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Summary In some plant–virus interactions plants show a sign of healing from virus infection, a phenomenon called symptom recovery. It is assumed that the meristem exclusion of the virus is essential to this process. The discovery of RNA silencing provided a possible mechanism to explain meristem exclusion and recovery. Here we show evidence that silencing is not the reason for meristem exclusion in Nicotiana benthamiana plants infected with Cymbidium ringspot virus (CymRSV). Transcriptome analysis followed by in situ hybridization shed light on the changes in gene expression in the shoot apical meristem (SAM) on virus infection. We observed the down‐regulation of meristem‐specific genes, including WUSCHEL (WUS). However, WUS was not down‐regulated in the SAM of plants infected with meristem‐invading viruses such as turnip vein‐clearing virus (TVCV) and cucumber mosaic virus (CMV). Moreover, there is no connection between loss of meristem function and fast shoot necrosis since TVCV necrotized the shoot while CMV did not. Our findings suggest that the observed transcriptional changes on virus infection in the shoot are key factors in tip necrosis and symptom recovery. We observed a lack of GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDH) expression in tissues around the meristem, which likely stops virus replication and spread into the meristem.

This article belongs to the Special Issue Plant Defense Responses against Viruses., RNA silencing is a sequence specific post-transcriptional mechanism regulating important biological processes including antiviral defense in plants. Argonaute (AGO) proteins, the catalytic subunits of the silencing complexes, are loaded with small RNAs to execute the sequence specific RNA cleavage or translational inhibition. Plants encode several AGO proteins and a few of them, especially AGO1 and AGO2, have been shown to be required for antiviral silencing. Previously, we have shown that the P1 protein of the sweet potato mild mottle virus (SPMMV) suppresses the primary RNA silencing response by inhibiting AGO1. To analyze the role of AGO2 in antiviral defense against the SPMMV, we performed a comparative study using a wild type and ago2−/− mutant Nicotiana benthamiana. Here we show that the AGO2 of N. benthamiana attenuates the symptoms of SPMMV infection. Upon SPMMV infection the levels of AGO2 mRNA and protein are greatly increased. Moreover, we found that AGO2 proteins are loaded with SPMMV derived viral small RNAs as well as with miRNAs. Our results indicate that AGO2 protein takes over the place of AGO1 to confer antiviral silencing. Finally, we provide a plausible explanation for the AGO2 mediated recovery of an SPMMV-infected sweet potato., This study was supported by grants of the Hungarian Ministry of Technology and Innovation by grant numbers GINOP-2.3.2-15-2016-00015 and GINOP-2.3.2-15-2016-00032). Additional financial support at CRAG was provided by Generalitat de Catalunya CERCA institution and by the Ministry of Science and Innovation-State Research Agency (AEI, Spain) through the “Severo Ochoa Programme for Centres of Excellence in R&D” SEV-2015-0533 and CEX2019-000902-S and the grant PID2019-105692RB-100 to JLM.

Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here, we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which are produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. Here we show that NSD is required for the elimination of 5′ cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants.

RNA guided ribonuclease complexes play central role in RNA interference. Members of the evolutionarily conserved Argonaute protein family form the catalytic cores of these complexes. Unlike a number of other plant Argonautes, the role of AGO2 has been obscure until recently. Newer data, however, have indicated its involvement in various biotic and abiotic stress responses. Despite its suggested importance, there is no detailed characterization of this protein to date. Here we report cloning and molecular characterization of the AGO2 protein of the virological model plant Nicotiana benthamiana. We show that AGO2 can directly repress translation via various miRNA target site constellations (ORF, 3' UTR). Interestingly, although AGO2 seems to be able to silence gene expression in a slicing independent fashion, its catalytic activity is still a prerequisite for efficient translational repression. Additionally, mismatches between the 3' end of the miRNA guide strand and the 5' end of the target site enhance gene silencing by AGO2. Several functionally important amino acid residues of AGO2 have been identified that affect its small RNA loading, cleavage activity, translational repression potential and antiviral activity. The data presented here help us to understand how AGO2 aids plants to deal with stress.

RNA silencing is a homology-dependent gene inactivation mechanism that regulates a wide range of biological processes including antiviral defense. To deal with host antiviral responses viruses evolved mechanisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Besides working as silencing suppressors, these proteins may also fulfill other functions during infection. In many cases the interplay between the suppressor function and other “unrelated” functions remains elusive. We will present host factors implicated in antiviral pathways and summarize the current status of knowledge about the diverse viral suppressors’ strategies acting at various steps of antiviral silencing in plants. Besides, we will consider the multi-functionality of these versatile proteins and related biochemical processes in which they may be involved in fine-tuning the plant-virus interaction. Finally, we will present the current applications and discuss perspectives of the use of these proteins in molecular biology and biotechnology.

