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Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms., Double-stranded RNA immunoprecipitation allows the identification of Arabidopsis host proteins associated with tobacco rattle virus replication complexes.

Double-stranded RNA binding (DRB) proteins are generally considered as promoting cofactors of Dicer or Dicer-like (DCL) proteins that ensure efficient and precise production of small RNAs, the sequence-specificity guide of RNA silencing processes in both plants and animals. However, the characterization of a new clade of DRB proteins in Arabidopsis has recently challenged this view by showing that DRBs can also act as potent inhibitors of DCL processing. This is achieved through sequestration of a specific class of small RNA precursors, the endogenous inverted-repeat (endoIR) dsRNAs, thereby selectively preventing production of their associated small RNAs, the endoIR-siRNAs. Here, we concisely summarize the main findings obtained from the characterization of these new DRB proteins and discuss how the existence of such complexes can support a potential, yet still elusive, biological function of plant endoIR-siRNAs.

Plant RNA viruses form highly organized membrane-bound virus replication complexes (VRCs) to replicate their genome and multiply. This process requires both virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) intermediates of replication that trigger potent antiviral defenses in all eukaryotes. In this work, we describe the use of A. thaliana constitutively expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down viral replicating RNA and associated proteins in planta upon infection with tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from TRV-infected plants revealed the presence of (i) viral proteins such as the replicase, which attested to the successful isolation of VRCs, and (ii) a number of host proteins, some of which have previously been involved in virus infection. Among a set of nine selected such host candidate proteins, eight showed dramatic re-localization upon infection, and seven of these co-localized with B2-labeled TRV replication complexes, providing ample validation for the immunoprecipitation results. Infection of A. thaliana T-DNA mutant lines for eight of these factors revealed that genetic knock-out of the Double-stranded RNA-Binding protein 2 (DRB2) leads to increased TRV accumulation. In addition, over-expression of this protein caused a dramatic decrease in the accumulation of four unrelated plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We therefore propose B2:GFP-mediated pull down of dsRNA to be a novel and robust method to explore the proteome of VRCs in planta, allowing the discovery of key players in the viral life cycle.AUTHOR SUMMARYViruses are an important class of pathogens that represent a major problem for human, animal and plant health. They hijack the molecular machinery of host cells to complete their replication cycle, a process frequently associated with the production of double-stranded RNA (dsRNA) that is regarded as a universal hallmark of infection by RNA viruses. Here we exploited the capacity of a GFP-tagged dsRNA-binding protein stably expressed in transgenic Arabidopsis to pull down dsRNA and associated proteins upon virus infection. In this manner we specifically captured short and long dsRNA from tobacco rattle virus (TRV) infected plants, and successfully isolated viral proteins such as the replicase, which attested to the successful isolation of virus replication complexes (VRCs). More excitingly, a number of host proteins, some of which have previously been involved in virus infection, were also captured. Remarkably, among a set of nine host candidates that were analyzed, eight showed dramatic re-localization to viral factories upon infection, and seven of these co-localized dsRNA-labeled VRCs. Genetic knock-out and over-expression experiments revealed that one of these proteins, A. thaliana DRB2, has a remarkable antiviral effect on four plant RNA viruses belonging to different families, providing ample validation of the potential of this experimental approach in the discovery of novel defense pathways and potential biotech tools to combat virus infections in the field. Being compatible with any plant virus as long as it infects Arabidopsis, we propose our dsRNA-centered strategy to be a novel and robust method to explore the proteome of VRCs in planta.

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant&ndash, microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant&ndash, virus interactions in complement to Saccharomyces cerevisiae.

In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles1-3. In plants, RNA interference (RNAi) mediated by small interfering (si)RNA is the main antiviral defence mechanism. To protect themselves against the cell- and non-cell autonomous effects of RNAi, viruses produce viral suppressors of RNA silencing (VSR)4, whose study is crucial to properly understand the biological cycle of plant viruses and potentially find new solutions to control these pathogens. By combining biochemical approaches, cell-specific inhibition of RNAi movement and peroxisome isolation, we show here that one such VSR, the peanut clump virus (PCV)-encoded P15, isolates siRNA from the symplasm by delivering them into the peroxisomal matrix. Infection with PCV lacking this ability reveals that piggybacking of these VSR-bound nucleic acids into peroxisomes potentiates viral systemic movement by preventing the spread of antiviral siRNA. Collectively, these results highlight organellar confinement of antiviral molecules as a novel pathogenic strategy that may have its direct counterpart in other plant and animal viruses.

