Suppressors of RNA silencing encoded by plant viruses and their role in viral infections

RNA silencing as a robust host defense mechanism against plant viruses is generally countered by virus-encoded silencing suppressors. This strategy is now increasingly recognized to be used by animal viruses as well. We present here an overview of the common features shared by some of the better studied plant viral silencing suppressors. We then briefly describe the characteristics of the few reported animal viral suppressors, notably their extraordinary ability of cross-kingdom suppression. We next discuss the basis for biased protection of viral RNA and subviral parasites by silencing suppressors, the link between movement and silencing suppression, the influence of temperature on the outcome of viral infection and the effect of viral silencing suppressors on the microRNA pathway.     

Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1

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.     

StRemorin1.3 hampers Potato virus X TGBp1 ability to increase plasmodesmata permeability,but does not interfere with its silencing suppressor activity

The Triple Gene Block 1 (TGBp1) protein encoded by the Potato virus X is a multifunctional protein that acts as a suppressor of RNA silencing or facilitates the passage of virus from cell to cell by promoting the plasmodesmata opening. We previously showed that the membrane raft protein StRemorin1.3 is able to impair PVX infection. Here, we show that overexpressed StRemorin1.3 does not impair the silencing suppressor activity of TGBp1, but affects its ability to increase plasmodesmata permeability. A similar effect on plasmodesmata permeability was observed with other movement proteins, suggesting that REM is a general regulator of plasmodesmal size exclusion limit. These results add to our knowledge of the mechanisms underlying the StREM1.3 role in virus infection.     

Two amino acids near the N‐terminus of Cucumber mosaic virus 2b play critical roles in the suppression of RNA silencing and viral infectivity

Cucumber mosaic virus (CMV) 2b suppresses RNA silencing primarily through the binding of double‐stranded RNA (dsRNA) of varying sizes. However, the biologically active form of 2b remains elusive. Here, we demonstrate that the single and double alanine substitution mutants in the N‐terminal 15th leucine and 18th methionine of CMV 2b exhibit drastically attenuated virulence in wild‐type plants, but are efficiently rescued in mutant plants defective in RNA‐dependent RNA polymerase 6 (RDR6) and Dicer‐like 4 (DCL4). Moreover, the transgenic plants of 2b, but not 2blm (L15A/M18A), rescue the high infectivity of CMV‐Δ2b through the suppression of antiviral silencing. L15A, M18A or both weaken 2b suppressor activity on local and systemic transgene silencing. In contrast with the high affinity of 2b to short and long dsRNAs, 2blm is significantly compromised in 21‐bp duplex small interfering RNA (siRNA) binding ability, but maintains a strong affinity for long dsRNAs. In cross‐linking assays, 2b can form dimers, tetramers and oligomers after treatment with glutaraldehyde, whereas 2blm only forms dimers, rather than tetramers and oligomers, in vitro. Together, these findings suggest that L15 and M18 of CMV 2b are required for high affinity to ds‐siRNAs and oligomerization activity, which are essential for the suppression activity of 2b on antiviral silencing.     

dsRNA with 5′ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants

In plants, SGS3 and RNA‐dependent RNA polymerase 6 (RDR6) are required to convert single‐ to double‐stranded RNA (dsRNA) in the innate RNAi‐based antiviral response and to produce both exogenous and endogenous short‐interfering RNAs. Although a role for RDR6‐catalysed RNA‐dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA‐binding protein with unexpected substrate selectivity favouring 5′‐overhang‐containing dsRNA. The conserved XS and coiled‐coil domains are responsible for RNA‐binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi‐based host immune response, is a dsRNA‐binding protein with similar specificity to SGS3. In competition‐binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5′ overhang is required for subsequent steps in RNA‐mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs.     

Generation of tobacco lines with widely different reduction in nicotine levels via RNA silencing approaches

Issues related to the nicotine content of tobacco have been public concerns.Several reports have described decreasing nicotine levels by silencing the putrescine N-methyltransferase (PMT) genes, but the reported variations of nicotine levels among transgenic lines are relatively low in general. Here we describe the generation in tobacco (Nicotiana tabacum) lines with widely different, reduced nicotine levels using three kinds of RNA-silencing approaches.The relative efficacies of suppression were compared among the three approaches regarding the aspect of nicotine level in tobacco leaves.By suppressing expression of the PMT genes, over 200 transgenic lines were obtained with nicotine levels reduced by 9.1-96.7%. RNA interference (RNAi) was the most efficient method of reducing the levels of nicotine,whereas cosuppression and antisense methods were less effective. This report gives clues to the efficient generation of plants with a variety of metabolite levels, and the results demonstrate the relative efficiencies of various RNA-silencing methods.     

Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana

Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway.