Potato Virus Y NIb Multifunctional Protein Suppresses Antiviral Defense by Interacting with Several Protein Components of the RNA Silencing Pathway.

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

V2 protein encoded by Tomato yellow leaf curl China virus is an RNA silencing suppressor

Fate of hairpin transcript components during RNA silencing and its suppression in transgenic virus-resistant tobacco

The 2b proteins encoded by cucumovirus act as post-transcriptional gene silencing suppressors to counter host defence during infection. Here we report the crystal structure of Tomato aspermy virus 2b (TAV2b) protein bound to a 19 bp small interfering RNA (siRNA) duplex. TAV2b adopts an all alpha-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. TAV2b has a pair of hook-like structures to recognize simultaneously two alpha-helical turns of A-form RNA duplex by fitting its alpha-helix backbone into two adjacent major grooves of siRNA duplex. The conserved pi-stackings between tryptophan and the 5'-terminal base of siRNA duplex from both ends enhance the recognition. TAV2b further oligomerizes to form a dimer of dimers through the conserved leucine-zipper-like motif at its amino-terminal alpha-helix. Biochemical experiments suggest that TAV2b might interfere with the post-transcriptional gene silencing pathway by directly binding to siRNA duplex.

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.

Two proteins of the citrus tatter leaf virus (CTLV), a strain of the apple stem grooving virus (ASGV), capable of inducing citrus bud union disorders on commercially important trifoliate and citrange rootstocks, were identified as viral suppressors of RNA silencing (VSR). Both the coat protein (CP) and the movement protein (MP) suppressed RNA silencing in GFP-transgenic Nicotiana benthamiana 16c plants in agrobacterium-mediated co-infiltration assays; the MP acted as a local VSR, while the CP acted as a systemic VSR. When the potato virus X (PVX) infectious vector harbored either the CTLV CP or MP gene, viral infection and symptom development were promoted in N. benthamiana . Deletions of amino acids in the CP sequence or the MP sequence resulted in failure to promote PVX infections as well as suppression of silencing in agrobacterium-mediated co-infiltration assays. Mass spectrometry-based immunoprecipitation proteomics showed that neither the CTLV CP nor the MP interacts with cellular components directly involved in host antiviral RNA silencing pathways. RNA immunoprecipitation (RIP) and RNA-protein pull-down assays indicated that the CTLV MP interacts with double-stranded RNA (dsRNA) presumably through a protein complex or proteins containing RNA binding domains. It is possible that the MP prevents dsRNA cleavage through this mechanism, leading to suppression of host antiviral RNA silencing. These findings confirmed that CTLV uses VSRs as part of its overall strategy to overcome host antiviral defenses and are indicative of the ability of ASGV and CTLV to infect a wide range of hosts including different species of woody and herbaceous plants.

Contrasting effects of HC-Pro and 2b viral suppressors from Sugarcane mosaic virus and Tomato aspermy cucumovirus on the accumulation of siRNAs

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection.

The biological relationship between suppression of RNA silencing and virus movement poses an intriguing question in virus-plant interactions. Here, we have used a local RNA silencing assay, based on a movement-deficient Turnip crinkle virus TCV/GFPΔCP, to investigate the influence of silencing suppression by three different viral suppressors: the TCV 38K coat protein (CP), the 126K protein of Tobacco mosaic virus (TMV), and P19 of Tomato bushy stunt virus (TBSV) on cell-to-cell movement and long-distance spread of TCV/GFPΔCP. First, we found that TCV CP blocked the induction of local RNA silencing, but failed to support virus trafficking in silencing-suppressed transgenic plants, although it acted as a functional movement protein in non-transformed plants. Second, we demonstrated that the TMV 126K suppressor inhibited TCV/GFPΔCP-mediated RNA silencing, but did not facilitate intercellular spread of the chimaeric carmovirus. However, TMV and TMVΔCP prevented the initiation of RNA silencing by TCV/GFPΔCP and caused TCV/GFPΔCP to move between cells, although only TMV supported its long-distance spread. Third, TBSV P19 functioned as a movement protein for TCV/GFPΔCP and as a silencing suppressor in non-transformed and silencing-suppressed transgenic plants. We further identified three types of mutant P19 proteins that possessed no or varied functionality in silencing suppression and in the facilitation of carmovirus movement. These results suggest that, although suppression of local RNA silencing is essential for the maintenance of viral RNA, recovery of cell-to-cell movement and long-distance spread of movement-deficient carmoviruses is not a direct consequence of such silencing suppression.

RNA silencing is triggered by double-stranded RNAs (dsRNAs), and the dsRNAs are processed into 21to 24-nulceotide (nt) short interfering RNAs (siRNAs) by a host dsRNA-specific ribonuclease, Dicer. The siRNAs are subsequently incorporated into ARGONAUTE (AGO) proteins and serve as a guide for either sequence-specific cleavage or translational repression of a target RNA. Because viral dsRNAs originating from either replicative intermediates or hairpin structures on viral genomes can become inducers of RNA silencing, the RNA silencing machinery in plants is considered to be a natural antiviral defense mechanism. In contrast, plant viruses have evolved a counter defense strategy, producing RNA silencing suppressors (RSSs) that interfere with the RNA silencing pathway. Among RSSs of plant viruses, HC-Pro of potyviruses, 2b of Cucumber mosaic virus (CMV) and P19 of tombusviruses have been extensively studied. These RSSs are structurally diverse, but many use a common strategy to interfere with the RNA silencing pathway by binding to siRNAs. In this study, we first developed a protoplastbased system for RNA silencing to measure RSS activity more stringently than with the commonly used Agrobacterium-mediated transient expression system. Using the protoplast assay, we performed various functional analyses of the 2b protein (2b) of Cucumovirus. In addition, we established a strategy for screening and assessing chemical compounds that inhibit the interaction of viral RSS and siRNAs, leading to attenuation of viral disease symptoms.

The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b’s nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b’s C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b’s nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b’s efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm.

RNA silencing is an important mechanism of antiviral defence in plants. To counteract this resistance mechanism, many viruses have evolved RNA silencing suppressors. In this study, we analysed five proteins encoded by Sweet potato chlorotic fleck virus (SPCFV) for their abilities to suppress RNA silencing using a green fluorescent protein (GFP)-based transient expression assay in Nicotiana benthamiana line 16c plants. Our results showed that a putative nucleotide-binding protein (NaBp), but not other proteins encoded by the virus, could efficiently suppress local and systemic RNA silencing induced by either sense or double-stranded RNA (dsRNA) molecules. Deletion mutation analysis of NaBp demonstrated that the basic motif (an arginine-rich region) was critical for its RNA silencing suppression activity. Using confocal laser scanning microscopy imaging of transfected protoplasts expressing NaBp fused to GFP, we showed that NaBp accumulated predominantly in the nucleus. Mutational analysis of NaBp demonstrated that the basic motif represented part of the nuclear localization signal. In addition, we demonstrated that the basic motif in NaBp was a pathogenicity determinant in the Potato virus X (PVX) heterogeneous system. Overall, our results demonstrate that the basic motif of SPCFV NaBp plays a critical role in RNA silencing suppression, nuclear localization and viral pathogenesis.

Root-knot nematodes damage crops around the world by developing complex feeding sites from normal root cells of their hosts. The ability to initiate and maintain this feeding site (composed of individual "giant cells") is essential to their parasitism process. RNA silencing pathways in plants serve a diverse set of functions, from directing growth and development to defending against invading pathogens. Influencing a host's RNA silencing pathways as a pathogenicity strategy has been well-documented for viral plant pathogens, but recently, it has become clear that silencing pathways also play an important role in other plant pathosystems. To determine if RNA silencing pathways play a role in nematode parasitism, we tested the susceptibility of plants that express a viral suppressor of RNA silencing. We observed an increase in susceptibility to nematode parasitism in plants expressing viral suppressors of RNA silencing. Results from studies utilizing a silenced reporter gene suggest that active suppression of RNA silencing pathways may be occurring during nematode parasitism. With these studies, we provide further evidence to the growing body of plant-biotic interaction research that suppression of RNA silencing is important in the successful interaction between a plant-parasitic animal and its host.

The mixed infection of Cucumber mosaic virus (CMV) and a potyvirus has been known to increase CMV titer in Nicotiana benthamiana plants, resulting in synergistic viral symptoms. We found that among three potyviruses--Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Clover yellow vein virus (C1YVV)--synergistic effects on CMV (or a recombinant CMV vector) titers were most efficiently induced by a co-infection with PVY in N. benthamiana plants. In addition, the helper component-proteinase (HC-Pro) gene of PVY expressed by transgenic plants, which is a viral RNA silencing suppressor, was sufficient to cancel the cycling pattern of CMV titer, resulting in increased levels of overall CMV accumulation. Surprisingly, we found that the levels of CMV and the foreign protein expressed from the CMV vector were much higher in the HC-Pro-transgenic plants than the levels detected in the plants mixed-infected with CMV and PVY. The mechanism for canceling the cyclic infection of CMV by the HC-Pro protein alone is discussed in view of the interaction between RNA silencing and HC-Pro, as well as the possible involvement of the 3a protein.

Identification of Strawberry vein banding virus encoded P6 as an RNA silencing suppressor