK inverted repeat construct. We next asked whether reducing expression of the two cellular kinases known to be inhibited by AL2 and L2 affected GFP silencing. We adopted an RNA interference approach to explore this question, based on the observation that components of the silencing machinery, and by analogy other proteins that might play a supporting role, can be at least partially silenced (5). Inverted repeat constructs designed to express dsRNA were prepared using Arabidopsis ADK2 (dsADK) and SNF1 kinase AKIN11 (dsSNF1) sequences. ADK and SNF1 genes are highly conserved among plants, mammals, and yeasts, and both of the Arabidopsis proteins can functionally complement corresponding yeast mutants (21, 64). Thus, it was considered likely that these sequences would trigger silencing directed against N. benthamiana ADK and SNF1. Subsequent measurements of ADK and SNF1 mRNA levels in infiltrated tissue showed that this occurred (see below).

As was apparent from GFP fluorescence and RNA gel blot assays, expression of dsADK as the third component of the transient system (GFP, dsGFP, and dsADK) suppressed GFP-directed silencing by supporting the accumulation of GFP mRNA and reducing the accumulation of GFP siRNA in a manner similar to the geminivirus proteins. In contrast, the dsSNF1 construct failed to suppress GFP silencing when used as the third component (Fig. 1).

We confirmed that the dsADK and dsSNF1 constructs reduced their target mRNA levels by using RT-PCR to examine RNA extracts from infiltrated tissue. PCRs contained ADK- or SNF1-specific primer pairs designed from cloned N. benthamiana cDNA fragments, as well as a primer set to amplify 18S rRNA as an internal control. Nucleotide sequence comparisons revealed extensive identity between the cloned ADK and SNF1 fragments from N. benthamiana and the corresponding Arabidopsis mRNAs (data not shown). As evident from the ethidium bromide-stained agarose gel shown in Fig. 2A, ADK mRNA levels were substantially reduced by the dsADK construct but remained unaffected by other treatments. DNA gel blot hybridization confirmed the identity of the PCR-generated ADK fragment (data not shown). A substantial reduction in SNF1 mRNA levels was likewise observed in tissue infiltrated with dsSNF1, but as noted previously, DNA gel blot hybridization was necessary to detect the PCR fragment corresponding to this low-abundance transcript, even in control tissue (21) (Fig. 2B).



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  		FIG. 2. Reduction of ADK and SNF1 mRNA levels by dsADK and dsSNF1 constructs. N. benthamiana leaf tissues were infiltrated with Agrobacterium cultures delivering Ti plasmids expressing GFP, dsGFP and the indicated proteins or vector control, inverted repeat RNA constructs, or the adenosine inhibitor A-134974 (100 µM). ADK mRNA or SNF1 mRNA in RNA extracts prepared from infiltration zones 5 days postinfiltration was detected by RT-PCR. (A) Reaction mixtures included ADK and 18S rRNA (internal control) primer sets, and products derived from ADK mRNA (570 bp) and rRNA (1,290 bp) were detected by ethidium bromide staining following agarose gel electrophoresis. The first and last lanes show an ADK marker fragment generated by PCR from a plasmid containing N. benthamiana ADK cDNA (Nb ADK). Three times as much product was loaded in the first lane compared to the last lane (3X and 1X). (B) Reaction mixtures contained SNF1 and 18S rRNA primer sets, and products derived from SNF1 mRNA (699 bp) were detected by DNA gel blot hybridization using a 32P-labeled probe specific for SNF1 (top). The 18S rRNA products (1,290 bp), but not the SNF1 products, could be detected by ethidium bromide staining prior to gel blot analysis (bottom). The last lane shows an SNF1 marker fragment generated by PCR from a plasmid containing N. benthamiana SNF1 cDNA.

Thus, reducing ADK mRNA levels, but not SNF1 mRNA levels, resulted in suppression of RNA silencing in the transient system. However, because SNF1-related kinases constitute a small gene family in most plant species (11, 16), we cannot rule out the possibility that other family members could functionally substitute for the one targeted by our dsSNF1 construct.