RNA silencing is not only an evolutionarily conserved gene regulatory mechanism, but in plants also serves as the basis for robust adaptive antiviral immune responses. ARGONAUTE (AGO) proteins form the catalytic cores of the RNA-guided ribonuclease complexes, which play a central role in RNA silencing. Here we describe an in vivo assay system for analyzing the activities of AGO proteins in the virological model plant Nicotiana benthamiana .

RNA silencing is not only an evolutionarily conserved gene regulatory mechanism, but in plants also serves as the basis for robust adaptive antiviral immune responses. ARGONAUTE (AGO) proteins form the catalytic cores of the RNA-guided ribonuclease complexes, which play a central role in RNA silencing. Here we describe an in vivo assay system for analyzing the activities of AGO proteins in the virological model plant Nicotiana benthamiana .

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination., Author Summary To better understand the specific effect of p19 viral suppressor of RNA silencing (VSR) on antiviral silencing and endogenous small RNA pathways, we generated a N. benthamiana plant (p19syn) capable of sustaining the ectopic expression of the Cymbidium ringspot virus (CymRSV) p19 upon infection with a suppressor-deficient CymRSV (Cym19stop). By using wt and p19syn plants in combination with CymRSV and Cym19stop, we were able to analyze the effects of p19 provided “in trans” and “in cis” during the viral invasion of the plant. We have shown that p19 can efficiently sequester endogenous small RNAs (sRNAs) in mock-inoculated p19syn plants while it does not bind these sRNAs upon Cym19stop infection. Also, the presence of p19 in virus infection did not alter the expression of miRNAs significantly. These findings do not support the widely accepted assumption that viral symptoms are the direct consequence of the impact of VSRs on endogenous silencing pathways. We demonstrated that p19 preferentially sequesters positive:negative viral short interfering RNAs (vsiRNAs) pairs and that the binding by p19 is independent of vsiRNA sequence or the type of the 5’-end nucleotide. We have also found that 3’ truncation is induced on p19 bound sRNAs. Finally using AGO1- and AGO2- immunoprecipitation experiments we observed that p19 specifically compromises vsiRNAs’ loading into AGO1 but not AGO2. Since antiviral silencing is strongly inhibited by p19, this suggests that AGO1 is the main effector protein against CymRSV tombusvirus.

RNA silencing is an evolutionary conserved sequence-specific gene inactivation mechanism that contributes to the control of development, maintains heterochromatin, acts in stress responses, DNA repair and defends against invading nucleic acids like transposons and viruses. In plants RNA silencing functions as one of the main immune systems. RNA silencing process involves the small RNAs and trans factor components like Dicers, Argonautes and RNA-dependent RNA polymerases. To deal with host antiviral silencing responses viruses evolved mechanisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Due to the overlap between endogenous and antiviral silencing pathways while blocking antiviral pathways viruses also impact endogenous silencing processes. Here we provide an overview of antiviral silencing pathway, host factors implicated in it and the crosstalk between antiviral and endogenous branches of silencing. We summarize the current status of knowledge about the viral counter-defense strategies acting at various steps during virus infection in plants with the focus on representative, well studied silencing suppressor proteins. Finally we discuss future challenges of the antiviral silencing and counter-defense research field.

In plants, RNA silencing is a surveillance mechanism against invading viruses. It involves the production of virus-derived small interfering RNAs (vsiRNAs), which guide the RNA-induced silencing complex (RISC) to inactivate viruses. vsiRNAs may also promote the silencing of host mRNAs in a sequence-specific manner. In this work, vsiRNAs derived from two grapevine-infecting viruses (Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus) were selected from cDNA libraries of short RNAs and were cross-referenced with the remnants of both cleaved host transcripts and viral RNAs from a degradome dataset. We identified dozens of host transcripts targeted by vsiRNAs. Among them, several encode putative proteins involved in ribosome biogenesis and in biotic and abiotic stresses. Moreover, we identified vsiRNAs which explain the cleavage sites in viral genomes. A consistent fraction of vsiRNAs did not apparently account for cleavage, suggesting that only a low percentage of vsiRNAs are involved in the antiviral response.

Powdery mildew is one of the most important cereal diseases worldwide. Genetic analysis has revealed that mutant alleles of the Mlo gene cause broad-spectrum resistance against this pathogen in barley. In this study, the possibility of inducing broad-spectrum powdery mildew resistance against this pathogen by RNAi of the barley Mlo ortholog in wheat was examined using virus-induced gene silencing (VIGS). A clear correlation was found between resistance and accumulation of Mlo-specific siRNAs, raising the possibility of designing powdery mildew resistance in wheat by RNA silencing using both transgenic and non-transgenic approaches.

