RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants

In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.     

Suppression of antiviral silencing by cucumber mosaic virus 2b protein in Arabidopsis is associated with drastically reduced accumulation of three classes of viral small interfering RNAs

We investigated the genetic pathway in Arabidopsis thaliana targeted during infection by cucumber mosaic virus (CMV) 2b protein, known to suppress non-cell-autonomous transgene silencing and salicylic acid (SA)-mediated virus resistance. We show that 2b expressed from the CMV genome drastically reduced the accumulation of 21-, 22-, and 24-nucleotide classes of viral small interfering RNAs (siRNAs) produced by Dicer-like4 (DCL4), DCL2, and DCL3, respectively. The defect of a CMV 2b-deletion mutant (CMV-Delta2b) in plant infection was efficiently rescued in Arabidopsis mutants producing neither 21- nor 22-nucleotide viral siRNAs. Since genetic analysis further identifies a unique antiviral role for DCL3 upstream of DCL4, our data indicate that inhibition of the accumulation of distinct viral siRNAs plays a key role in 2b suppression of antiviral silencing. Strikingly, disease symptoms caused by CMV-Delta2b in Arabidopsis mutants defective in antiviral silencing were as severe as those caused by CMV, demonstrating an indirect role for the silencing suppressor activity in virus virulence. We found that production of CMV siRNAs without 2b interference depended largely on RNA-dependent RNA polymerase 1 (RDR1) inducible by SA. Given the known role of RDR6-dependent transgene siRNAs in non-cell-autonomous silencing, our results suggest a model in which 2b inhibits the production of RDR1-dependent viral siRNAs that confer SA-dependent virus resistance by directing non-cell-autonomous antiviral silencing.     

Arabidopsis RNA-Dependent RNA Polymerases and Dicer-Like Proteins in Antiviral Defense and Small Interfering RNA Biogenesis during Turnip Mosaic Virus Infection

Plants respond to virus infections by activation of RNA-based silencing, which limits infection at both the single-cell and system levels. Viruses encode RNA silencing suppressor proteins that interfere with this response. Wild-type Arabidopsis thaliana is immune to silencing suppressor (HC-Pro)-deficient Turnip mosaic virus, but immunity was lost in the absence of DICER-LIKE proteins DCL4 and DCL2. Systematic analysis of susceptibility and small RNA formation in Arabidopsis mutants lacking combinations of RNA-dependent RNA polymerase (RDR) and DCL proteins revealed that the vast majority of virus-derived small interfering RNAs (siRNAs) were dependent on DCL4 and RDR1, although full antiviral defense also required DCL2 and RDR6. Among the DCLs, DCL4 was sufficient for antiviral silencing in inoculated leaves, but DCL2 and DCL4 were both involved in silencing in systemic tissues (inflorescences). Basal levels of antiviral RNA silencing and siRNA biogenesis were detected in mutants lacking RDR1, RDR2, and RDR6, indicating an alternate route to form double-stranded RNA that does not depend on the three previously characterized RDR proteins.     

Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs

In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout the TRV genome to siRNA formation. Indirect evidence suggests that DCL2 is responsible for production of a subset of siRNAs derived from the 3' end region of TRV. TRV siRNA biogenesis and antiviral silencing are strongly dependent on the combined activity of the host-encoded RNA-dependent RNA polymerases RDR1, RDR2, and RDR6, thus providing evidence that perfectly complementary double-stranded RNA serves as a substrate for siRNA production. We conclude that the overall composition of viral siRNAs in TRV-infected plants reflects the combined action of several interconnected pathways involving different DCL and RDR activities.     

An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis

The silencing phenotype in Arabidopsis thaliana lines with an inverted repeat transgene under the control of a phloem-specific promoter was manifested in regions around veins due to a mobile signal of silencing. Genetic analysis implicates RNA-DEPENDENT RNA POLYMERASE2 (RDR2) and an RNA polymerase IVa subunit gene (NRPD1a) in the signaling mechanism. We also identified an SNF2 domain-containing protein (CLASSY1) that acts together with RDR2 and NRPD1a in the spread of transgene silencing and in the production of endogenous 24-nucleotide short interfering RNAs (siRNAs). Cytochemical analysis indicates that CLASSY1 may act in the nucleus with NRPD1a and RDR2 in the upstream part of RNA silencing pathways that generate a double-stranded RNA substrate for Dicer-like (DCL) nucleases. DCL3 and ARGONAUTE4 act in a downstream part of the pathway, leading to endogenous 24-nucleotide siRNA production, but are not required for intercellular signaling. From genetic analysis, we conclude that another downstream part of the pathway associated with intercellular signaling requires DCL4 and at least one other protein required for 21-nucleotide trans-acting siRNAs. We interpret the effect of polymerase IVa and trans-acting siRNA pathway mutations in terms of a modular property of RNA silencing pathways.     