RNA silencing is suppressed by an ADK inhibitor. The results obtained with the dsADK construct prompted a search for another means to inhibit ADK. By employing an assay that uses thin layer chromatography to measure adenosine phosphorylation in the presence of {gamma}-32P-ATP (64), it was determined that the adenosine analogue A-134974 (38), a selective inhibitor of rat ADK, also inhibits ADK activity in a dose-dependent manner when added to protein extracts from N. benthamiana plants (Fig. 3).



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  		FIG. 3. The adenosine analogue A-134974 inhibits ADK activity in N. benthamiana extracts. Reaction mixtures contained the substrates adenosine (1 µM) and [{gamma}-32P]ATP, with 400 ng of total protein extract from stem tissue and the indicated concentrations of A-134974. It was previously determined that the level of ADK activity is proportional to the amount of added extract over a range of 100 to 500 ng (64). Labeled AMP and ATP were resolved by thin-layer chromatography, and ADK activity (AMP/AMP and ATP) in each reaction was calculated following phosphorimager quantitation of radioactivity in individual spots. The graph shows relative ADK activity plotted against increasing A-134974 concentrations.

In subsequent experiments, coinfiltration of A-134974 with Agrobacterium cultures harboring GFP and dsGFP constructs (GFP, dsGFP, and A-134974) resulted in suppression of GFP silencing similar to the geminivirus proteins and dsADK. GFP mRNA was observed to accumulate in infiltrated tissue, whereas the accumulation of siRNAs derived from GFP sequences was reduced relative to the GUS negative control (Fig. 1B and C). Silencing suppression was observed over the range of A-134974 concentrations tested (0.01 to 100 µM), although some variability was observed at lower concentrations (data not shown).

Although A-134974 has been shown to be a selective inhibitor of rat ADK (38), its possible effects on plant enzymes other than ADK are not known. Nevertheless, it can be said that reducing ADK mRNA levels by introducing a dsADK construct, or reducing ADK activity by adding an enzyme inhibitor, leads to silencing suppression in this transient system (Fig. 1 and 2A). Together, these results indicate that ADK activity is needed either for the initiation of RNA silencing or for its maintenance over the 5-day course of the experiment.

ADK activity is reduced in infiltrated leaf tissues expressing AL2 or L2 protein and exhibiting silencing suppression. We previously showed that AL2 and L2 are effective inhibitors of ADK activity both in vitro and in vivo, including geminivirus-infected tissue (64). Thus, we asked whether suppression of RNA silencing by these proteins and other agents used in these studies could be correlated with reductions in ADK activity. ADK activity levels in protein extracts obtained from tissues infiltrated 5 days previously with various test constructs were determined as previously described (64). In tissues receiving GFP-dsGFP and AL2, L2, dsADK, or A-134974, we found that ADK activity was markedly reduced, by more than 50 to 90% (Fig. 4). Comparable tissues contained relatively high levels of GFP mRNA and low levels of GFP-specific siRNAs compared to the GUS negative control (Fig. 1B and C). In contrast, in tissues infiltrated with GFP-dsGFP and GUS, dsSNF1, or HC-Pro, ADK activity was not significantly reduced relative to control tissue infiltrated with vector DNA, regardless of whether the constructs did (HC-Pro) or did not (GUS, dsSNF1) suppress RNA silencing (Fig. 4 and 1B and C). Thus, silencing suppression by AL2, L2, dsADK, and A-134974 was strongly correlated with substantial reductions in ADK activity, and the inability of HC-Pro to significantly inhibit the activity of this enzyme indicates that this is not a general feature of tissues exhibiting silencing suppression.



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  		FIG. 4. ADK activity in infiltration zones. N. benthamiana leaves were agroinfiltrated with GFP, dsGFP and the indicated constructs, empty vector, or the adenosine analogue A-134974 (100 µM). Protein extracts were prepared from infiltrated tissue 5 days postinfiltration, and ADK assays were performed by incubating samples (250 ng) with adenosine and [{gamma}-32P]ATP. Products were fractionated by thin-layer chromatography, and labeled AMP was quantitated by phosphorimaging as described in the legend to Fig. 3. The graph shows ADK activity relative to the vector control. The data are averages of two experiments; the error bars indicate ranges.