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 infection and replication of viruses in the host induce diverse mechanisms for combating viral infection. One of the best-studied antiviral defence mechanisms is based on RNA silencing. Consistently, several viral suppressors of RNA silencing (VSRs) have been identified from almost all plant virus genera, which are surprisingly diverse within and across kingdoms, exhibiting no obvious sequence similarities. VSRs efficiently inhibit host antiviral responses by interacting with the key components of cellular silencing machinery, often mimicking their normal cellular functions. Recent findings have revealed that the impact of VSRs on endogenous pathways is more complex and profound than had been estimated thus far. This review highlights the current understanding of and new insights into the mechanisms and functions of plant VSRs.

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.

Plant viruses are inducers and targets of RNA silencing. Viruses counteract with RNA silencing by expressing silencing-suppressor proteins. Many of the identified proteins bind siRNAs, which prevents assembly of silencing effector complexes, and also interfere with their 3′ methylation, which protects them against degradation. Here, we investigated the 3′ modification of silencing-related small RNAs in Nicotiana benthamiana plants infected with viruses expressing RNA silencing suppressors, the p19 protein of Carnation Italian ringspot virus (CIRV) and HC-Pro of Tobacco etch virus (TEV). We found that CIRV had only a slight effect on viral siRNA 3′ modification, but TEV significantly inhibited the 3′ modification of si/miRNAs. We also found that p19 and HC-Pro were able to bind both 3′ modified and non-modified small RNAs in vivo. The findings suggest that the 3′ modification of viral siRNAs occurs in the cytoplasm, though miRNA 3′ modification likely takes place in the nucleus as well. Both silencing suppressors inhibited the 3′ modification of si/miRNAs when they and small RNAs were transiently co-expressed, suggesting that the inhibition of si/miRNA 3′ modification requires spatial and temporal co-expression. Finally, our data revealed that a HEN1-like methyltransferase might account for the small RNA modification at the their 3′-terminal nucleotide in N. benthamiana.

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants.

The multiplicity of infection (MOI), i.e., the number of viral genomes that infect a cell, is an important parameter in virus evolution, which for each virus and environment may have an optimum value that maximizes virus fitness. Thus, the MOI might be controlled by virus functions, an underexplored hypothesis in eukaryote-infecting viruses. To analyze if the MOI is controlled by virus functions, we estimated the MOI in plants coinfected by two genetic variants of Tomato bushy stunt virus (TBSV); by TBSV and a TBSV-derived defective interfering RNA (DI-RNA); or by TBSV and a second tombusvirus, Cymbidium ringspot virus (CymRSV). The MOI was significantly larger in TBSV-CymRSV coinfections (∼4.0) than in TBSV-TBSV or TBSV–DI-RNA coinfections (∼1.7 to 2.2). Coinfections by CymRSV or TBSV with chimeras in which an open reading frame (ORF) of one virus species was replaced by that of the other identified a role of viral proteins in determining the MOI, which ranged from 1.6 to 3.9 depending on the coinfecting genotypes. However, no virus-encoded protein or genomic region was the sole MOI determinant. Coinfections by CymRSV and TBSV mutants in which the expression of the gene-silencing suppressor protein p19 was abolished also showed a possible role of gene silencing in MOI determination. Taken together, these results demonstrate that the MOI is a quantitative trait showing continuous variation and that as such it has a complex determination involving different virus-encoded functions. IMPORTANCE The number of viral genomes infecting a cell, or the multiplicity of infection (MOI), is an important parameter in virus evolution affecting recombination rates, selection intensity on viral genes, evolution of multipartite genomes, or hyperparasitism by satellites or defective interfering particles. For each virus and environment, the MOI may have an optimum value that maximizes virus fitness, but little is known about MOI control in eukaryote-infecting viruses. We show here that in plants coinfected by two genotypes of Tomato bushy stunt virus (TBSV), the MOI was lower than in plants coinfected by TBSV and Cymbidium ringspot virus (CymRSV). Coinfections by CymRSV or TBSV with TBSV-CymRSV chimeras showed a role of viral proteins in MOI determination. Coinfections by CymRSV and TBSV mutants not expressing the gene-silencing suppressor protein also showed a role of gene silencing in MOI determination. The results demonstrate that the MOI is a quantitative trait with a complex determination involving different viral functions.