International audience; In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA. These data suggest a possible role for TSL phosphorylation in the specific regulation of exogenous and antiviral RNA silencing in Arabidopsis and identify TSL as an intrinsic regulator of RNA interference., Nucleic Acids Research, 42 (12), ISSN:1362-4962, ISSN:0301-5610

We show, with miR171, that plant miRNA genes are modular independent transcription units in which the fold-back pre-miRNA is sufficient for miRNA processing, and that the upstream region contains highly specific promoter elements. Processing depends on flanking sequences within the miRNA stem-loop precursor rather than the miRNA sequence itself, and mutations affecting target pairing at the center and 5′ but not 3′ region of the miRNA compromise its function in vivo. Inactivation of the SDE1 RNA-dependent-RNA-polymerase was mandatory for accurate representation of miRNA activity by sensor constructs in Arabidopsis. Work in sde1 background revealed a near-perfect spatial overlap between the patterns of miR171 transcription and activity, supporting the idea that plant miRNAs enable cell differentiation.

In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.

Le RNA silencing est un mecanisme opere par de petites molecules d’ARN qui inhibent l’expression de genes tant au niveau transcriptionnel que post-transcriptionnel via des interactions sequence-specifiques. Conserve chez la plupart des eucaryotes, ce phenomene est implique dans le maintien de l’integrite des genomes ou dans la regulation de facteurs specifiques au cours du developpement, aussi bien chez les animaux que chez les plantes. Chez ces dernieres, le RNA silencing joue egalement un role crucial dans la defense antivirale. Pour contrer cette reponse, les virus ont developpe une batterie de strategies pour inhiber ce mecanisme. Cette revue detaille les mecanismes du RNA silencing et de son inhibition au cours des interactions plante/ virus et suggere les possibles consequences de cette bataille moleculaire sur l’evolution de leur genomes respectifs.

In RNA interference (RNAi), double-stranded RNA (dsRNA) is processed into short interfering RNA (siRNA) to mediate sequence-specific gene knockdown. The genetics of plant RNAi is not understood, nor are the bases for its spreading between cells. Here, we unravel the requirements for biogenesis and action of siRNAs directing RNAi in Arabidopsis thaliana and show how alternative routes redundantly mediate this process under extreme dsRNA dosages. We found that SMD1 and SMD2, required for intercellular but not intracellular RNAi, are allelic to RDR2 and NRPD1a, respectively, previously implicated in siRNA-directed heterochromatin formation through the action of DCL3 and AGO4. However, neither DCL3 nor AGO4 is required for non-cell autonomous RNAi, uncovering a new pathway for RNAi spreading or detection in recipient cells. Finally, we show that the genetics of RNAi is distinct from that of antiviral silencing and propose that this experimental silencing pathway has a direct endogenous plant counterpart.

We show, with miR171, that plant miRNA genes are modular independent transcription units in which the fold-back pre-miRNA is sufficient for miRNA processing, and that the upstream region contains highly specific promoter elements. Processing depends on flanking sequences within the miRNA stem-loop precursor rather than the miRNA sequence itself, and mutations affecting target pairing at the center and 5′ but not 3′ region of the miRNA compromise its function in vivo. Inactivation of the SDE1 RNA-dependent-RNA-polymerase was mandatory for accurate representation of miRNA activity by sensor constructs in Arabidopsis. Work in sde1 background revealed a near-perfect spatial overlap between the patterns of miR171 transcription and activity, supporting the idea that plant miRNAs enable cell differentiation.

RNA sequences required for assembly into rod-shaped virions of RNA-1 and RNA-2 of Peanut clump virus (PCV) were mapped by testing the ability of different RNA-1 and -2 deletion mutants to be encapsidated in vivo in an RNase-resistant form. Encapsidation of RNA-1 was found to require a sequence domain in the 5'-proximal part of the P15 gene, the 3'-proximal gene of RNA-1. On the other hand, the subgenomic RNA which encodes P15 was not encapsidated, suggesting that other features of RNA-1 are important as well. Two sequences which could drive encapsidation of RNA-2 deletion mutants were located. One was in the 5'-proximal coat protein gene and the other in the P14 gene near the RNA 3' terminus. There were no obvious sequence homologies between the different assembly initiation sequences.