AL2 does not bind siRNA. It has been demonstrated that some plant viral silencing suppressors, including p19 and Beet yellows virus p21, bind siRNA (and similar miRNA) duplexes (10, 46). The influenza virus NS1 protein also has siRNA binding activity (32). TGMV AL2 protein is known to bind ssDNA and dsDNA in a sequence-nonspecific manner, although the significance of this activity is unknown (22). The possibility that this orphan nucleic acid binding activity might extend to siRNA was directly tested.

Electrophoretic mobility shift experiments were carried out to assess the binding activity of recombinant GST-AL2 fusion protein expressed in E. coli and purified as previously described (22). Similarly prepared GST-p19 and GST served as positive and negative controls, respectively. The proteins were incubated with an siRNA population isolated from wheat germ extract primed with GFP-derived, 32P-labeled dsRNA (53). In the absence of added protein or in the presence of GST or GST-AL2, the labeled siRNA migrated near the bottom of the gel, whereas in the presence of GST-p19 nearly all of it was found in slower-migrating complexes (Fig. 5). GST and GST-AL2 were additionally incubated with a labeled ~500-nt ssDNA fragment from pUC18 to verify biological activity of the viral fusion protein (22). This experiment demonstrated that the AL2 protein was active as GST-AL2, but not GST, and was able to form slower-migrating complexes with ssDNA (Fig. 5). Thus, AL2 does not have significant siRNA binding activity under conditions that support its efficient binding by the p19 protein.



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  		FIG. 5. AL2 does not bind siRNA. Autoradiographs of nondenaturing polyacrylamide gels illustrating electrophoretic mobility shift experiments with an siRNA probe (left panel) and an ssDNA probe (right panel) are presented. Labeled siRNA or ssDNA (~500 bp) was incubated without added protein (–) or with GST (negative control), GST-p19, or GST-AL2 in duplicate reactions as indicated. The inability of GST-AL2 to bind siRNA was confirmed twice with each of two different protein preparations.


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DISCUSSION
 
We previously found that the AL2 and L2 proteins interact with and inactivate ADK in vitro and in vivo and that ADK activity is reduced in TGMV- and BCTV-infected tissue in an AL2/L2-dependent manner (64). In this report, we showed that the TGMV AL2 and BCTV L2 proteins similarly act as suppressors of RNA silencing directed against a GFP reporter gene in a transient three-component system and that silencing suppression does not require the AL2 transcriptional activation domain. Since the BCTV protein is not required for viral gene expression and does not activate transcription in yeast, it is likely that suppression by L2 likewise does not depend on transcriptional activation (21, 24, 48). We further demonstrated that reducing ADK mRNA levels by expression of an inverted repeat construct directed against ADK (dsADK), or reducing ADK activity by adding an inhibitor (A-134974), results in a similar suppression of RNA silencing. GFP mRNA accumulated to relatively high levels, and GFP-derived siRNAs were reduced in tissues infiltrated with GFP-dsGFP and AL2, AL21-100, L2, dsADK, and A-134974 compared with the GUS negative control. Direct measurement of ADK activity in comparable tissues following infiltration with these same constructs revealed that ADK activity was significantly reduced (>50% to 90%) relative to control tissue infiltrated with vector or GUS. Thus, ADK activity is required to initiate or maintain RNA silencing in the transient assay, and ADK inhibition by AL2 and L2 is at least part of the mechanism by which these related proteins suppress silencing.

It has been reported that mutational disruption of a likely nuclear localization signal of Tomato yellow leaf curl virus-China C2 (AL2) abolishes silencing suppression, implying that suppression by this protein involves events that occur in the nucleus (13). In contrast, we have shown that AL2 exists in both the nuclear and cytoplasmic compartments in TGMV-infected cells, providing an opportunity for interaction with ADK, which is believed to be a cytoplasmic protein (64). It is possible that some of the mutations introduced in the Tomato yellow leaf curl virus-China study affected amino acid residues required both for suppression and nuclear localization or interfered with ADK interaction, or perhaps rendered the C2 protein inactive. It is also possible that AL2 proteins suppress silencing through multiple mechanisms by impacting steps that occur in the cytoplasm or the nucleus.