p2 of rice stripe virus (RSV) interacts with OsSGS3 and is a silencing suppressor

A rice cDNA library was screened by a galactosidase 4 (Gal4)-based yeast two-hybrid system with Rice stripe virus (RSV) p2 as bait. The results revealed that RSV p2 interacted with a rice protein exhibiting a high degree of identity with Arabidopsis thaliana suppressor of gene silencing 3 (AtSGS3). The interaction was confirmed by bimolecular fluorescence complementation assay. SGS3 has been shown to be involved in sense transgene-induced RNA silencing and in the biogenesis of trans-acting small interfering RNAs (ta-siRNAs), possibly functioning as a cofactor of RNA-dependent RNA polymerase 6 (RDR6). Given the intimate relationships between virus and RNA silencing, further experiments were conducted to show that p2 was a silencing suppressor. In addition, p2 enhanced the accumulation and pathogenicity of Potato virus X in Nicotiana benthamiana. Five genes that have been demonstrated to be targets of TAS3-derived ta-siRNAs were up-regulated in RSV-infected rice. The implications of these findings are discussed.     

Satellite RNA-derived small interfering RNA satsiR-12 targeting the 3' untranslated region of Cucumber mosaic virus triggers viral RNAs for degradation

RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.     

Viroid-induced symptoms in Nicotiana benthamiana plants are dependent on RDR6 activity

Viroids are small self-replicating RNAs that infect plants. How these noncoding pathogenic RNAs interact with hosts to induce disease symptoms is a long-standing unanswered question. Recent experimental data have led to the suggestive proposal of a pathogenic model based on the RNA silencing mechanism. However, evidence of a direct relation between key components of the RNA silencing pathway and symptom expression in infected plants remains elusive. To address this issue, we used a symptomatic transgenic line of Nicotiana benthamiana that expresses and processes dimeric forms of Hop stunt viroid (HSVd). These plants were analyzed under different growing temperature conditions and were used as stocks in grafting assays with the rdr6i-Nb line, in which the RNA-dependent RNA polymerase 6 (RDR6) is constitutively silenced. Here, we show that the symptom expression in N. benthamiana plants is independent of HSVd accumulation levels but dependent on an active state of the viroid-specific RNA silencing pathway. The scion of rdr6i-Nb plants remained asymptomatic when grafted onto symptomatic plants, despite an accumulation of a high level of mature forms of HSVd, indicating the requirement of RDR6 for viroid-induced symptom production. In addition, the RDR6 requirement for symptom expression was also observed in wild-type N. benthamiana plants mechanically infected with HSVd. These results provide biological evidence of the involvement of the viroid-specific RNA silencing pathway in the symptom expression associated with viroid pathogenesis.     

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

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.     

The CaMV transactivator/viroplasmin interferes with RDR6-dependent trans-acting and secondary siRNA pathways in Arabidopsis

Several RNA silencing pathways in plants restrict viral infections and are suppressed by distinct viral proteins. Here we show that the endogenous trans-acting (ta)siRNA pathway, which depends on Dicer-like (DCL) 4 and RNA-dependent RNA polymerase (RDR) 6, is suppressed by infection of Arabidopsis with Cauliflower mosaic virus (CaMV). This effect was associated with overaccumulation of unprocessed, RDR6-dependent precursors of tasiRNAs and is due solely to expression of the CaMV transactivator/viroplasmin (TAV) protein. TAV expression also impaired secondary, but not primary, siRNA production from a silenced transgene and increased accumulation of mRNAs normally silenced by the four known tasiRNA families and RDR6-dependent secondary siRNAs. Moreover, TAV expression upregulated DCL4, DRB4 and AGO7 that mediate tasiRNA biogenesis. Our findings suggest that TAV is a general inhibitor of silencing amplification that impairs DCL4-mediated processing of RDR6-dependent double-stranded RNA to siRNAs. The resulting deficiency in tasiRNAs and other RDR6-/DCL4-dependent siRNAs appears to trigger a feedback mechanism that compensates for the inhibitory effects.