Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.interpathway regulation ͉ plant antiviral defense R NA silencing is a cellular mechanism that uses small RNA molecules (21-30 nt in length) as sequence-specific mediators to regulate the expression of a diverse array of genes at the transcriptional, posttranscriptional, or translational levels (1). In plants, these very small RNA species are termed small interfering RNAs (siRNAs) or micro RNAs (miRNAs) depending on the source of their precursors. They are generated by a family of double-stranded RNA (dsRNA)-specific RNases called Dicerlike ribonucleases (DCLs) (2). Once produced, the siRNAs and miRNAs are recruited by Argonaute proteins (AGOs) into RNA-induced silencing complexes (RISCs) to direct the cleavage or translational repression of homologous mRNAs or to remodel the homologous chromosomal DNA to achieve transcriptional silencing (3). Another family of dsRNA-binding proteins (DRBs) has been found to modulate the function of DCLs (4). Plants also encode RNA-dependent RNA polymerases (RDRs) to produce some of the dsRNA precursors that serve as templates for DCLs (2). In Arabidopsis, 4 DCLs, 10 AGOs, 5 DRBs, and up to 6 RDRs have been identified. They participate in at least four different endogenous RNA silencing pathways to achieve spatial and temporal regulation of gene expression throughout the plant life cycle and to condition the plant response to biotic and abiotic stresses (5).Although the plant RNA silencing mechanism was first revealed through studies aimed to unravel the complexity of plant antiviral defense strategies, the details of plant antiviral RNA silencing pathway(s) are far...

Posttranscriptional gene silencing (PTGS), or RNA silencing, is a sequence-specific RNA degradation process that targets foreign RNA, including viral and transposon RNA for destruction. Several RNA plant viruses have been shown to encode suppressors of PTGS in order to survive this host defense. We report here that the coat protein (CP) of Turnip crinkle virus (TCV) strongly suppresses PTGS. The Agrobacterium infiltration system was used to demonstrate that TCV CP suppressed the local PTGS as strongly as several previously reported virus-coded suppressors and that the action of TCV CP eliminated the small interfering RNAs associated with PTGS. We have also shown that the TCV CP must be present at the time of silencing initiation to be an effective suppressor. TCV CP was able to suppress PTGS induced by sense, antisense, and doublestranded RNAs, and it prevented systemic silencing. These data suggest that TCV CP functions to suppress RNA silencing at an early initiation step, likely by interfering the function of the Dicer-like RNase in plants.Posttranscriptional gene silencing (PTGS) is a sequencespecific RNA degradation process that leads to the elimination of the targeted RNA and loss of the function(s) encoded by the targeted RNA (1,6,62,69). This phenomenon was first observed and intensively studied in plant systems (see reference 64 for a review), where it has been associated with several processes, including cosuppression (44), repeat induced gene silencing (70), RNA-mediated resistance (35, 58), or homology-dependent gene silencing (43). Similar mechanisms were later discovered in other organisms, including quelling in filamentous fungus Neurospora crassa (9) and RNA interference (RNAi) in Caenorhabditis elegans (18) and Drosophila melanogaster (30). Recent research has revealed that all of these different phenomena have many common features and are now considered to be manifestations of an RNA-targeting pathway, whose natural functions include protecting hosts from invading viral RNAs and transposons (see references 45, 54, and 72 for reviews). RNA silencing has been proposed as a more general term to describe these related processes (1).Initiation and maintenance stages have been identified as distinct phases of the PTGS or RNA silencing process (10,50,65). In the initiation stage, the invading RNA triggers a pathway that results in its being degraded into a small RNA species of discrete size (21 to 25 nucleotides [nt]) called small interfering RNAs (siRNAs) that function as a guide for further degradation in the maintenance stage (22, 71). The most potent initiator of PTGS is thought to be double-stranded RNA (dsRNA) (8,18,30,68), although single-stranded RNA (ssRNA), both sense and antisense orientations, or even DNA trigger RNA silencing (15,65,66). ssRNA is most likely converted to a double-stranded form with the help of a host RNAdependent RNA polymerase (RdRP) in order to be effective (9,11,42,52,57). The dsRNA initiators are then degraded by an RNase III-like RNase (e.g., Dicer in Drosophila [3])...

SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.RNA silencing is a surveillance system in eukaryotic organisms triggered by double-stranded RNA (dsRNA) that is subsequently digested by a dsRNA-specific RNase (Dicer or Dicer-like) into a small RNA species of 21 to 25 nucleotides (nt) long, called small interfering RNA (siRNA). The resultant siRNAs are then recruited into the RNA-induced silencing complex to direct the degradation of other RNAs with sequence complementarity to siRNAs (13). RNA silencing is thought to function primarily in defending eukaryotic cells against RNA molecular parasites, such as RNA viruses and transposon RNAs. Plant viruses, as well as some animal viruses, counteract this host defense mechanism by encoding suppressors of RNA silencing, which act at different steps of the pathway and with various strengths to ensure their successful systemic invasion of specific hosts (26,31).In addition to guarding the host against parasitic RNAs, recent studies have shown that processes highly related to RNA silencing are also involved in developmental regulation (22, 28), methylation of chromosomal DNA and histones, and chromatin maintenance (20,43). miRNA-mediated regulation of gene expression in both animal and plant systems is a particularly interesting discovery. Unlike siRNAs, miRNAs are encoded by genomes of eukaryotes in the form of partially double-stranded precursor molecules, which are processed by Dicer-like RNase(s) to release mature miRNAs. The miRNAs then mediate degradation or translational repression of the target RNAs (15). One well-studied example in plants is miR165/166. This miRNA targets the mRNA of three class III homeodomain leucine zipper (HD-ZIP III) transcription factors, PHABULOSA (PHB), PHAVOLUTA (PHV), and REVOLUTA (REV), for cleavage (10,...