One manifestation of RNA silencing, known as post-transcriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in animals, is a nucleotide sequence-specific RNA turnover mechanism with the outstanding property of propagating throughout the organism, most likely via movement of nucleic acids. Here, the cell-to-cell movement of RNA silencing in plants is investigated. We show that a short-distance movement process, once initiated from a small group of cells, can spread over a limited and nearly constant number of cells, independent of the presence of homologous transcripts. There is also a long-range cell-to-cell movement process that occurs as a relay amplification, which requires the combined activity of SDE1, a putative RNA-dependent RNA polymerase, and SDE3, a putative RNA helicase. Extensive and limited cell-to-cell movements of silencing are triggered by the same molecules, occur within the same tissues and likely recruit the same plasmodesmata channels. We propose that they are in fact manifestations of the same process, and that extensive cell-to-cell movement of RNA silencing results from re-iterated short-distance signalling events. The likely nature of the nucleic acids involved is presented.

RNA silencing is a regulatory mechanism that controls the expression of endogenous genes and exogenous molecular parasites such as viruses, transgenes, and transposable elements. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. In plants, one of the most fascinating aspects of RNA silencing is its mobile nature, in other words its ability to spread from the cell where it has been initiated to neighboring cells, through movement of sRNA molecules. To study this process, a key step is to directly monitor the spread of these nucleic acid species. Here we describe how this can be achieved through biolistic delivery of fluorescently labeled siRNA.

RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

Plants employ multiple layers of innate immunity to fight pathogens. For both RNA and DNA viruses, RNA silencing plays a critical role in plant resistance. To escape this antiviral silencing-based immune response, viruses have evolved various counterdefense strategies, the most widespread being production of viral suppressors of RNA silencing (VSRs) that target various stages of the silencing mechanisms. Recent findings from in planta analyses have provided new insights into the mode of action of VSRs and revealed that plants react to the perturbation of the silencing pathways brought by viral infection by deploying a battery of counter-counterdefense measures. As well as discussing which experimental approaches have been most effective in delivering clear and unambiguous results, this review provides a detailed account of the surprising variety of offensive and defensive strategies set forth by both viruses and hosts in their struggle for survival.

RNA silencing mediated by small RNAs (sRNAs) is a conserved regulatory process with key antiviral and antimicrobial roles in eukaryotes. A widespread counter-defensive strategy of viruses against RNA silencing is to deploy viral suppressors of RNA silencing (VSRs), epitomized by the P19 protein of tombusviruses, which sequesters sRNAs and compromises their downstream action. Here, we provide evidence that specific Nicotiana species are able to sense and, in turn, antagonize the effects of P19 by activating a highly potent immune response that protects tissues against Tomato bushy stunt virus infection. This immunity is salicylate- and ethylene-dependent, and occurs without microscopic cell death, providing an example of “extreme resistance” (ER). We show that the capacity of P19 to bind sRNA, which is mandatory for its VSR function, is also necessary to induce ER, and that effects downstream of P19-sRNA complex formation are the likely determinants of the induced resistance. Accordingly, VSRs unrelated to P19 that also bind sRNA compromise the onset of P19-elicited defense, but do not alter a resistance phenotype conferred by a viral protein without VSR activity. These results show that plants have evolved specific responses against the damages incurred by VSRs to the cellular silencing machinery, a likely necessary step in the never-ending molecular arms race opposing pathogens to their hosts., Author Summary Multiple and complex layers of defense help plants to combat pathogens. A first line of defense relies on the detection, via dedicated host-encoded receptors, of signature molecules (so called pathogen-associated molecular patterns, PAMPs) produced by pathogens. In turn, this PAMP-triggered immunity (PTI) may be itself antagonized by adapted pathogens that have evolved virulence effectors to target key PTI components. Host plants react to PTI suppression by producing disease resistance (R) proteins that recognize virulence effectors and activate highly specific resistance called Effector Triggered Immunity (ETI). It has been noted that RNA silencing, a sequence-specific antiviral defense response based on the production of virus-derived 21–24 nt small RNAs on the one hand, and its suppression by virulence effectors, called viral suppressors of RNA silencing (VSRs) on the other, are conceptually similar to PTI. Here we provide strong support to this hypothesis by showing that extreme resistance is indeed activated following detection, in specific host species, of the VSR activity of a viral virulence effector. The ensuing antiviral immunity displays many characteristics of ETI, suggesting that one or several R proteins must sense the integrity of the host silencing machinery.

RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species.

Recent work on metazoans has uncovered the existence of an endogenous RNA-silencing pathway that functionally recapitulates the effects of experimental RNA interference (RNAi) used for gene knockdown in organisms such as Caenorhabditis elegans and Drosophila. The endogenous short interfering (si)RNA involved in this pathway are processed by Dicer-like nucleases from genomic loci re-arranged to form extended inverted repeats (IRs) that produce perfect or near-perfect dsRNA molecules. Although such IR loci are commonly detected in plant genomes, their genetics, evolution and potential contribution to plant biology through endogenous silencing have remained largely unexplored. Through an exhaustive analysis performed using Arabidopsis, we provide here evidence that at least two such endogenous IRs are genetically virtually indistinguishable from the transgene constructs commonly used for RNAi in plants. We show how these loci can be useful probes of the cellular mechanism and fluidity of RNA-silencing pathways in plants, and provide evidence that they may arise and disappear on an ecotype scale, show highly cell-specific expression patterns and respond to various stresses. IR loci thus have the potential to act as molecular sensors of the local environments found within distinct ecological plant niches. We further show that the various siRNA size classes produced by at least one of these IR loci are functionally loaded into cognate effector proteins and mediate both post-transcriptional gene silencing and RNA-directed DNA methylation (RdDM) of endogenous as well as exogenous targets. Finally, and as previously reported during plant experimental RNAi, we provide evidence that endogenous IR-derived siRNAs of all size classes are not cell-autonomous and can be transported through graft junctions over long distances, in target tissues where they are functional, at least in mediating RdDM. Collectively, these results define the existence of a bona fide, endogenous and systemic RNAi pathway in plants that may have implications in adaptation, epiallelism and trans-generational memory.

siRNA Movement in Plant Tissues Long-distance movement of RNA interference (RNAi)–derived signals in plants plays an important role in development and in defense against viral attack. The nature of the signals that spread from cell to cell is not known, although evidence suggests that they are nucleic acids of some sort (see the Perspective by Martienssen ). Molnar et al. (p. 872 , published online 22 April) and Dunoyer et al. (p. 912 , published online 22 April) now show that in Arabidopsis , both exogenous and endogenous small interfering RNAs (siRNAs), rather than their long double-stranded precursor RNAs, are the molecules that transfer information between plant cells. A viral protein that counters RNAi though sequestering siRNAs blocked spreading of a transgene RNAi silencing signal. Furthermore, siRNA-processing enzymes were required in the source, and not the recipient, cells for spreading, and bombardment of plants with double-stranded siRNAs directly showed siRNA spread between cells. Endogenous siRNAs also spread between tissues and were capable of directing DNA methylation of target sequences in distant tissues.

RNA silencing is a conserved eukaryotic process mediated by small RNA molecules that inhibit gene expression at the transcriptional, mRNA-stability or translational level through sequence-specific interactions. Diverse roles have been identified for RNA silencing such as genome defense against mobile DNA elements or down-regulation of specific factors during plant and animal development. In plants, RNA silencing plays a crucial role in antiviral defense by inhibiting viral accumulation and sometimes preventing systemic infection. As a counter-defense mechanism, viruses have evolved anti-silencing strategies through the production of viral suppressors of RNA silencing. Here we review the mechanism of RNA silencing and its inhibition during plant/virus interactions and suggest the possible consequences of this molecular arms race on the evolution of both viral and host genomes.