A comparison of viral suppressor protein phenotypes is informative. In the system employed here, GFP mRNA accumulation was highest with p19, whereas the geminivirus proteins were weaker suppressors that allowed much less GFP mRNA to accumulate even though they effectively reduced siRNA accumulation over a 5-day period. There is sound evidence that p19 acts by binding and sequestering siRNAs (10, 29, 46). Thus, the disparity in suppression efficiency, coupled with our observation that AL2/L2 proteins do not bind siRNA, argues that the geminivirus and tombusvirus proteins target different steps in the silencing pathway. HC-Pro also supported more robust GFP mRNA accumulation than AL2 and L2, but unlike the geminivirus proteins, it did not prevent the accumulation of GFP-derived siRNAs through 5 days postinfiltration and did not cause a significant reduction in ADK activity in infiltrated tissue. Thus, the mechanism by which AL2 and L2 suppress silencing appears to be different from those of HC-Pro and p19.

Various transient silencing protocols sometimes yield different outcomes with the same suppressor proteins. This is particularly true for siRNA accumulation, where variability may be attributed to the time points examined (days postinfiltration), the presence or absence of a strong silencing inducer (e.g., a dsRNA-generating construct), and whether the silenced locus is present on a transient template or is a transgene (18, 25, 35, 36, 46, 56). Nevertheless, it is interesting that we and others have observed p19 and HC-Pro to differentially affect siRNA accumulation given recent evidence indicating that both proteins can block siRNA/siRNA* duplex unwinding (10, 29). One interpretation of p19's ability to reduce siRNA accumulation, originally proposed by Silhavy and colleagues, suggests that the p19-siRNA interaction additionally interferes with an amplification step mediated by siRNA and RNA-dependent RNA polymerase (23, 34, 46, 54). We speculate that the ability of AL2 and L2 proteins to reduce siRNA accumulation also results from interference with an amplification step but one that requires ADK activity and does not involve sequestration of siRNA.

Our data demonstrating that ADK activity is required for RNA silencing in intact leaf tissues appear to explain previous observations that transgenic plants with reduced ADK activity due to silencing revert at high frequency (39, 64). Further, with the revelation that this nucleoside kinase activity is important for silencing, ADK inactivation by the geminivirus pathogenicity factors AL2 and L2 may now be viewed as a counterdefense against this antiviral pathway. However, this does not contradict our previous hypothesis that ADK inactivation contributes to the suppression of SNF1-mediated responses (21, 64). It would be interesting if, by targeting a single metabolic enzyme, geminivirus AL2 proteins managed to interfere with two distinct antiviral defenses.

Why is ADK required for RNA silencing? It seems most likely that this housekeeping enzyme plays an indirect, supporting role in the process. Beginning from this premise, one possible answer to the question is that the energy requirements of the silencing pathway demand efficient, ADK-mediated adenosine salvage. Perhaps a more attractive answer arises from recent evidence showing that ADK plays a critical role in sustaining S-adenosylmethionine-dependent methyltransferase activity and that ADK-deficient yeast and Arabidopsis exhibit reduced methylation (30, 39, 45, 66). This suggests a link with viral pathogenesis because DNA and specific types of histone methylation (e.g., H3K9) are known to be associated with RNA silencing and other, predominantly negative, epigenetic pathways (for reviews see references 3, 4, and 40). Methylation would be important in an Agrobacterium-based transient system like the one employed in our studies if T-DNA templates were subject to epigenetic modification. If this is the case, then the recent observation that TGMV AL2 protein cannot suppress silencing directed against a GFP reporter gene in a protoplast transfection assay might reflect differences in system requirements and differences in the structures of plasmid and T-DNA-derived templates (42). T-DNA templates may well be associated with chromatin and could possibly be methylated. Plasmid-borne silencing loci may be inaccessible to methyltransferase activities, and thus methylation (and ADK activity) may not be a significant factor in protoplasts.

That plants might use methylation as a defense implies that the geminivirus minichromosome is also a target for DNA and/or histone methyltransferases. In support of this idea, we previously found that in vitro methylation of TGMV DNA impairs its ability to replicate in tobacco protoplasts. However, we also found that progeny viral DNA, and DNA isolated from N. benthamiana plants infected with wild-type TGMV, was not densely methylated (7). A more thorough examination of the methylation state of the geminivirus minichromosome in inoculated tissues from host and nonhost plants is ongoing, as are studies to investigate the role of ADK activity in RNA silencing.


