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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–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–virus interactions in complement to Saccharomyces cerevisiae.

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.

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.

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

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.

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.

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.

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.

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, 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.

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.

Background Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed. Conclusions/Significance Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4., PLoS ONE, 7 (4), ISSN:1932-6203

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.