Plant virus‐mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation

Virus infections induce the expression of ARGONAUTE1 (AGO1) mRNA and in parallel enhance the accumulation of miR168 (regulator of AGO1 mRNA). Here, we show that in virus‐infected plants the enhanced expression of AGO1 mRNA is not accompanied by increased AGO1 protein accumulation. We also show that the induction of AGO1 mRNA level is a part of the host defence reaction, whereas the induction of miR168, which overlaps spatially with virus‐occupied sectors, is mediated mainly by the Tombusvirus p19 RNA‐silencing suppressor. The absence of p19 results in the elimination of miR168 induction and accompanied with the enhanced accumulation of AGO1 protein. In transient expression study, p19 mediates the induction of miR168 and the down‐regulation of endogenous AGO1 level. P19 is not able to efficiently bind miR168 in virus‐infected plants, indicating that this activity is uncoupled from the small RNA‐binding capacity of p19. Our results imply that plant viruses can inhibit the translational capacity of AGO1 mRNA by modulating the endogenous miR168 level to alleviate the anti‐viral function of AGO1 protein.     

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.     

The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation

Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.     

Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing

Viruses express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing-based host defenses. Although virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, small RNAs (sRNAs) and Argonaute (AGO) proteins seem to be the most frequent targets. Recently, GW/WG motifs of some VSRs have been proposed to dictate their suppressor function by mediating interaction with AGO(s). Here we have studied the VSR encoded by Pelargonium line pattern virus (family Tombusviridae). The results show that p37, the viral coat protein, blocks RNA silencing. Site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed generation of suppressor-competent and -incompetent molecules and uncoupling of the VSR and particle assembly capacities. The engineered mutants were used to assess the importance of p37 functions for viral infection and the relative contribution of diverse molecular interactions to suppressor activity. Two main conclusions can be drawn: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic Pelargonium line pattern virus infection, and (ii) the suppressor activity of p37 relies on the ability to bind sRNAs rather than on interaction with AGOs. The data also caution against potential misinterpretations of results due to overlap of sequence signals related to distinct protein properties. This is well illustrated by mutation of the GW motif in p37 that concurrently affects nucleolar localization, efficient interaction with AGO1, and sRNA binding capability. These concomitant effects could have been overlooked in other GW motif-containing suppressors, as we exemplify with the orthologous p38 of turnip crinkle virus.     

Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection

Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well-characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant-encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two-hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co-localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N-terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP-interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3-overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans-acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA-binding protein 4) and AGO2 (ARGONAUTE protein 2), to achieve efficient suppression of RNA silencing-mediated antiviral defence.     

Satellite RNA-derived small interfering RNA satsiR-12 targeting the 3' untranslated region of Cucumber mosaic virus triggers viral RNAs for degradation

RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.     

Characterization of silencing suppressor p24 of Grapevine leafroll‐associated virus 2

Grapevine leafroll‐associated virus 2 (GLRaV‐2) p24 has been reported to be an RNA silencing suppressor (RSS). However, the mechanisms underlying p24's suppression of RNA silencing are unknown. Using Agrobacterium infiltration‐mediated RNA silencing assays, we showed that GLRaV‐2 p24 is a strong RSS triggered by positive‐sense green fluorescent protein (GFP) RNA, and that silencing suppression by p24 effectively blocks the accumulation of small interfering RNAs. Deletion analyses showed that the region of amino acids 1–188, which contains all predicted α‐helices and β‐strands, is required for the RSS activity of p24. Hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, previously shown to be critical for p24 self‐interaction, are also crucial for silencing suppression, and western blotting results suggested that a lack of self‐interaction ability results in decreased p24 accumulation in plants. The mutants showed greatly weakened or a lack of RSS activity. Substitution with two basic residues at positions 2 or 86, putatively involved in RNA binding, totally abolished the RSS activity of p24, suggesting that p24 uses an RNA‐binding strategy to suppress RNA silencing. Our results also showed that W54 in the WG/GW‐like motif (W54/G55) is crucial for the RSS activity of p24, whereas p24 does not physically interact with AGO1 of Nicotiana benthamiana. Furthermore, p24 did not promote AGO1 degradation, but significantly up‐regulated AGO1 mRNA expression, and this effect was correlated with the RSS activity of p24, indicating that p24 may interfere with microRNA‐directed processes. The presented results contribute to our understanding of viral suppression of RNA silencing and the molecular mechanisms underlying GLRaV‐2 infection.     

Cytoplasmic Arabidopsis AGO7 accumulates in membrane-associated siRNA bodies and is required for ta-siRNA biogenesis

Formation of trans-acting small interfering RNAs (ta-siRNAs) from the TAS3 precursor is triggered by the AGO7/miR390 complex, which primes TAS3 for conversion into double-stranded RNA by the RNA-dependent RNA polymerase RDR6 and SGS3. These ta-siRNAs control several aspects of plant development. The mechanism routing AGO7-cleaved TAS3 precursor to RDR6/SGS3 and its subcellular organization are unknown. We show that AGO7 accumulates together with SGS3 and RDR6 in cytoplasmic siRNA bodies that are distinct from P-bodies. siRNA bodies colocalize with a membrane-associated viral protein and become positive for stress-granule markers upon stress-induced translational repression, this suggests that siRNA bodies are membrane-associated sites of accumulation of mRNA stalled during translation. AGO7 congregates with miR390 and SGS3 in membranes and its targeting to the nucleus prevents its accumulation in siRNA bodies and ta-siRNA formation. AGO7 is therefore required in the cytoplasm and membranous siRNA bodies for TAS3 processing, revealing a hitherto unknown role for membrane-associated ribonucleoparticles in ta-siRNA biogenesis and AGO action in plants.     

The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways

One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3' overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised.     

Polerovirus protein P0 prevents the assembly of small RNA‐containing RISC complexes and leads to degradation of ARGONAUTE1

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double‐stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein–protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA‐induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre‐programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high‐molecular‐weight complex, suggesting the existence of a multi‐protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA–RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co‐expressed AGO1 in both the presence and absence of P0.     

Temperature-dependent survival of Turnip crinkle virus-infected arabidopsis plants relies on an RNA silencing-based defense that requires dcl2, AGO2, and HEN1

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.     

Convergent Evolution of Argonaute-2 Slicer Antagonism in Two Distinct Insect RNA Viruses

RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.     

The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana

Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.     

Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis

Micro RNAs (miRNAs) are processed from precursor RNA molecules with precisely defined secondary stem-loop structures. ARGONAUTE1 (AGO1) is the main executor component of miRNA pathway and its expression is controlled via the auto-regulatory feedback loop activity of miR168 in plants. Previously we have shown that AGO1 loading of miR168 is strongly restricted leading to abundant cytoplasmic accumulation of AGO-unbound miR168. Here, we report, that intrinsic RNA secondary structure of MIR168a precursor not only defines the processing of miR168, but also precisely adjusts AGO1 loading efficiency determining the biologically active subset of miR168 pool. Our results show, that modification of miRNA duplex structure of MIR168a precursor fragment or expression from artificial precursors can alter the finely adjusted loading efficiency of miR168. In dcl1-9 mutant where, except for miR168, production of most miRNAs is severely reduced this mechanism ensures the elimination of unloaded AGO1 proteins via enhanced AGO1 loading of miR168. Based on this data, we propose a new competitive loading mechanism model for miR168 action: the miR168 surplus functions as a molecular buffer for controlled AGO1 loading continuously adjusting the amount of AGO1 protein in accordance with the changing size of the cellular miRNA pool.