Defective interfering (DI) RNAs of tombusviruses are short, non-coding, symptom-modulating RNAs originating from the viral genome. The presence of homologous DI RNA in virus infection attenuates the otherwise lethal viral symptoms. Nicotiana benthamiana plants infected with tomato bushy stunt tombusvirus pepper isolate (TBSV-P) show severe symptoms, which culminate in the death of the plant. In contrast, plants co-inoculated with TBSV-P and TBSV-P-derived DI RNA display attenuated symptoms. However, co-inoculation of TBSV-P with heterologous DI RNA, originating from Carnation Italian ringspot tombusvirus results in development of apical necrotic symptoms. To localize the symptom-determining factors on DI RNA genome, chimeras of protective and non-protective DI RNAs have been constructed. All chimeras were biologically active and accumulated to a high level in the presence of helper virus. We identified a 5' proximal sequence element of the DI RNA as the most important symptom determinant region. However, our results demonstrated that the symptom modulating ability of this region is also influenced by the sequence composition of whole DI RNAs.

RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected with Cymbidium ringspot tombusvirus (CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show that Tobacco mosaic virus (TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation of PDS siRNAs than the corresponding antisense PDS sequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.

RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus . We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.

Defective interfering (DI) RNAs are subviral replicons originating from the viral genome and are associated with many plant RNA viruses and nearly all animal RNA viruses. The presence of DI RNAs in tombusvirus-infected plants reduces the accumulation of helper virus RNA and results in the development of attenuated symptoms similar to those caused by tombusviruses defective in p19, the posttranscriptional gene silencing (PTGS) suppressor. In situ analysis of infected plants containing DI RNAs revealed that the extent of virus infection was spatially restricted as was found for p19-defective tombusvirus. Previously, p19 was shown to suppress PTGS by sequestering the small interfering RNAs (siRNAs), which act as the specificity determinant for PTGS. Our results demonstrate that DI RNAs dramatically elevate the level of virus-specific siRNAs in viral infections, resulting in the saturation of p19 and the accumulation of unbound siRNAs. Moreover, we showed that, at low temperature, where PTGS is inhibited, DI RNAs are not able to efficiently interfere with virus accumulation and protect the plants. These data show that the activation of PTGS plays a pivotal role in DI RNA-mediated interference. Our data also support a role for 21-nucleotide siRNAs in PTGS signaling.

RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19–siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing.

In plants, posttranscriptional gene silencing (PTGS) is an ancient and effective defense mechanism against viral infection. A number of viruses encode proteins that suppress virus-activated PTGS. The p19 protein of tombusviruses is a potent PTGS suppressor which interferes with the onset of PTGS-generated systemic signaling and is not required for viral replication or for viral movement in Nicotiana benthamiana. This unique feature of p19 suppressor allowed us to analyze the mechanism of PTGS-based host defense and its viral suppression without interfering with other viral functions. In contrast to the necrotic symptoms caused by wild-type tombusvirus, the infection of p19-defective mutant virus results in the development of a typical PTGS-associated recovery phenotype in N. benthamiana . In this report we show the effect of PTGS on the viral infection process for N. benthamiana infected with either wild-type Cymbidium Ringspot Tombusvirus (CymRSV) or a p19-defective mutant (Cym19stop). In situ analyses of different virus-derived products revealed that PTGS is not able to reduce accumulation of virus in primary infected cells regardless of the presence of p19 PTGS suppressor. We also showed that both CymRSV and Cym19stop viruses move systemically in the vasculature, with similar efficiencies. However, in contrast to the uniform accumulation of CymRSV throughout systemically infected leaves, the presence of Cym19stop virus was confined to and around the vascular bundles. These results suggest that the role of p19 is to prevent the onset of mobile signal-induced systemic PTGS ahead of the viral infection front, leading to generalized infection.

RNA interference (RNAi) is a double-stranded (ds)RNA-inducible, sequence-specific RNA-degradation mechanism that operates as a natural antiviral system in plants and animals. Successful virus infection requires evasion or suppression of RNAi. Indeed, RNAi suppressor proteins have been identified in plant and animal viruses, although the molecular mechanism of silencing inhibition is still poorly understood. Because many RNA viruses encode dsRNA-binding proteins (dsRBPs) and as RNAi is triggered by the accumulation of dsRNAs, dsRBPs were examined to see if they inhibit RNAi. Here, it is shown that heterologous dsRBPs suppressed RNAi in plants, indicating that in natural host-virus interactions, pathogen-encoded dsRBPs could inactivate RNAi-mediated host defences.

Temperature dramatically affects plant–virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well-known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing-based phenotypes of transgenic plants are lost. Consistently, the levels of virus- and transgene-derived small (21–26 nucleotide) interfering (si) RNAs—the central molecules of RNA silencing-mediated defence pathways—are dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not influence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes.

