Dicer-Like Protein 4 and RNA-Dependent RNA Polymerase 6 Are Involved in Tomato Torrado Virus Pathogenesis in Nicotiana benthamiana.

Tomato torrado virus (ToTV) is a type member of the Torradovirus genus in the Secoviridae family known to cause severe necrosis in susceptible tomato varieties. ToTV also infects other Solanaceae plants, including Nicotiana benthamiana, where it induces distinctive disease symptoms: plant growth drop with the emergence of spoon-like malformed systemic leaves. Virus-induced post-transcriptional gene silencing (PTGS) is significant among plant defense mechanisms activated upon virus invasion. The PTGS, however, can be counteracted by suppressors of RNA silencing commonly found in viruses, which efficiently disrupt the antiviral defense of their host. Here, we addressed the question of PTGS antiviral activity and its suppression in N. benthamiana during ToTV infection-a phenomenon not described for any representative from the Torradovirus genus so far. First, we showed that neither the Vp26-a necrosis-inducing pathogenicity determinant of ToTV-nor other structural viral proteins limited the locally induced PTGS similar to p19, a well-characterized potent suppressor of RNA silencing of tombusviruses. Moreover, by employing wild-type and transgenic lines of N. benthamiana with suppressed Dicer-like 2 (DCL2), Dicer-like 4 (DCL4), Argonaute 2 and RNA-dependent RNA polymerase 6 (RDR6) proteins, we proved their involvement in anti-ToTV defense. Additionally, we identified DCL4 as the major processor of ToTV-derived siRNA. More importantly, our results indicate the essential role of the Suppressor of Gene Silencing 3 (SGS3)/RDR6 pathway in anti-ToTV defense. Finally, we conclude that ToTV might not require a potent RNA silencing suppressor during infection of the model plant N. benthamiana.

The nonstop decay and the RNA silencing systems operate cooperatively in plants.

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.

Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants.

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.

Functional dissection of a plant Argonaute.

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 May Play a Role in but Is Not the Only Determinant of the Multiplicity of Infection.

UNLABELLED: 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.

Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis.

BACKGROUND: Nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. It can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. To discover and characterize the miRNAs and their cleaved target mRNAs in N. benthamiana, we sequenced small RNA transcriptomes and degradomes of two N. benthamiana accessions and validated them by Northern blots. RESULTS: We used a comprehensive molecular approach to detect and to experimentally validate N. benthamiana miRNAs and their target mRNAs from various tissues. We identified 40 conserved miRNA families and 18 novel microRNA candidates and validated their target mRNAs with a genomic scale approach. The accumulation of thirteen novel miRNAs was confirmed by Northern blot analysis. The conserved and novel miRNA targets were found to be involved in various biological processes including transcription, RNA binding, DNA modification, signal transduction, stress response and metabolic process. Among the novel miRNA targets we found the mRNA of REPRESSOR OF SILENCING (ROS1). Regulation of ROS1 by a miRNA provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ROS1 activity. CONCLUSIONS: The identified conserved and novel miRNAs along with their target mRNAs also provides a tissue specific atlas of known and new miRNA expression and their cleaved target mRNAs of N. benthamiana. Thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.

AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation.

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.

RNA interference-mediated intrinsic antiviral immunity in plants.

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.

The 5' regulatory sequences of active miR-146a promoters are hypomethylated and associated with euchromatic histone modification marks in B lymphoid cells.

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.

A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery.

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.

Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs.

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.

Structural and functional analysis of viral siRNAs.

A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.

Virus-induced gene silencing of Mlo genes induces powdery mildew resistance in Triticum aestivum.

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.

CRISPR-Cas9 Targeting of the eIF4E1 Gene Extends the Potato Virus Y Resistance Spectrum of the Solanum tuberosum L. cv. Desirée.

Translation initiation factors and, in particular, the eIF4E family are the primary source of recessive resistance to potyviruses in many plant species. However, no eIF4E-mediated resistance to this virus genus has been identified in potato (Solanum tuberosum L.) germplasm. As in tomato, the potato eIF4E gene family consists of eIF4E1, its paralog eIF4E2, eIF(iso)4E, and nCBP. In tomato, eIF4E1 knockout (KO) confers resistance to a subset of potyviruses, while the eIF4E1/2 double KO, although conferring a broader spectrum of resistance, leads to plant developmental defects. Here, the tetraploid potato cv. Desirée owning the dominant Ny gene conferring resistance to potato virus Y (PVY) strain O but not NTN was used to evaluate the possibility to expand its PVY resistance spectrum by CRISPR-Cas9-mediated KO of the eIF4E1 susceptibility gene. After a double process of plant protoplast transfection-regeneration, eIF4E1 KO potatoes were obtained. The knockout was specific for the eIF4E1, and no mutations were identified in its eIF4E2 paralog. Expression analysis of the eIF4E family shows that the disruption of the eIF4E1 does not alter the RNA steady-state level of the other family members. The eIF4E1 KO lines challenged with a PVYNTN isolate showed a reduced viral accumulation and amelioration of virus-induced symptoms suggesting that the eIF4E1 gene was required but not essential for its multiplication. Our data show that eIF4E1 editing can be usefully exploited to broaden the PVY resistance spectrum of elite potato cultivars, such as Desirée, by pyramiding eIF4E-mediated recessive resistance.

Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1.

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.

Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis.

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at the post-transcriptional level. Many plant miRNAs are conserved, and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have only been found in certain species. Here, we use deep-sequencing, computational and molecular methods to identify, profile, and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. A total of 24 conserved miRNA families were identified in all four tissues, and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs, we also found 21 new grapevine-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNA candidates have been described. We have found that many known and new miRNAs showed tissue-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine-specific miRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.

Argonaute 2 Controls Antiviral Activity against Sweet Potato Mild Mottle Virus in Nicotiana benthamiana.

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.

Inhibition of 3' modification of small RNAs in virus-infected plants require spatial and temporal co-expression of small RNAs and viral silencing-suppressor proteins.

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.