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ACKNOWLEDGMENTS
 
We thank Biao Ding, Erich Grotewold, Deborah Parris, and Yijun Qi for helpful discussions.

This work was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grants 00-35301-9084 and 04-35301-14508, and in part by a grant from the Biotechnology Research and Development Corporation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Biotechnology Center, Ohio State University, 201 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210. Phone: (614) 292-3281. Fax: (614) 292-5379. E-mail: bisaro.1{at}osu.edu. Back


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REFERENCES
 
    1
  1. Anandalakshmi, R., G. J. Pruss, X. Ge, R. Marathe, A. C. Mallory, T. H. Smith, and V. B. Vance. 2000. A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science 290:142-144.[Abstract/Free Full Text]
    2. 2
  2. Anandalakshmi, R., G. J. Pruss, X. Ge, R. Marathe, T. H. Smith, and V. B. Vance. 1998. A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. USA 95:13079-13084.[Abstract/Free Full Text]
    3. 3
  3. Bender, J. 2004. DNA methylation and epigenetics. Annu. Rev. Plant Biol. 55:41-68.[CrossRef][Medline]
    4. 4
  4. Bender, J. 2001. A vicious cycle: RNA silencing and DNA methylation in plants. Cell 106:129-132.[CrossRef][Medline]
    5. 5
  5. Bernstein, E., A. Caudy, S. M. Hammond, and G. J. Hannon. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363-366.[CrossRef][Medline]
    6. 6
  6. Brigneti, G., O. Voinnet, W.-X. Li, L.-H. Ji, S.-W. Ding, and D. C. Baulcombe. 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17:6739-6746.[CrossRef][Medline]
    7. 7
  7. Brough, C. L., W. E. Gardiner, N. Inamdar, X. Y. Zhang, M. Ehrlich, and D. M. Bisaro. 1992. DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts. Plant Mol. Biol. 18:703-712.[CrossRef][Medline]
    8. 8
  8. Carrington, J. C., and V. Ambros. 2003. Role of miRNAs in plant and animal development. Science 301:336-338.[Abstract/Free Full Text]
    9. 9
  9. Chan, S. W.-L., D. Zilberman, Z. Xie, L. K. Johansen, J. C. Carrington, and S. E. Jacobsen. 2004. RNA silencing genes control de novo DNA methylation. Science 303:1336.[Free Full Text]
    10. 10
  10. Chapman, E. J., A. I. Prokhnevsky, K. Gopinath, V. V. Dolja, and J. C. Carrington. 2004. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev. 18:1179-1186.[Abstract/Free Full Text]
    11. 11
  11. Chikano, H., M. Ogawa, Y. Ikeda, N. Koizumi, T. Kusano, and H. Sano. 2001. Two novel genes encoding SNF1-related protein kinases from Arabidopsis thaliana: differential accumulation of AtSR1 and AtSR2 transcripts in response to cytokinins and sugars, and phosphorylation of sucrose synthase by AtSR2. Mol. Gen. Genet. 264:674-681.[CrossRef][Medline]
    12. 12
  12. Denli, A. M., and G. J. Hannon. 2003. RNAi: an ever-growing puzzle. Trends Biochem. Sci. 28:196-201.[CrossRef][Medline]
    13. 13
  13. Dong, X., R. van Wezel, J. Stanley, and Y. Hong. 2003. Functional characterization of the nuclear localization signal for a suppressor of posttranscriptional gene silencing. J. Virol. 77:7026-7033.[Abstract/Free Full Text]
    14. 14
  14. Dunoyer, P., C.-H. Lecellier, E. A. Parizotto, C. Himber, and O. Voinnet. 2004. Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16:1235-1250.[Abstract/Free Full Text]
    15. 15
  15. Gutierrez, C. 1999. Geminivirus DNA replication. Cell. Mol. Life Sci. 56:313-329.[CrossRef][Medline]
    16. 16