Posttranscriptional gene silencing (PTGS) processes double-stranded (ds) RNAs into 21–25 nucleotide (nt) RNA fragments that direct ribonucleases to target cognate mRNAs. In higher plants, PTGS also generates mobile signals conferring sequence-specific silencing in distant organs. Since PTGS acts as an antiviral system in plants, successful virus infection requires evasion or suppression of gene silencing. Here we report that the 19 kDa protein (p19) of tombusviruses is a potent silencing suppressor that prevents the spread of mobile silencing signal. In vitro, p19 binds PTGS-generated, 21–25 nt dsRNAs and 21-nt synthetic dsRNAs with 2-nt 3′ overhanging end(s), while it barely interacts with single-stranded (ss) RNAs, long dsRNAs or blunt-ended 21-nt dsRNAs. We propose that p19 mediates silencing suppression by sequestering the PTGS-generated 21–25 nt dsRNAs, thus depleting the specificity determinants of PTGS effector complexes. Moreover, the observation that p19-expressing transgenic plants show altered leaf morphology might indicate that the p19-targeted PTGS pathway is also important in the regulation of plant development.

Co-inoculation of Nicotiana benthamiana plants with in vitro transcripts of both genomic and short defective interfering (DI) RNAs of Cymbidium ringspot virus results in an accumulation of de novo generated DI RNA dimers. Time-course analysis of DI RNA accumulation in the inoculated leaves showed early accumulation of DI RNA dimers followed by increased levels of DI RNA monomers. In contrast, DI RNA dimers were barely detectable in systems where cell-to-cell movement does not take place (protoplasts) or is less important (monomeric DI RNA-expressing transgenic plants). Our results also demonstrated that the size of DI RNAs is important in the colonization of inoculated leaves, suggesting that DI RNA dimers are quickly selected for cell-to-cell movement if short DI RNA monomers are used for infection.

Post-transcriptional gene silencing (PTGS) is a sequence-specific degradation mechanism that operates in almost all eukaryotic cells. In plants, double-stranded RNA triggers PTGS, generating 21- to 25-nucleotide guide RNAs responsible for specific degradation of cognate mRNA. The double stranded RNA intermediates of replicating plant viruses often induce PTGS, leading to symptom attenuation. Here we demonstrate the role of PTGS in defective interfering (DI) RNA-mediated symptom attenuation in plants infected with Cymbidium ringspot tombusvirus (CymRSV). Analysis of 21- to 25-nucleotide RNAs in Nicotiana benthamiana infected with CymRSV indicated that PTGS was not spread homogeneously along the viral genome. The 21- to 25-nucleotide RNAs derived mainly from plus-stranded RNA and likely arose from local basepaired structures. In contrast to helper viral RNA, short DI RNAs were not accessible to helper virus-induced RNA degradation guided by the 21- to 25-nucleotide RNAs. Our results suggest a model in which PTGS plays an important role in the selective accumulation and symptom attenuation mediated by DI RNAs. Because PTGS operates in a wide variety of different organisms, this model is applicable to DI RNA generation and accumulation in both plant and animal cells.

Avirulent genes either directly or indirectly produce elicitors that are recognized by specific receptors of plant resistance genes, leading to the induction of host defense responses such as hypersensitive reaction (HR). HR is characterized by the development of a necrotic lesion at the site of infection which results in confinement of the invader to this area. Artificial chimeras and mutants of cymbidium ringspot (CymRSV) and the pepper isolate of tomato bushy stunt (TBSV-P) tombusviruses were used to determine viral factors involved in the HR resistance phenotype of Datura stramonium upon infection with CymRSV. A series of constructs carrying deletions and frameshifts of the CymRSV coat protein (CP) undoubtedly clarified that an 860-nucleotide (nt)-long RNA sequence in the CymRSV CP coding region (between nt 2666 and 3526) is the elicitor of a very rapid HR-like response of D. stramonium which limits the virus spread. This finding provides the first evidence that an untranslatable RNA can trigger an HR-like resistance response in virus-infected plants. The effectiveness of the resistance response might indicate that other nonhost resistance could also be due to RNA-mediated HR. It is an appealing explanation that RNA-mediated HR has evolved as an alternative defense strategy against RNA viruses.

The complete nucleotide sequences of the genome of the pepper isolate of tomato bushy stunt Tombusvirus (TBSV-P), and its defective interfering (DI) RNAs were determined. The genome of TBSV-P is a linear single-stranded monopartite RNA molecule of positive polarity, 4776 nucleotides long and has an organisation identical to that reported for other tombusviruses. In vitro transcripts of the genome were highly infectious, and it could support replication of the DI RNAs associated with the wild type virus. Two DI RNAs were found in the infected leaves of Nicotiana clevelandii, whose sequences were completely derived from the genomic RNA. The longest DI RNA (DI-5) has 550 nucleotides (nt), while the shorter DI RNA (DI-4) composed of 463 nt, both of them were formed by essentially the same genomic sequence blocks. Since host specificity of TBSV-P and other tombusviruses with available infectious cDNA clones is different, it is feasible to carry out gene exchange studies to determine viral host specificity factors for tombusviruses.