High complementarity between plant microRNAs (miRNAs) and their messenger RNA targets is thought to cause silencing, prevalently by endonucleolytic cleavage. We have isolated Arabidopsis mutants defective in miRNA action. Their analysis provides evidence that plant miRNA–guided silencing has a widespread translational inhibitory component that is genetically separable from endonucleolytic cleavage. We further show that the same is true of silencing mediated by small interfering RNA (siRNA) populations. Translational repression is effected in part by the ARGONAUTE proteins AGO1 and AGO10. It also requires the activity of the microtubule-severing enzyme katanin, implicating cytoskeleton dynamics in miRNA action, as recently suggested from animal studies. Also as in animals, the decapping component VARICOSE (VCS)/Ge-1 is required for translational repression by miRNAs, which suggests that the underlying mechanisms in the two kingdoms are related.

In plants, silencing of mRNA can be transmitted from cell to cell and also over longer distances from roots to shoots. To investigate the long-distance mechanism, WT and mutant shoots were grafted onto roots silenced for an mRNA. We show that three genes involved in a chromatin silencing pathway, NRPD1a encoding RNA polymerase IVa, RNA-dependent RNA polymerase 2 (RDR2), and DICER-like 3 (DCL3), are required for reception of long-distance mRNA silencing in the shoot. A mutant representing a fourth gene in the pathway, argonaute4 (ago4), was also partially compromised in the reception of silencing. This pathway produces 24-nt siRNAs and resulted in decapped RNA, a known substrate for amplification of dsRNA by RDR6. Activation of silencing in grafted shoots depended on RDR6, but no 24-nt siRNAs were detected in mutant rdr6 shoots, indicating that RDR6 also plays a role in initial signal perception. After amplification of decapped transcripts, DCL4 and DCL2 act hierarchically as they do in antiviral resistance to produce 21- and 22-nt siRNAs, respectively, and these guide mRNA degradation. Several dcl genotypes were also tested for their capacity to transmit the mobile silencing signal from the rootstock. dcl1–8 and a dcl2 dcl3 dcl4 triple mutant are compromised in micro-RNA and siRNA biogenesis, respectively, but were unaffected in signal transmission.

At the request of the authors, PLOS Pathogens is retracting this publication following an investigation into concerns about the origin and assembly of Figure 6 and a mounting mistake in Figure 1B. The Northern blot depicted in Figure 6 contains several band duplications affecting the panels labelled 'IP@HA' and 'total RNA'. The figure was provided by Patrice Dunoyer during revision and was extracted from the Master thesis of a former student working under his supervision, without the prior consultation or consent of this student. The other authors of Sansregret et al. were not informed about the origin of this figure and, regrettably, its erroneous content escaped their attention both at the final revision and proofreading stages. During inspection of the original blots, we realised that the loading control of Figure 1B was not the correct one; we have found the cognate one and the loadings are comparable. Importantly, the cross-reacting band visible on Figure 1B also provides an internal loading control. This mistake was made by co-authors Raphael Sansregret and Kamal Bouarab. Further analysis of the raw material underlying the results depicted in Figure 6 was found to support the original conclusions drawn from it. The other conclusions of the published article also remain valid. However, given the nature and extent of data manipulation in Figure 6, the authors have collectively decided to retract the study. All authors concur with this statement and apologise for not having detected these errors. Kamal Bouarab and Olivier Voinnet, as the corresponding authors, take full responsibility for the publication of this erroneous paper and regret deeply the inconvenience caused.

The above article from The EMBO Journal , published online on 22 April 2010, has been retracted by agreement between the authors, the journal Chief Editor and Head of Scientific Publications, EMBO, Bernd Pulverer, and John Wiley & Sons Ltd. The authors' and editors' statements are as follows. Following an investigation conducted by the The EMBO Journal , the corresponding authors were alerted of several image irregularities found in this paper. Patrice Dunoyer, who assembled the figures, acknowledges making the following errors …

Regulation of gene expression through microRNAs (miRNAs) and antiviral defense through small interfering RNAs (siRNAs) are aspects of RNA silencing, a process originally discovered as an unintended consequence of plant transformation by disarmed Agrobacterium tumefaciens strains. Although RNA silencing protects cells against foreign genetic elements, its defensive role against virulent, tumor-inducing bacteria has remained unexplored. Here, we show that siRNAs corresponding to transferred-DNA oncogenes initially accumulate in virulent A. tumefaciens-infected tissues and that RNA interference-deficient plants are hypersusceptible to the pathogen. Successful infection relies on a potent antisilencing state established in tumors whereby siRNA synthesis is specifically inhibited. This inhibition has only modest side effects on the miRNA pathway, shown here to be essential for disease development. The similarities and specificities of the A. tumefaciens RNA silencing interaction are discussed and contrasted with the situation encountered with plant viruses.

