Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase

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.     

Suppressor of RNA silencing encoded by Beet yellows virus

Using an Agrobacterium-mediated transient assay, we screened the 15.5-kb genome of the Beet yellows virus for proteins with RNA silencing suppressor activity. Among eight proteins tested, only a 21-kDa protein (p21) was able to suppress double-stranded (ds) RNA-induced silencing of the green fluorescent protein (GFP) mRNA. Restoration of GFP expression by p21 under these conditions had no apparent effect on accumulation of the small interfering RNAs. In addition, p21 elevated the transient expression level of the GFP mRNA in the absence of dsRNA inducer. Similar activities were detected using homologs of p21 encoded by other members of the genus Closterovirus. Computer analysis indicated that p21-like proteins constitute a novel protein family that is unrelated to other recognized suppressors of RNA silencing. Examination of the subcellular distribution in BYV-infected plants revealed that p21 is partitioned between soluble cytoplasmic form and proteinaceous inclusion bodies at the cell periphery.     

Characterization of virus-induced gene silencing in tobacco plants infected with apple latent spherical virus

Summary Apple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.     

Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent long distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.     

Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon

Launching the Beet yellows virus (BYV) minireplicon by agrobacterial delivery resulted in an unexpectedly low number of infected cells per inoculated leaf. This effect was due to a strong RNA silencing response in the agroinfiltrated leaves. Strikingly, ectopic co-expression of p21, a BYV RNA silencing suppressor, increased minireplicon infectivity by three orders of magnitude. Mutational analysis demonstrated that this effect correlates with suppressor activity of p21. Five diverse, heterologous viral suppressors were also active in this system, providing a useful approach for a dramatic, up to 10,000-fold, increase of the efficiency of agroinfection. The minireplicon agroinfection assay was also used to identify a new suppressor, a homolog of BYV p21, derived from Grapevine leafroll-associated virus-2. In addition, we report preliminary data on the suppressor activity of the p10 protein of Grapevine virus A and show that this protein belongs to a family of Zn-ribbon-containing proteins encoded by filamentous plant RNA viruses from three genera. The members of this family are predicted to have RNA silencing suppressor activity.     

Non-structural protein 1 from avian influenza virus H9N2 is an efficient RNA silencing suppressor with characteristics that differ from those of <Emphasis Type="Italic">Tomato bushy stunt virus</Emphasis> p19

Non-structural protein 1 (NS1) of influenza A virus is a multifunctional dimeric protein that contains a conserved N-terminal RNA binding domain. Studies have shown that NS1 suppresses RNA silencing and the NS1 proteins encoded by different influenza A virus strains exhibit differential RNA silencing suppression activities. In this study, we showed that the NS1 protein from avian influenza virus (AIV) H9N2 suppressed systemic RNA silencing induced by sense RNA or dsRNA. It resulted in more severe Potato virus X symptom, but could not reverse established systemic green fluorescent protein silencing in Nicotiana benthamiana. In addition, its systemic silencing suppression activity was much weaker than that of p19. The local silencing suppression activity of AIV H9N2 NS1 was most powerful at 7 dpi and was even stronger than that of p19. And the inhibition ability to RNA silencing of NS1 is stronger than that of p19 in human cells. Collectively, these results indicate that AIV H9N2 NS1 is an effective RNA silencing suppressor that likely targets downstream step(s) of dsRNA formation at an early stage in RNA silencing. Although NS1 and p19 both bind siRNA, their suppression mechanisms seem to differ because of differences in their suppression activities at various times post-infiltration and because p19 can reverse established systemic RNA silencing, but NS1 cannot.     

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

RNA silencing and suppression of silencing are host and virus interactions for defense and counter-defense. Here, we explored the function effect of HC-Pro encoded by Sugarcane mosaic virus (SCMV) on the suppression of RNA silencing. siRNA northern blotting assay indicated that the replication of SCMV was regulated by host RNA silencing machinery. Co-expression assay demonstrated that the HC-Pro encoded by SCMV suppressed the RNA silencing induced by sense RNA and dsRNA. Transitive RNA silencing assay showed that HC-Pro down-regulated the accumulation of 3' secondary siRNA, but not 5' secondary and primary siRNA. Meanwhile, the 2b gene of Tomato aspermy cucumovirus (Tav) evidently down-regulated the accumulation of 5' secondary siRNA. Importantly, we found that HC-Pro and Tav2b down-regulated the accumulation of RDR6 mRNA. Thus, HC-Pro, an RNA silencing suppressor encoded by SCMV, regulates the accumulation of different siRNAs and has more than one target in the RNA silencing pathway.     

