Modelling the dynamics of viral suppressors of RNA silencing
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Abstract:
Virus infection in plants is limited by RNA silencing. In turn, viruses can counter RNA silencing with silencing suppressors. Viral suppressors of RNA silencing have been shown to play a role in symptom development in plants. We here study four different strategies employed by silencing suppressors: small interfering RNA (siRNA) binding, double-strand RNA (dsRNA) binding and degrading or inactivating Argonaute. We study the effect of the suppressors on viral accumulation within the cell as well as its spread on a tissue with mathematical and computational models. We find that suppressors which target Argonaute are very effective in a single cell, but that targeting dsRNA or siRNA is much more effective at the tissue level. Although targeting Argonaute can be beneficial for viral spread, it can also cause hindrance in some cases owing to raised levels of siRNAs that can spread to other cells.Keywords:
Argonaute
RNA Silencing
Trans-acting siRNA
RNA-induced silencing complex
Piwi-interacting RNA
RNA-induced transcriptional silencing
Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.
Argonaute
RNA-induced silencing complex
Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
Dicer
Piwi-interacting RNA
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Argonaute
Trans-acting siRNA
RNA-induced silencing complex
RNA Silencing
Dicer
RNA-induced transcriptional silencing
Piwi-interacting RNA
RasiRNA
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Plants utilize a variety of defense mechanisms against invading pathogens. RNA-interference (RNAi) is a defense mechanism that plants use to combat viruses1. Double-stranded virus RNA is recognized by host Dicer, an endonuclease that is part of the plant’s immune system, and cleaved at different positions creating 21-25 base pair small interfering RNAs (siRNAs)2. The siRNA becomes part of the RNA-induced silencing complex (RISC) with the protein Argonaute, and is used to locate complementary viral or host mRNAs. Once the complementary strand is located, it is cleaved by Argonaute, a protein component of RISC(Fig. 1), effectively silencing the gene. This phenomenon allows RNA viruses to be used as vectors for silencing host genes in a process termed virus-induced gene silencing (VIGS).
Argonaute
RNA-induced silencing complex
Trans-acting siRNA
RNA Silencing
Dicer
RNA-induced transcriptional silencing
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Abstract Several distinct pathways of RNA silencing operate in plants with roles including the suppression of virus accumulation, control of endogenous gene expression, and direction of DNA and chromatin modifications. Proteins of the Dicer-Like and Argonaute (AGO) families have key roles within these silencing pathways and have distinct biochemical properties. We are interested in the relationships between different silencing pathways and have used Nicotiana benthamiana as a model system. While not being an amenable plant for traditional genetics, N. benthamiana is extensively used for RNA-silencing studies. Using virus-induced gene silencing technology we demonstrate that both NbAGO1- and NbAGO4-like genes are required for full systemic silencing but not for silencing directed by an inverted repeat transgene. Phenotypic differences between the virus-induced gene silencing plants indicate that NbAGO1 and NbAGO4 like act at different stages of the silencing pathways. Suppression of NbAGO1 expression recapitulated the hypomorphic mutant phenotype of certain Arabidopsis (Arabidopsis thaliana) ago1 alleles, however, suppression of NbAgo4 like resulted in phenotypes differing in some respects from those reported for Arabidopsis ago4. We suggest that the small interfering RNA amplification step required for full systemic silencing is dependent upon a nuclear event requiring the activity of NbAGO4 like.
Argonaute
Trans-acting siRNA
RNA-induced silencing complex
Dicer
RNA Silencing
RNA-induced transcriptional silencing
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The RNAi Pathway RNA interference (RNAi) or RNA silencing is a gene regulatory system, widely conserved in eukaryotes, that represses gene expression through a homology-dependent mechanism.This repressive effect is mediated by small non-coding RNAs (sRNAs) of about 20-30 nucleotides, derived from double-stranded RNA (dsRNA) precursors that are recognized and processed by the RNaseIII Dicer.These sRNAs are loaded into an RNA-induced silencing complex (RISC), where the Argonaute protein plays a main role.Upon loading, the sRNAs selectively guide RISC to the target RNAs, causing their degradation or preventing their translation.In certain organisms, including fungi and parasitic protozoa, the silencing mechanism requires RNA-dependent RNA polymerases (RdRPs) to generate dsRNA from single-stranded RNA (ssRNA) or to amplify sRNA signals [1,2].Originally described as a defense mechanism against invasive nucleic acids and viruses, RNAi and related pathways play many fundamental roles in metazoans, including regulation of mRNA accumulation and translation, chromatin silencing, programmed DNA rearrangements, and genome surveillance.
Argonaute
RNA Silencing
RNA-induced silencing complex
Dicer
RNA-induced transcriptional silencing
Trans-acting siRNA
DNA-directed RNA interference
RasiRNA
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Abstract RNA interference/silencing mechanisms triggered by double‐stranded RNA (dsRNA) have been described in many eukaryotes, including fungi. These mechanisms have in common small RNA molecules (siRNAs or microRNAs) originating from dsRNAs that, together with the effector protein Argonaute, mediate silencing. The genome of the fungal pathogen Candida albicans harbours a well‐conserved Argonaute and a non‐canonical Dicer, essential members of silencing pathways. Prototypical siRNAs are detected as members of the C. albicans transcriptome, which is potential evidence of RNA interference/silencing pathways in this organism. Surprisingly, expression of a dsRNA a hairpin ADE2 dsRNA molecule to interfere with the endogenous ADE2 mRNA did not result in down‐regulation of the message or produce adenine auxotrophic strains. Cell free assays showed that the hairpin dsRNA was a substrate for the putative C. albicans Dicer, discounting the possibility that the nature of the dsRNA trigger affects silencing functionality. Our results suggested that unknown cellular events govern the functionality of siRNAs originating from transgenes in RNA interference/silencing pathways in C. albicans . Copyright © 2010 John Wiley & Sons, Ltd.
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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.
RNA-induced silencing complex
Argonaute
Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
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Citations (96)
Argonaute
Dicer
Trans-acting siRNA
RNA Silencing
RNA-induced silencing complex
RNA-induced transcriptional silencing
RasiRNA
DNA-directed RNA interference
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Citations (1)
In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine.
Argonaute
RNA-induced silencing complex
Trans-acting siRNA
RNA Silencing
RNA-induced transcriptional silencing
Piwi-interacting RNA
RasiRNA
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Citations (34)
JMJ14 is a histone H3 Lys4 (H3K4) trimethyl demethylase that affects mobile RNA silencing in an Arabidopsis transgene system. It also influences CHH DNA methylation, abundance of endogenous transposon transcripts, and flowering time. JMJ14 acts at a point in RNA silencing pathways that is downstream from RNA-dependent RNA polymerase 2 (RDR2) and Argonaute 4 (AGO4). Our results illustrate a link between RNA silencing and demethylation of histone H3 trimethylysine. We propose that JMJ14 acts downstream from the Argonaute effector complex to demethylate histone H3K4 at the target of RNA silencing.
Argonaute
RNA-induced transcriptional silencing
RNA-induced silencing complex
Trans-acting siRNA
RNA Silencing
Piwi-interacting RNA
Demethylase
RasiRNA
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Citations (111)