Regulation of gene expression through microRNAs (miRNAs) and antiviral defense through small interfering RNAs (siRNAs) are aspects of RNA silencing, a process originally discovered as an unintended consequence of plant transformation by disarmed Agrobacterium tumefaciens strains. Although RNA silencing protects cells against foreign genetic elements, its defensive role against virulent, tumor-inducing bacteria has remained unexplored. Here, we show that siRNAs corresponding to transferred-DNA oncogenes initially accumulate in virulent A. tumefaciens-infected tissues and that RNA interference-deficient plants are hypersusceptible to the pathogen. Successful infection relies on a potent antisilencing state established in tumors whereby siRNA synthesis is specifically inhibited. This inhibition has only modest side effects on the miRNA pathway, shown here to be essential for disease development. The similarities and specificities of the A. tumefaciens RNA silencing interaction are discussed and contrasted with the situation encountered with plant viruses.

Plants and animals activate defenses after perceiving pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin. In Arabidopsis , perception of flagellin increases resistance to the bacterium Pseudomonas syringae , although the molecular mechanisms involved remain elusive. Here, we show that a flagellin-derived peptide induces a plant microRNA (miRNA) that negatively regulates messenger RNAs for the F-box auxin receptors TIR1, AFB2, and AFB3. Repression of auxin signaling restricts P. syringae growth, implicating auxin in disease susceptibility and miRNA-mediated suppression of auxin signaling in resistance.

In RNA interference1,2, the RNase-III enzyme Dicer3 processes exogenous double-stranded RNA into small interfering RNAs (siRNAs). siRNAs guide RNA-induced silencing complexes to cleave homologous transcripts, enabling gene-specific knock-down4. In plants, double-stranded RNA is processed into siRNA species of 21 nucleotides (nt) and 24 nt (ref. 5), but, unlike in nematodes6, the Dicer enzymes involved in this processing have not been identified. Additionally, in both plants and nematodes, systemic signals7,8,9,10 with RNA components convey the sequence-specific effects of RNA interference between cells. Here, we describe Arabidopsis thaliana mutants with altered silencing cell-to-cell movement beyond the vasculature. At least three SILENCING MOVEMENT DEFICIENT genes (SMD1, SMD2 and SMD3) are required for trafficking, the extent of which correlates with siRNA levels in the veins. Five alleles defective in synthesis of 21-nt, but not 24-nt, siRNAs carry mutations in Dicer-like 4 (DCL4) that are involved in biogenesis of trans-acting siRNAs11,12. We show that the biogenesis and function of trans-acting siRNA can be genetically uncoupled from a bona fide DCL4-dependent pathway that accounts for RNA interference and for production of the 21-nt siRNA component of the plant cell-to-cell silencing signal.

During infection, Beet necrotic yellow vein virus (BNYVV) particles localize transiently to the cytosolic surfaces of mitochondria. To understand the molecular basis and significance of this localization, we analyzed the targeting and membrane insertion properties of the viral proteins. ORF1 of BNYVV RNA-2 encodes the 21-kDa major coat protein, while ORF2 codes for a 75-kDa minor coat protein (P75) by readthrough of the ORF1 stop codon. Bioinformatic analysis highlighted a putative mitochondrial targeting sequence (MTS) as well as a major (TM1) and two minor (TM3 and TM4) transmembrane regions in the N-terminal part of the P75 readthrough domain. Deletion and gain-of-function analyses based on the localization of green fluorescent protein (GFP) fusions showed that the MTS was able to direct a reporter protein to mitochondria but that the protein was not persistently anchored to the organelles. GFP fused either to MTS and TM1 or to MTS and TM3-TM4 efficiently and specifically associated with mitochondria in vivo. The actual role of the individual domains in the interaction with the mitochondria seemed to be determined by the folding of P75. Anchoring assays to the outer membranes of isolated mitochondria, together with in vivo data, suggest that the TM3-TM4 domain is the membrane anchor in the context of full-length P75. All of the domains involved in mitochondrial targeting and anchoring were also indispensable for encapsidation, suggesting that the assembly of BNYVV particles occurs on mitochondria. Further data show that virions are subsequently released from mitochondria and accumulate in the cytosol.