RNA viruses which do not have a poly(A) tail or a tRNA-like structure for the protection of their vulnerable 3′ termini may have developed a different strategy to maintain their genome integrity. We provide evidence that deletions of up to 7 nucleotides from the 3′ terminus of cucumber mosaic cucumovirus (CMV) satellite RNA (satRNA) were repaired in planta in the presence of the helper virus (HV) CMV. Sequence comparison of 3′-end-repaired satRNA progenies, and of satRNA and HV RNA, suggested that the repair was not dependent on a viral template. The 3′ end of CMV satRNA lacking the last three cytosines was not repaired in planta in the presence of tomato aspermy cucumovirus (TAV), although TAV is an efficient helper for the replication of CMV satRNA. With use of pseudorecombinants constructed by the interchange of RNAs 1 and 2 of TAV and CMV, evidence was provided that the 3′-end repair was controlled by RNAs 1 and 2 of CMV, which encode subunits of the viral RNA replicase. These results, and the observation of short repeated sequences close to the 3′ terminus of repaired molecules, suggest that the HV replicase maintains the integrity of the satRNA genome, playing a role analogous to that of cellular telomerases.

RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene-inactivation system that plays an essential role in many biological processes, such as genome defense against mobile DNA elements or regulation of factors involved in plant and animal development. In higher plants and invertebrates, it also functions as a powerful antiviral mechanism. To overcome antiviral RNAi, viruses have evolved suppressor proteins which counteract host RNAi-based antiviral processes and target one or more key points in the RNAi machinery. Here, we review recent progress in our understanding of the mechanism and function of antiviral RNAi in plants and on the viral responses through the expression of silencing suppressor proteins. As a counter-attack RNAi may also regulate innate immunity in plants and contribute to a novel layer of defense against pathogen attack. We also discuss emerging evidence that viruses use RNAi to manipulate host gene expression to modify the cellular environment for the benefit of invading viruses.

Although the microRNA miR-146a is an important regulator of immunological processes and contributes to the pathogenesis of certain B cell lymphoma types, in B cells the epigenetic regulation of miR-146a expresion has not been studied yet. To elucidate the mechanisms controlling miR-146a expression in B lymphoid cells we analysed epigenetic marks, including CpG methylation and histone modifications, at the miR-146a promoter in well characterized Epstein-Barr virus (EBV) positive and EBV negative B cell lines. In addition, EBV positive epithelial cell lines were also studied as controls. In cells with a silent miR-146a promoter the 5' regulatory sequences comprising a CpG island were devoid of activating histone modifications, independently of the methylation pattern of the regulatory region. The regulatory sequences flanking the inactive miR-146 promoter were hypermethylated at CpG dinucleotides in the EBV positive Burkitt's lymphoma (BL) cell lines of memory B cell phenotype (Rael and Akata), partially methylated in the mammary carcinoma cell lines C2G6 and C4A3, and completely unmethylated in the nasopharyngeal carcinoma cell line C666-1. In contrast, in EBV positive cell lines of activated B cell phenotype, and EBV negative BL cell lines the invariably unmethylated 5' regulatory sequences of active miR-146a promoters were enriched in the euchromatic histone modification marks acetylated histone H3, acetylated histone H4, and histone H3 dimethylated at lysine 4. The euchromatic histone modification marks extended over the immediate vicinity of the transcriptional initiation site to the 3' intron, too. We concluded that similarly to the promoters of protein coding genes, both DNA methylation and histone modifications contribute to the host cell dependent expression of miR-146a.

RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene-inactivation system that plays an essential role in many biological processes, such as genome defense against mobile DNA elements or regulation of factors involved in plant and animal development. In higher plants and invertebrates, it also functions as a powerful antiviral mechanism. To overcome antiviral RNAi, viruses have evolved suppressor proteins which counteract host RNAi-based antiviral processes and target one or more key points in the RNAi machinery. Here, we review recent progress in our understanding of the mechanism and function of antiviral RNAi in plants and on the viral responses through the expression of silencing suppressor proteins. As a counter-attack RNAi may also regulate innate immunity in plants and contribute to a novel layer of defense against pathogen attack. We also discuss emerging evidence that viruses use RNAi to manipulate host gene expression to modify the cellular environment for the benefit of invading viruses.