c While RNA silencing is a potent antiviral defense in plants, well-adapted plant viruses are known to encode suppressors of RNA silencing (VSR) that can neutralize the effectiveness of RNA silencing. As a result, most plant genes involved in antiviral silencing were identified by using debilitated viruses lacking silencing suppression capabilities. Therefore, it remains to be resolved whether RNA silencing plays a significant part in defending plants against wild-type viruses. We report here that, at a higher plant growth temperature (26°C) that permits rigorous replication of Turnip crinkle virus (TCV) in Arabidopsis, plants containing loss-of-function mutations within the Dicer-like 2 (DCL2), Argonaute 2 (AGO2), and HEN1 RNA methyltransferase genes died of TCV infection, whereas the wild-type Col-0 plants survived to produce viable seeds. To account for the critical role of DCL2 in ensuring the survival of wild-type plants, we established that higher temperature upregulates the activity of DCL2 to produce viral 22-nucleotide (nt) small interfering RNAs (vsRNAs). We further demonstrated that DCL2-produced 22-nt vsRNAs were fully capable of silencing target genes, but that this activity was suppressed by the TCV VSR. Finally, we provide additional evidence supporting the notion that TCV VSR suppresses RNA silencing through directly interacting with AGO2. Together, these results have revealed a specialized RNA silencing pathway involving DCL2, AGO2, and HEN1 that provides the host plants with a competitive edge against adapted viruses under environmental conditions that facilitates robust virus reproduction.

RNA silencing as a robust host defense mechanism against plant viruses is generally countered by virus-encoded silencing suppressors. This strategy is now increasingly recognized to be used by animal viruses as well. We present here an overview of the common features shared by some of the better studied plant viral silencing suppressors. We then briefly describe the characteristics of the few reported animal viral suppressors, notably their extraordinary ability of cross-kingdom suppression. We next discuss the basis for biased protection of viral RNA and subviral parasites by silencing suppressors, the link between movement and silencing suppression, the influence of temperature on the outcome of viral infection and the effect of viral silencing suppressors on the microRNA pathway.

The capsid protein (CP) of Turnip crinkle virus (TCV) is a multifunctional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long-distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV CP and evaluated the interdependence of these functions. A series of TCV mutants containing alterations in the CP coding region were generated. These alterations range from single-amino-acid substitutions and domain truncations to knockouts of CP translation. The latter category also contained two constructs in which the CP coding region was replaced by either the cDNA of a silencing suppressor of a different virus or that of green fluorescent protein. These mutants were used to infect Arabidopsis plants with diminished antiviral silencing capability (dcl2 dcl3 dcl4 plants). There was a strong correlation between the ability of mutants to reach systemic leaves and the silencing suppressor activity of mutant CP. Virus particles were not essential for entry of the viral genome into vascular bundles in the inoculated leaves in the absence of antiviral silencing. However, virus particles were necessary for egress of the viral genome from the vasculature of systemic leaves. Our experiments demonstrate that TCV CP not only allows the viral genome to access the systemic movement channel through silencing suppression but also ensures its smooth egress by way of assembled virus particles. These results illustrate that efficient long-distance movement of TCV requires both functions afforded by the CP.

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana benthamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. benthamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.

Previous studies on turnip crinkle virus (TCV) have suggested that the two small, centrally located ORFs, conserved in all Carmoviruses, are both required for cell-to-cell movement (Hacker et al., 1992). We now demonstrate that the cell-to-cell movement of TCV is mediated by in trans complementation of the two proteins. First, both of the putative movement proteins (MPs p8 and p9) were shown to be translated in vitro from transcripts representing the 1.7-kb subgenomic RNA. Western blot analysis, using antisera prepared against GST fusion proteins of both genes, was then used to show that the p8 but not the p9 protein accumulated to detectable levels in particulate fractions of infected cells. Cell-to-cell movement of various MP mutants in Arabidopsis was evaluated by in situ hybridization of inoculated leaves. Changes in either of the two MP genes resulted in failure of the mutants to move cell-to-cell. Coat protein was found to be unnecessary for cell-to-cell movement. Complementation of cell-to-cell movement by co-inoculating p8-defective mutants with a p9-defective mutant resulted in delayed systemic infection. In contrast, efficient cell-to-cell movement was achieved when the MP mutants were inoculated into transgenic plants expressing the corresponding functional gene(s). These experiments provide further evidence that both MP genes encoded by Carmoviruses must function in trans in the same cell in order to mediate cell-to-cell movement.