RNA silencing was originally identified as an immune system targeted against transposons and viruses, but is now also recognized as a major regulatory process that affects all layers of host gene expression through the activities of various small RNA species. Recent work in plants and animals indicates that viruses not only suppress, but can also exploit, endogenous RNA silencing pathways to redirect host gene expression. There are also indications that cellular, as opposed to virus-derived small RNAs, might well constitute an unsuspected defense layer against foreign nucleic acids. This complex interplay has implications in the context of disease resistance and evolution of viral genomes.

In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing.

More than 30 years ago, it was suggested from electron microscopic and/or biochemical analyses that replication of positive-strand RNA viruses, whether in plants or in animals, takes place in association with intracellular membranes (for reviews, see references 13 and 14). The type of membranes involved in replication depends on the virus considered: tymoviruses, for instance, induce vesicular structures by invagination of chloroplast membranes in which both viral RNA and nonstructural proteins were detected (24, 25), whereas infection by tombusviruses leads to the formation of multivesicular bodies derived from mitochondria or peroxisomes (9, 56, 57). Several plant viruses belonging to different groups, such as tobamoviruses (30), bromoviruses (50), potyviruses (59), and comoviruses (11), similarly appear to strongly modify the endomembrane compartments. For Cowpea mosaic virus (CPMV), virus-induced accumulation of vesicles derived from the endomembrane system was described as early as 1974 by de Zoeten et al. (15), and in several instances fibrilar material tentatively identified as double-stranded RNA was shown to accumulate in such vesicles (20, 28). Viral proteins involved in replication were immunodetected on these vesicles in CPMV-infected cells (66). Similar information is available with animal viruses and has been described extensively in the literature, more particularly for poliovirus, the type member of picornaviridae. Evidence for the involvement of virus-induced vesicles in poliovirus replication was obtained already in the sixties (10), and the molecular mechanisms underlying the formation of virus-induced vesicles, their origin, and their role in viral replication were recently unraveled (19, 58, 62). The fact that distinct types of membranes are involved in the replication of different viruses implies the establishment of specific interactions between such host membranes and virus-encoded proteins. In some cases, the transmembrane domains responsible for their anchoring on specific membranes could be identified (18, 35, 56) allowing the formation of dedicated structures where coupled translation and synthesis of both minus- and plus-strand RNA take place (8). Such complexes probably ensure protection of the viral RNA being synthesized from degradation by cellular RNases. In the case of poliovirus, it was shown that specific viral proteins were responsible for vesicle formation (6, 7, 62) and that formation of the poliovirus replication complex is a process that requires coupled viral translation, vesicle production, and viral RNA synthesis (19). Grapevine fanleaf nepovirus (GFLV) has a bipartite RNA genome of positive polarity and, like all members of the Comoviridae, carries a small protein, VPg, covalently linked to the 5′ end of the viral genomic RNAs, which are both polyadenylated at their 3′ end. Each RNA encodes a single polyprotein, and this family of plant viruses shares therefore many common features with Picornaviridae. RNA1 encodes polyprotein P1 (253 kDa), which is processed by an embedded proteinase activity into five proteins required for replication, namely, 1A (of unknown function), 1B (probably the helicase), 1C (VPg), 1D (proteinase), and 1E (polymerase) (38, 40). These proteins are the only proteins required for RNA1 replication (65), and they function in trans to ensure RNA2 replication. RNA2 encodes polyprotein P2 (122 kDa), which is processed in trans by 1D into three proteins (39). Protein 2A is necessary for RNA2 replication, together with RNA1-encoded proteins (23). It is associated with membranous structures and is recruited by the RNA1-encoded replication machinery. We hypothesized that the 2A moiety of polyprotein P2 could mediate the transport of the nascent P2-RNA2 complexes from their initial location in the cytosol to the perinuclear replication sites where RNA2 replication and P2 cleavage take place (23). Therefore, protein 2A could play the role of a “homing protein.” Protein 2B is the movement protein (MP) forming tubules through which viral particles are delivered to uninfected adjacent cells (54). Finally, protein 2C is the coat protein (CP). Using epifluorescence microscopy, we have previously described the formation of a perinuclear complex where viral RNA was synthesized and viral proteins accumulated (23), but this could not be further analyzed due to technical limitations. In the present study, the structural preservation of cells and the intensity of immunolabelings were markedly enhanced, and image resolution was further improved by using high-resolution confocal microscopy. Moreover, the use of transgenic T-BY2 cell lines expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi apparatus (GA) provided a direct means to follow the virus-induced modifications of the endomembrane system and allowed us to conclude that GFLV replicates in association with specific membranes mainly derived from ER.