  16. Halford, N., and D. G. Hardie. 1998. SNF1-related protein kinases: global regulators of carbon metabolism in plants? Plant Mol. Biol. 37:735-748.[CrossRef][Medline]
    17. 17
  17. Hamilton, A., and D. C. Baulcombe. 1999. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950-952.[Abstract/Free Full Text]
    18. 18
  18. Hamilton, A., O. Voinnet, L. Chappell, and D. C. Baulcombe. 2002. Two classes of short interfering RNA in RNA silencing. EMBO J. 21:4671-4679.[CrossRef][Medline]
    19. 19
  19. Hammond, S., E. Bernstein, D. Beach, and G. Hannon. 2000. An RNA-directed nuclease mediates PTGS in Drosophila cells. Nature 404:293-296.[CrossRef][Medline]
    20. 20
  20. Hanley-Bowdoin, L., S. Settlage, B. M. Orozco, S. Nagar, and D. Robertson. 1999. Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit. Rev. Plant Sci. 18:71-106.[CrossRef]
    21. 21
  21. Hao, L., H. Wang, G. Sunter, and D. M. Bisaro. 2003. Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase. Plant Cell 15:1034-1048.[Abstract/Free Full Text]
    22. 22
  22. Hartitz, M. D., G. Sunter, and D. M. Bisaro. 1999. The geminivirus transactivator (TrAP) is a single-stranded DNA and zinc-binding phosphoprotein with an acidic activation domain. Virology 263:1-14.[CrossRef][Medline]
    23. 23
  23. Himber, C., P. Dunoyer, G. Moissard, C. Ritzenthaler, and O. Voinnet. 2003. Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J. 22:4523-4533.[CrossRef][Medline]
    24. 24
  24. Hormuzdi, S. G., and D. M. Bisaro. 1995. Genetic analysis of beet curly top virus: examination of the roles of L2 and L3 genes in viral pathogenesis. Virology 206:1044-1054.[CrossRef][Medline]
    25. 25
  25. Johansen, L. K., and J. C. Carrington. 2001. Silencing on the spot: induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol. 126:930-938.[Abstract/Free Full Text]
    26. 26
  26. Jones, L., A. J. Hamilton, O. Voinnet, C. L. Thomas, A. J. Maule, and D. C. Baulcombe. 1999. RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell 11:2291-2302.[Abstract/Free Full Text]
    27. 27
  27. Kasschau, K. D., and J. C. Carrington. 1998. A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95:461-470.[CrossRef][Medline]
    28. 28
  28. Kasschau, K. D., Z. Xie, E. Allen, C. Llave, E. J. Chapman, K. A. Krizan, and J. C. Carrington. 2003. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev. Cell 4:205-217.[CrossRef][Medline]
    29. 29
  29. Lakatos, L., G. Szittya, D. Silhavy, and J. Burgyan. 2004. Molecular mechanism of RNA silencing suppression mediated by the p19 protein of tombusviruses. EMBO J. 23:876-884.[CrossRef][Medline]
    30. 30
  30. Lecoq, K., I. Belloc, C. Desgranges, and B. Daignan-Fornier. 2001. Role of adenosine kinase in Saccharomyces cerevisiae: identification of the ADO1 gene and study of the mutant phenotypes. Yeast 18:335-342.[CrossRef][Medline]
    31. 31
  31. Li, H., W.-X. Li, and S.-W. Ding. 2002. Induction and suppression of RNA silencing by an animal virus. Science 296:1319-1321.[Abstract/Free Full Text]
    32. 32
  32. Li, W.-X., H. Li, R. Lu, F. Li, M. Dus, P. Atkinson, E. W. A. Brydon, K. L. Johnson, A. Garcia-Sastre, L. A. Ball, P. Palese, and S.-W. Ding. 2004. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl. Acad. Sci. USA 101:1350-1355.[Abstract/Free Full Text]
    33. 33
  33. Li, W.-X., and S.-W. Ding. 2001. Viral suppressors of RNA silencing. Curr. Opin. Biotech. 12:150-154.[CrossRef][Medline]
    34. 34
  34. Lipardi, C., Q. Wei, and B. M. Paterson. 2001. RNAi as random degradative PCR. siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107:297-307.[CrossRef][Medline]
    35. 35
  35. Llave, C., K. D. Kasschau, and J. C. Carrington. 2000. Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc. Natl. Acad. Sci. USA 97:13401-13406.[Abstract/Free Full Text]
    36. 36
  36. Mallory, A. C., L. Ely, T. H. Smith, R. Marathe, R. Anandalakshmi, M. Fagard, H. Vaucheret, G. Pruss, L. Bowman, and V. B. Vance. 2001. HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. Plant Cell 13:571-583.[Abstract/Free Full Text]
    37. 37
  37. Mallory, A. C., B. J. Reinhardt, D. Bartel, V. B. Vance, and L. H. Bowman. 2002. A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco. Proc. Natl. Acad. Sci. USA 99:15228-15233.[Abstract/Free Full Text]
    38. 38
  38. McGaraughty, S., K. L. Chu, C. T. Wismer, J. Mikusa, C. Z. Zhu, M. Cowart, E. A. Kowaluk, and M. F. Jarvis. 2001. Effects of A-134974, a novel adenosine kinase inhibitor, on carrageenan-induced inflammatory hyperalgesia and locomotor activity in rats: evaluation of the sites of action. J. Pharmacol. Exp. Ther. 296:501-509.[Abstract/Free Full Text]
    39. 39
  39. Moffatt, B. A., Y. Y. Stevens, M. S. Allen, J. D. Snider, L. A. Periera, M. I. Todorova, P. S. Summers, E. A. Weretilnyk, L. Martin-Caffrey, and C. Wagner. 2002. Adenosine kinase deficiency is associated with developmental abnormalities and reduced transmethylation. Plant Physiol. 128:812-821.[Abstract/Free Full Text]
    40. 40
  40. Paszkowski, J., and S. A. Whitham. 2001. Gene silencing and DNA methylation processes. Curr. Opin. Plant Biol. 4:123-129.[CrossRef][Medline]
    41. 41
  41. Pilartz, M., and H. Jeske. 2003. Mapping of Abutilon mosaic geminivirus minichromosomes. J. Virol. 77:10808-10818.[Abstract/Free Full Text]
    42. 42
  42. Qi, Y., X. Zhong, A. Itaya, and B. Ding. 2004. Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level. Nucleic Acids Res. 32:e179.[Abstract/Free Full Text]
    43. 43
  43. Restrepo, M. A., D. D. Freed, and J. C. Carrington. 1990. Nuclear transport of plant potyviral proteins. Plant Cell 2:987-998.[Abstract/Free Full Text]
    44. 44
  44. Ruiz, M. T., O. Voinnet, and D. C. Baulcombe. 1998. Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937-946.[Abstract/Free Full Text]
    45. 45
  45. Schoor, S., and B. A. Moffatt. 2004. Applying high throughput techniques in the study of adenosine kinase in plant metabolism and development. Front. Biosci. 9:1771-1781.[Medline]
    46. 46
  46. Silhavy, D., A. Molnar, A. Lucioli, G. Szittya, C. Hornyik, M. Tavazza, and J. Burgyan. 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]
    47. 47
  47. Smith, N. A., S. P. Singh, M.-B. Wang, P. A. Stoutjesdijk, A. G. Green, and P. M. Waterhouse. 2000. Total silencing by intron-spliced RNAs. Nature 407:319-320.[CrossRef][Medline]
    48. 48
  48. Stanley, J., J. Latham, M. S. Pinner, I. Bedford, and P. G. Markham. 1992. Mutational analysis of the monopartite geminivirus beet curly top virus. Virology 191:396-405.[CrossRef][Medline]
    49. 49
  49. Sunter, G., and D. M. Bisaro. 2003. Identification of a minimal sequence required for activation of the tomato golden mosaic virus coat protein promoter in protoplasts. Virology 305:452-462.[CrossRef][Medline]
    50. 50
  50. Sunter, G., and D. M. Bisaro. 1997. Regulation of a geminivirus coat protein promoter by AL2 protein (TrAP): evidence for activation and derepression mechanisms. Virology 232:269-280.[CrossRef][Medline]
    51. 51
  51. Sunter, G., and D. M. Bisaro. 1992. Transactivation of geminivirus AR1 and BR1 gene expression by the viral AL2 gene product occurs at the level of transcription. Plant Cell 4:1321-1331.[Abstract/Free Full Text]