Multiple suppressors of RNA silencing encoded by both genomic RNAs of the crinivirus, Tomato chlorosis virus

Viruses express proteins with silencing suppression activity to counteract the RNA silencing-mediated defense response of the host. In the family Closteroviridae, examples of multiple-component RNA silencing suppression systems have been reported. To ascertain if this is a general strategy in this group of viruses, we have explored the bipartite genome of Tomato chlorosis virus (ToCV, genus Crinivirus). We have identified the RNA1-encoded p22 protein as an effective silencing suppressor by using a Agrobacterium co-infiltration assay. p22 suppressed local RNA silencing induced either by sense RNA or dsRNA very efficiently, but did not interfere with short or long-distance systemic spread of silencing. We have also demonstrated by using the heterologous vector PVX the silencing suppression activity of the RNA-2 encoded coat protein (CP) and minor coat protein (CPm). In this study, we demonstrate an even greater complexity of silencing suppressor activity for a plant virus, and for the first time we show the presence of RNA silencing suppressor genes encoded by both genomic RNA molecules of a bipartite genome in the complex family Closteroviridae.     

HC-Pro hypo- and hypersuppressor mutants: differences in viral siRNA accumulation in vivo and siRNA binding activity in vitro

Viruses have evolved mechanisms to suppress the RNA silencing defense of their hosts, allowing replication and systemic colonization. In a recent study, we found that the effect of mutations in the RNA silencing suppressor of tobacco etch virus (TEV) was variable, ranging from complete abolition of suppressor activity to significantly stronger suppression. Whereas hyposuppressor mutants were less virulent and accumulated fewer viral particles than the wild type, hypersuppressors induced symptoms similar to those of the wild type and accumulated particles to similar levels. Here, we further characterize a set of these mutants in terms of their ability to bind in vitro and induce accumulation in vivo of virus-derived siRNAs. Hyposuppressor alleles are less efficient at binding siRNAs than hypersuppressors, whereas the latter are not different from the wild type. As a consequence of lower viral accumulation, plants infected with virus bearing a hyposuppressor allele also accumulate less virus-derived siRNA.     

The multifunctional plant viral suppressor of gene silencing P19 interacts with itself and an RNA binding host protein

Tomato bushy stunt virus (TBSV) is an RNA plant virus encoding a protein of approximately 19 kDa (P19) that is involved in various activities important for pathogenicity, including virus transport and suppression of gene silencing. In this study, we provide evidence in vivo and in vitro that P19 specifically interacts with itself to predominantly form dimers, and with a novel host protein, Hin19. Hin19 has a high degree of similarity with a class of RNA-binding proteins of which many are involved in RNA processing. The binding of P19 to itself and to Hin19 both depend on a structurally important central region of P19 that was previously shown critical for its biological function in plants. Our findings provide evidence for a model in which virus spread through suppression of defense-related gene silencing involves the formation of a complex that includes P19 dimers and a newly identified host RNA-binding protein.     

Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms

Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of helper component-proteinase (HC-Pro) has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses RNA silencing. RNA silencing suppression assays using Nicotiana benthamiana 16C plants expressing GFP were conducted with each WSMV protein; only P1 suppressed RNA silencing. Accumulation of GFP siRNAs was markedly reduced in leaves infiltrated with WSMV P1 at both 3 and 6 days post infiltration relative to WSMV HC-Pro and the empty vector control. On the other hand, helper component-proteinase (HC-Pro) of two species in the mite-transmitted genus Rymovirus, family Potyviridae was demonstrated to be a suppressor of RNA silencing. Symptom enhancement assays were conducted by inoculating Potato virus X (PVX) onto transgenic N. benthamiana. Symptoms produced by PVX were more severe on transgenic plants expressing WSMV P1 or potyvirus HC-Pro compared to transgenic plants expressing GFP or WSMV HC-Pro.     

