Resistant plants respond rapidly to invading avirulent plant viruses by triggering a hypersensitive response (HR). An HR is accompanied by a restraint of virus multiplication and programmed cell death (PCD), both of which have been observed in systemic necrosis triggered by a successful viral infection. Here, we analyzed signaling pathways underlying the HR in resistance genotype plants and those leading to systemic necrosis. We show that systemic necrosis in Nicotiana benthamiana, induced by Plantago asiatica mosaic virus (PlAMV) infection, was associated with PCD, biochemical features, and gene expression patterns that are characteristic of HR. The induction of necrosis caused by PlAMV infection was dependent on SGT1, RAR1, and the downstream mitogen-activated protein kinase (MAPK) cascade involving MAPKKKalpha and MEK2. However, although SGT1 and RAR1 silencing led to an increased accumulation of PlAMV, silencing of the MAPKKKalpha-MEK2 cascade did not. This observation indicates that viral multiplication is partly restrained even in systemic necrosis induced by viral infection, and that this restraint requires SGT1 and RAR1 but not the MAPKKKalpha-MEK2 cascade. Similarly, although both SGT1 and MAPKKKalpha were essential for the Rx-mediated HR to Potato virus X (PVX), SGT1 but not MAPKKKalpha was involved in the restraint of PVX multiplication. These results suggest that systemic necrosis and HR consist of PCD and a restraint of virus multiplication, and that the latter is induced through unknown pathways independent from the former.

Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.Viruses are obligate intracellular pathogens that manipulate and exploit the molecular mechanism of the host to survive in a hostile cellular environment. The presence of viruses and their propagation induces diverse mechanisms in the host for combating viral infection at both the single-cell and the wholeorganism levels. In plants, one of the most important defense mechanisms against viruses is RNA silencing, which is a sequence-specific RNA degradation process regulated by small RNA molecules (11,12). Since RNA viruses go through singlestranded RNA or double-stranded RNA (dsRNA) stages at a given point in their replication, they are both active initiators and targets of host RNA silencing. When plant RNA viruses invade host cells, dsRNAs derived from their replication intermediates or highly structured genomic RNAs are recognized by plant dicer-like nucleases and processed into small interfering RNAs (siRNAs) of 21 to 24 nucleotides in length. The siRNAs are then recruited to a multiprotein effector complex called the RNA-induced silencing complex (RISC), which includes the slicer endonuclease Argonaute and subsequently mediates the cleavage of cognate viral RNAs. RNA silencing is a non-cell-autonomous event in higher plants and, once RNA silencing is induced in the initial cell, it spreads over the whole organism through the vasculature and from cell to cell presumably via plasmodesmata, mediated by mobile small RNA signals (3,14,22,36,48,51). The cell-to-cell and long-distance movement of virus-derived small RNA signals likely serves to immunize surrounding naive cells ahead of the infection front.

RNA silencing is an important defence mechanism against virus infection, and many plant viruses encode RNA silencing suppressors as a counter defence. In this study, we analysed the RNA silencing suppression ability of multiple virus species of the genus Potexvirus. Nicotiana benthamiana plants exhibiting RNA silencing of a green fluorescent protein (GFP) transgene showed reversal of GFP fluorescence when systemically infected with potexviruses. However, the degree of GFP fluorescence varied among potexviruses. Agrobacterium-mediated transient expression assay in N. benthamiana leaves demonstrated that the triple gene block protein 1 (TGBp1) encoded by these potexviruses has drastically different levels of silencing suppressor activity, and these differences were directly related to variations in the silencing suppression ability during virus infection. These results suggest that suppressor activities differ even among homologous proteins encoded by viruses of the same genus, and that TGBp1 contributes to the variation in the level of RNA silencing suppression by potexviruses. Moreover, we investigated the effect of TGBp1 encoded by Plantago asiatica mosaic virus (PlAMV), which exhibited a strong suppressor activity, on the accumulation of microRNA, virus genomic RNA and virus-derived small interfering RNAs. INTRODUCTIONRNA silencing is an RNA-guided gene regulatory mechanism that operates in a wide variety of eukaryotic organisms. RNA silencing begins with processing of an RNA trigger into small RNAs (~21-30 nt) by the RNase III-type enzyme Dicer (Bernstein et al., 2001). In plants, small RNAs are divided into two classes: small interfering RNAs (siRNAs) and microRNAs (miRNAs) (Mallory & Vaucheret, 2006;Brodersen & Voinnet, 2006). siRNAs are generated from double-stranded RNAs (dsRNAs) which are usually derived from transgenes, viruses or endogenous non-coding RNA genes. On the other hand, miRNAs are generated from genome-encoded precursor RNAs with imperfect stem-loop structures. These small RNAs are then incorporated into multicomponent RNA-induced silencing complexes (RISC), which contain an Argonaute (AGO) family protein and lead to homologous RNA cleavage or translational repression, and (or) DNA/chromatin methylation (Hammond, 2005;Xie et al., 2004;Chapman & Carrington, 2007).One of the important roles of RNA silencing in plants is antiviral defence (Ratcliff et al., 1997;Vance & Vaucheret, 2001;Baulcombe, 2004;Ding & Voinnet, 2007). During virus infection, dsRNAs derived from viral replication intermediates or highly structured viral genomic RNAs trigger RNA silencing directed against the viruses. Consequently, initially symptomatic plants recover from infection and become resistant to secondary infection with homologous viruses. To counteract RNA silencing, viruses have evolved RNA silencing suppressors. More than 40 RNA silencing suppressors have been identified in plant, animal, insect and fungal viruses (Li & Ding, 2006). These suppressors do not have obvious sequence similarity to one another, and t...

RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/ RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs.

Plant virus expression vectors provide a powerful tool for basic research as well as for practical applications. Here, we report the construction of an expression vector based on plantago asiatica mosaic virus (PlAMV), a member of the genus Potexvirus. Modification of a vector to enhance the expression of a foreign gene, combined with the use of the foot-and-mouth disease virus 2A peptide, allowed efficient expression of the foreign gene in two model plant species, Arabidopsis thaliana and Nicotiana benthamiana. Comparison with the widely used potato virus X (PVX) vector demonstrated that the PlAMV vector retains an inserted foreign gene for a longer period than PVX. Moreover, our results showed that the GFP expression construct PlAMV-GFP exhibits stronger RNA silencing suppression activity than PVX-GFP, which is likely to contribute to the stability of the PlAMV vector.