In plants, post-transcriptional gene silencing (PTGS) is part of a defence mechanism against virus infection. Several plant viruses have been shown to encode proteins which can counteract PTGS. In this paper it is demonstrated that P15 of peanut clump pecluvirus (PCV) has anti-PTGS activity. P15 is a small cysteine-rich protein with no sequence similarity to previously described PTGS-suppressor proteins which has several novel properties. It possesses four C-terminal proximal heptad repeats that can potentially mediate a coiled-coil interaction and is targeted to peroxisomes via a C-terminal SKL motif. The coiled-coil sequence is necessary for the anti-PTGS activity of P15, but the peroxisomal localization signal is not, although it is required for efficient intercellular movement of the virus.

The role of valine aminoacylation of the two genomic RNAs of Peanut clump virus (PCV) was studied by comparing the amplification in vivo of RNAs with GAC, GΔC, or CCA anticodons in the tRNA-like structure (TLS) present at the 3′ end of each viral RNA. The PCV RNA1 TLS of isolate PCV2 possesses a GAC anticodon and is capable of highly efficient valylation, whereas the RNA2 TLS has a GΔC anticodon that does not support valylation. The presence in RNA1 of GΔC or CCA anticodons that conferred nonvalylatability resulted in about 2- to 4-fold and a 14- to 24-fold reduction, respectively, in RNA accumulations in tobacco BY-2 protoplasts inoculated with the RNA1 variants together with wild-type RNA2(GΔC). No differences in RNA levels were observed among protoplasts inoculated with the three variant RNA2s in the presence of wild-type RNA1(GAC). All combinations of valylatable and nonvalylatable RNAs 1 and 2 were similarly infectious in Nicotiana benthamiana plants, and viral RNAs accumulated to similar levels; all input TLS sequences were present unchanged in apical leaves. In direct competition experiments in N. benthamiana plants, however, both RNA1 and RNA2 with GAC valylatable anticodons outcompeted the nonvalylatable variants. We conclude that valylation provides a small but significant replicational advantage to both PCV RNAs. Sequence analysis of the TLS from RNA2 of a second PCV isolate, PO2A, revealed the presence of an intact GAC valine anticodon, suggesting that the differential valylation of the genomic RNAs of isolate PCV2 is not a general characteristic of PCV.

In RNA silencing, small-interfering (si)RNAs and micro (mi)RNAs are processed from double-stranded (ds)RNA by the RNaseIII Dicer [1]. siRNAs derive from long, near-perfectly base-paired dsRNAs of endogenous or exogenous origin, including viruses and transgenic hairpins used in experimental RNA interference (RNAi, Figure 1a) [2]. Endogenous miRNAs originate from DNA-encoded primary transcripts with imperfect fold-back structures; cellular targets of miRNAs include transcription factors important for cell-fate determination [3].

In plants, RNA silencing is a fundamental regulator of gene expression, heterochromatin formation, suppression of transposable elements, and defense against viruses. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. Although the spreading of RNA silencing across the plant has been recognized for nearly two decades, only recently have sRNAs been formally demonstrated as the mobile silencing signals. Here, we discuss the various types of mobile sRNA molecules, their short- and long-range movement, and their function in recipient cells.

Fluorescent beet necrotic yellow vein virus (BNYVV) particles were produced by replacing part of the readthrough domain of the minor coat protein P75 with the green fluorescent protein (GFP). The recombinant virus was functional in plants and P75–GFP was incorporated at one end of the rod-shaped virions. Laser scanning confocal microscopy and transmission electron microscopy showed that virus-like particles, almost certainly authentic BNYVV virions, localized to the cytoplasmic surface of mitochondria at early times postinfection but relocated at later times to semiordered clusters in the cytoplasm. This is the first report of specific targeting of plant virus particles to the mitochondria in vivo.