    52. 52
  52. Sunter, G., J. Sunter, and D. M. Bisaro. 2001. Plants expressing tomato golden mosaic virus AL2 or beet curly top virus L2 transgenes show enhanced susceptibility to infection by DNA and RNA viruses. Virology 285:59-70.[CrossRef][Medline]
    53. 53
  53. Tang, G., B. J. Reinhart, D. P. Bartel, and P. D. Zamore. 2003. A biochemical framework for RNA silencing in plants. Genes Dev. 17:49-63.[Abstract/Free Full Text]
    54. 54
  54. Vaistij, F. E., L. Jones, and D. C. Baulcombe. 2002. Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14:857-867.[Abstract/Free Full Text]
    55. 55
  55. Vance, V., and H. Vaucheret. 2001. RNA silencing in plants: defense and counterdefense. Science 292:2277-2280.[Abstract/Free Full Text]
    56. 56
  56. Vanitharani, R., P. Chellappan, J. S. Pita, and C. Fauquet. 2004. Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and suppression of posttranscriptional gene silencing. J. Virol. 78:9487-9498.[Abstract/Free Full Text]
    57. 57
  57. van Wezel, R., H. Liu, P. Tien, J. Stanley, and Y. Hong. 2002. Mutation of three cysteine residues in tomato yellow leaf curl virus-China C2 protein causes dysfunction in pathogenesis and posttranscriptional gene silencing-suppression. Mol. Plant-Microbe Interact. 15:203-208.[Medline]
    58. 58
  58. Vargason, J. M., G. Szittya, J. Burgyan, and T. M. Tanaka Hall. 2003. Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115:799-811.[CrossRef][Medline]
    59. 59
  59. Voinnet, O. 2001. RNA silencing as a plant immune system against viruses. Trends Genet. 17:449-459.[CrossRef][Medline]
    60. 60
  60. Voinnet, O. 2002. RNA silencing: small RNAs as ubiquitous regulators of gene expression. Curr. Opin. Plant Biol. 5:444-451.[CrossRef][Medline]
    61. 61
  61. Voinnet, O., Y. M. Pinto, and D. C. Baulcombe. 1999. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc. Natl. Acad. Sci. USA 96:14147-14152.[Abstract/Free Full Text]
    62. 62
  62. Voinnet, O., S. Rivas, P. Mestre, and D. C. Baulcombe. 2003. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33:949-956.[CrossRef][Medline]
    63. 63
  63. Volpe, T. A., C. Kidner, I. M. Hall, G. Teng, S. I. S. Grewal, and R. A. Martienssen. 2002. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297:1833-1837.[Abstract/Free Full Text]
    64. 64
  64. Wang, H., L. Hao, C.-Y. Shung, G. Sunter, and D. M. Bisaro. 2003. Adenosine kinase is inactivated by geminivirus AL2 and L2 proteins. Plant Cell 15:3020-3032.[Abstract/Free Full Text]
    65. 65
  65. Waterhouse, P. M., M. B. Wang, and T. Lough. 2001. Gene silencing as an adaptive defense against viruses. Nature 411:834-842.[CrossRef][Medline]
    66. 66
  66. Weretilnyk, E. A., K. J. Alexander, M. Drebenstedt, J. D. Snider, P. S. Summers, and B. A. Moffatt. 2001. Maintaining methylation activities during salt stress: the involvement of adenosine kinase. Plant Physiol. 125:856-865.[Abstract/Free Full Text]
    67. 67
  67. Ye, K., L. Malinina, and D. J. Patel. 2003. Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426:874-878.[CrossRef][Medline]
    68. 68
  68. Zamore, P. D., T. Tuschl, P. A. Sharp, and D. P. Bartel. 2000. RNAi: dsRNA directs the ATP-dependent cleavage of mRNA at 21-23 nt intervals. Cell 101:25-33.[CrossRef][Medline]

Journal of Virology, June 2005, p. 7410-7418, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7410-7418.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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