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CymRSV) was used to identify the cis -acting nature of the last 77-nt sequence of the viral genome which is required for DI RNA accumulation. The 3′-terminal cis -essential domain of both genomic and DI RNAs can be folded into a stable stem–loop structure composed of three hairpins and two short non-base-paired regions. None of the three conserved stem–loops can be deleted without abolishing the infectivity of DI RNA. Similarly, those mutants in which base-paired stem regions were disrupted by single-, double-, or triple-base substitutions were unable to replicate. However, when the original structures were reconstructed by compensatory mutations the viability of the molecules was also restored. Limited mutation (1 or 2 nt) in the non-base-paired region did not show any significant effect on viral replication. Our results strongly suggest that the proposed structure for the 3′ terminus of the viral genome is very important for viral RNA replication. It is very likely that the function of this structure is to promote the minus-strand synthesis of CymRSV DI RNA. Evidence is provided that the proposed 3′-terminal structure is relevant not only for CymRSV DI but for genomic RNA as well.

The nucleotide sequence of a cDNA clone corresponding to the entire RNA 3 of a pepper isolate of tomato aspermy cucumovirus (P-TAV) was determined. The 2222 nucleotides (nt) long RNA 3 is dicistronic and codes a 3a (280 amino acids) and a coat protein (cp, 218 amino acids). The nucleotide sequence of P-TAV RNA 3 shows a high sequence identity (99%) with that of C-TAV RNA 3 and the amino acid sequences of the 3a and the coat proteins between these two TAV strains are also very similar. The transcription start site of the subgenomic RNA 4 was mapped to the G at the position of 1148 of RNA 3. A full-length cDNA copy of P-TAV RNA-3 was cloned downstream of the phage T7 promoter. Transcripts synthesized in vitro had no extra nucleotides at the 5' end, but there were nine non-viral nucleotides at the 3' terminus. The transcripts of TAV RNA 3 were infectious when coinoculated into test plants in the presence of purified RNA 1 and 2 of P-TAV or Trk7-CMV respectively. Northern blot analysis of RNA extracted from inoculated plants showed the expected genomic and subgenomic RNAs.

The 33-kDa and its 92-kDa readthrough protein genes (open reading frames (ORFs) 1 and 2) of cymbidium ringspot tombusvirus (CyRSV) were introduced into Nicotiana benthamiana plants. Protoplasts derived from transgenic plants expressing the first two ORFs were inoculated with in vitro transcripts of defective interfering (DI) RNA. The efficient replication of input DI RNA, which is incapable of autonomous replication, confirmed the biological activity of the expressed viral replicase. In addition, in vitro transcripts of a genomic RNA deletion mutant clone encoding only ORFs 1 and 2 were able to replicate on their own and could support the replication of CyRSV DI RNAs in N. benthamiana protoplasts.

The complete nucleotide sequence (2209 nucleotides) of the RNA 3 of the cucumber mosaic virus strain Trk7 was determined. Trk7-CMV possesses two open reading frames which encode the 3a protein (279 amino acids) and the coat protein (218 amino acids). Sequence analysis proved the earlier biological and serological classification of the RNA 3 molecule of Trk7-CMV in subgroup II. A high degree of homology was found in the strains Q and Kin of CMV, while the average similarity between the two different subgroups is much lower.

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5′-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene., Author Summary Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is an interesting subviral RNA because it changes the green mosaic induced by CMV into a bright yellow mosaic in Nicotiana tabacum. The molecular basis underlying the induction of symptoms by viruses is not well understood, and this Y-sat-mediated modification of symptoms has been a long-standing mystery. In this study, we discovered the molecular mechanism involved in the Y-sat-induced yellowing. First, we showed that transgenic N. benthamiana plants that expressed the inverted-repeat sequence of Y-sat also developed a yellow phenotype, similar to the Y-sat-infected plants. Then, we found that tobacco magnesium protoporphyrin chelatase subunit I gene (ChlI, the key gene involved in chlorophyll synthesis) was downregulated in the transgenic plants and in the Y-sat-infected plants. We then identified a 22-nt long sequence that is complementary to the Y-sat including four G-U pairs in the ChlI mRNA. Finally, we demonstrated that a short interfering RNA (siRNA) derived from Y-sat specifically targeted and downregulated the ChlI mRNA, thus impairing the chlorophyll biosynthesis pathway. This discovery of the molecular basis of the symptom modification induced by Y-sat is the first demonstration that a subviral RNA can induce disease symptoms by regulating host gene expression through the RNA silencing machinery.