Defective-interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts

Tomato bushy stunt virus (TBSV) genomic RNA and one of its defective interfering (DI) RNAs were inoculated in various combinations to protoplasts of Nicotiana benthamiana. Ethidium bromide staining of electrophoretically separated RNAs from infected protoplasts, incorporation of [3H]uridine into TBSV and DI RNAs, and Northern hybridization at different times after inoculation clearly demonstrated reduced accumulation of genomic RNA in the presence of DI RNA. Accumulation of genomic RNA was very rapid between 3 and 9 hr postinfection. The presence of equimolar amounts of genomic and DI RNA in the inoculum resulted in a 65% suppression of genomic RNA accumulation. Suppression of genomic RNA was mediated by a reduction in the rate at which genomic RNA accumulated. Analysis of protoplasts inoculated with increasing ratios of DI:genomic RNA suggested that DI RNA-mediated suppression of genomic RNA synthesis results from competition for factors essential for viral replication. Incubation of protoplasts at different temperatures also had a profound effect on replication of both genomic and DI RNAs. Both replicated well at 27 degrees but were barely detectable at 32 degrees. Suppression of genomic RNA synthesis by DI RNA was similar at all temperatures tested. Thus, this study suggests that DI suppression of TBSV symptoms in whole plants and symptom attenuation at elevated temperatures are primarily the result of reduced viral replication.     

Development of Bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean

Plant virus-based vectors provide valuable tools for expression of foreign proteins in plants and for gene function studies. None of the presently available virus vectors is suitable for use in soybean. In the present study, we produced Bean pod mottle virus (BPMV)-based vectors that are appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The genes of interest were inserted into the RNA2-encoded polyprotein open reading frame between the movement protein (MP) and the large coat protein (L-CP) coding regions. Additional proteinase cleavage sites were created to flank the foreign protein by duplicating the MP/L-CP cleavage site. To minimize the chances of homologous recombination and thus insert instability, we took advantage of the genetic code degeneracy and altered the nucleotide sequence of the duplicated regions without affecting amino acid sequences. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. These included the reporter proteins GFP and DsRed, phosphinothricin acetyltransferase (encoded by the herbicide resistance bar gene), and the RNA silencing suppressors encoded by Tomato bushy stunt virus, Turnip crinkle virus, Tobacco etch virus, and Soybean mosaic virus. The possible use of BPMV as a VIGS vector to study gene function in soybean was also demonstrated with the phytoene desaturase gene. Our results suggest that the BPMV-based vectors are suitable for expression of foreign proteins in soybean and for functional genomics applications.     

Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins

Previous genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92(pol) replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92(pol) replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92(pol) accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92(pol) as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92(pol) levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination.     

Systemic antiviral silencing in plants

RNA silencing controls numerous developmental processes in eukaryotic organisms from fungi, plants, to animals. In plants as well as in animals, this system of RNA regulation functions as part of an immune response against invading viruses. From transitive RNA silencing to virus-induced gene silencing (VIGS), the systemic effects are proven to be the core of RNA silencing. This article reviews the latest advances in view of the effect of cellular RDR6, an RNA-dependent RNA polymerase (RdRp), on systemic RNA silencing, systemic virus silencing, and discusses the abilities of viral suppressors in modulating RNA silencing efficiency to establish effective infection.     

Inducible viral inoculation system with cultured plant cells facilitates a biochemical approach for virus-induced RNA silencing

An inducible virus infection system was demonstrated to be an efficient protein expression system for inducing synchronous virus vector multiplication in suspension-cultured plant cells. A GFP-tagged tomato mosaic virus (ToMV-GFP) derivative that has a defect in its 130 K protein, a silencing suppressor of ToMV, was synchronously infected to tobacco BY2 cultured cells using this system. In the infection-induced cells, viral RNA was degraded rapidly, and a cytosol extract prepared from the infected cells showed RNA degradation activity specific for ToMV- or GFP-related sequences. In lysate prepared from cells infected by ToMV-GFP carrying the wild-type 130 K protein, sequence-specific RNA degradation activity was suppressed, although siRNA derived from the virus was generated. Furthermore, the 130 K protein interfered with 3′-end methylation of siRNA. The inducible virus infection system may provide a method for biochemical analysis of antiviral RNA silencing and silencing suppression by ToMV.