The host-virus interaction is a continuous coevolutionary race involving both host defence strategies and virus escape mechanisms. RNA silencing is one of the main processes employed by eukaryotic organisms to fight viruses. However, viruses encode suppressor proteins to counteract this antiviral mechanism. Virtually all plant viruses encode at least one suppressor. In spite of being highly diverse at the protein level, a large group of these proteins inhibit RNA silencing very similarly, by sequestration of double-stranded RNA or small-interfering RNA molecules, the central players of the pathway. The RNA binding capacity of virus suppressor proteins can be studied by the electrophoretic mobility shift assay method. Also known as gel retardation assay, gel mobility assay, gel shift assay or band shift assay, EMSA is an in vitro technique used to characterize protein:DNA or protein:RNA interactions. The method had been developed based on the observation that protein: nucleic acid complexes migrate slower through a non-denaturing polyacrylamide gel than the free nucleic acid fragments. Here, we provide a detailed protocol for the analysis of crucifer-infecting Tobacco mosaic tobamovirus (cr-TMV) silencing suppressor protein p122 RNA binding capacity.

The host-virus interaction is a continuous coevolutionary race involving both host defence strategies and virus escape mechanisms. RNA silencing is one of the main processes employed by eukaryotic organisms to fight viruses. However, viruses encode suppressor proteins to counteract this antiviral mechanism. Virtually all plant viruses encode at least one suppressor. In spite of being highly diverse at the protein level, a large group of these proteins inhibit RNA silencing very similarly, by sequestration of double-stranded RNA or small-interfering RNA molecules, the central players of the pathway. The RNA binding capacity of virus suppressor proteins can be studied by the electrophoretic mobility shift assay method. Also known as gel retardation assay, gel mobility assay, gel shift assay or band shift assay, EMSA is an in vitro technique used to characterize protein:DNA or protein:RNA interactions. The method had been developed based on the observation that protein: nucleic acid complexes migrate slower through a non-denaturing polyacrylamide gel than the free nucleic acid fragments. Here, we provide a detailed protocol for the analysis of crucifer-infecting Tobacco mosaic tobamovirus (cr-TMV) silencing suppressor protein p122 RNA binding capacity.

The complete nucleotide sequence of plum pox virus (PPV) strain SK 68 was determined from a series of overlapping cDNA clones. The exact 5′ terminus was determined by direct RNA sequencing. The RNA sequence was 9786 nucleotides in length, excluding a 3′ terminal poly(A) sequence. The large open reading frame starts at nucleotide position 147 and is terminated at position 9568. Comparison of cistrons from other plum pox virus strains with those predicted for the SK 68 strain indicated the same genomic organizations. Comparison of sequences leads to the following conclusions: (1) The genetic organization of all four PPV strains is identical, containing one large polyprotein gene and two noncoding regions at the 5′ and 3′ ends; (2) pairwise comparison of the genomic sequence of PPV SK 68 with other PPV strains shows 11% alteration. Sequence differences among strains are spread in a uniform manner upon the genome, except for the P1, HC-pro, and two noncoding regions, which are more conserved (with a 4% and 6.6% change). The stability of the noncoding regions is probably linked to their role in replication. The sequence variation has little effect on the amino acid sequence of the corresponding polypeptides, as changes occur preferentially in the third position of the reading frame triplets, except in the case of the 5′ end of the coat protein gene (2.7% average difference in amino acid level, while in the case of coat protein it is 7.7%). The sequence analysis of the coat protein region of the four complete and one partial sequence indicates that the Hungarian plum pox virus strain diverges at the larger extent, similar to the El Amar strain, from which only less than half of the sequence is available.

Genomic RNA of cymbidium ringspot tombusvirus (CyRSV) contains five large open reading frames (ORFs). The two 5′ proximal ORFs encode proteins of 33 and 92 kDa and the three 3′ proximal ORFs encode proteins of 41,22, and 19 kDa. In addition, a small reading frame encoding a protein of 4 kDa was recently observed by computer analysis of the 3′ nontranslated region of CyRSV and other tombusvirus RNAs (ORF 6). The function of putative gene products was investigated with the use of several mutants. Mutants in ORFs 1 and 2 were not viable indicating that both 33- and 92-kDa proteins are required for replication. Deletion of a large portion of the coat protein gene, encoding a 41-kDa protein, did not prevent replication of viral RNA and cell-to-cell movement, but interfered severely with long-distance translocation. No virus particles or viral RNA could be detected in inoculated or upper leaves of plants inoculated with transcripts obtained from mutants not expressing the 22-kDa protein. However, replication and encapsidation occurred normally in inoculated protoplasts indicating that this gene product is a transport protein. Conversely, no role could be assigned to putative gene products of ORF 5 (19-kDa protein) and QRF 6. It was also shown that factors other than gene expression can influence the replication of RNA mutants, probably due to instability of RNA molecules.