Comparative analysis of RNA interference and pattern-triggered immunity induced by dsRNA in response to virus infection
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ABSTRACT: The main goal of this study was to compare the differential efficiency of the antiviral activity triggered by externally delivered virus-specific and nonspecific dsRNAs in plants. RNAi mediated by virus-specific dsRNA is the main antiviral defense mechanism in plants, but nonspecific dsRNA-triggered responses have been documented to play a role in antiviral defense. Using PVX as a model, we have determined that co-inoculation with either virus-specific or nonspecific dsRNA reduced virus accumulation in both inoculated and systemic leaves although at different extent. While the administration of dsRNA specific for the targeted virus induced a potent RNAi-based antiviral response that resulted in highly effective control of viral disease, the degree of interference with PVX-GFP infection afforded by nonspecific dsRNA (PTI) was limited. To identify early biological processes and pathways associated with dsRNA-based immunity, we conducted a global transcriptome RNAseq assay from N. benthamiana leaves inoculated with four different treatments, i.e., dsGFP alone (dsG), PVX-GFP combined with dsGFP (dsG_VG), wild-type (Wt) PVX combined with dsGFP (dsG_V) and bacterial nucleic acid extracts not expressing dsRNA as a control (Ctr). KEGG analysis of differentially expressed genes showed a significant enrichment of terms related to plant-pathogen signaling pathways (KEGG terms Plant-pathogen interaction and MAPK signaling) in all the three treatments with dsRNA. Our results further indicate that the transcriptomic response triggered by dsRNA alone includes canonical immune pathways or genes known to be involved in defense responses, i.e., Ca+2 signaling, ethylene signaling, MAPK signaling, WRKY transcription factors, PR transcriptional factors, NBS-LRR resistance genes, EDS1, and LRR receptor-like kinases, many of which are typical of antimicrobial PTI . Moreover, the transcriptomic response to the homologous combination (dsGFP plus PVX-GFP) had a greater overrepresentation of genes involved in plant-pathogen signaling pathways than the heterologous combination (dsGFP plus Wt PVX), highlighting a quantitative difference between RNAi and PTI immune responses.
Project description:Plants deploy cell surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens. LRR receptor kinases (LRR-RKs) and LRR receptor proteins (LRR-RPs) recognise microbe-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR (NLR) proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes, suggesting pathway convergence upstream of nuclear events. We report that PTI triggered by Arabidopsis LRR-RP (RLP23) requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and ADR1-family helper NLRs, which are all components of ETI. The cell surface LRR-RK SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting formation of constitutive supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane.
Project description:RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates, however whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner, loaded into AGO, and were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer-deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals.
Project description:We report the cloning and sequencing of both endogenous small RNAs and virus-derived siRNAs produced by the antiviral RNAi pathway in Drosophila. We find that a diverse panel of viruses are targeted by the RNAi pathway in Drosophila to produce abundant virus-derived siRNAs, and these siRNAs map to various locations within the viral genomes. Knockdown of various RNAi and miRNA pathway components alters the levels of these viral small RNAs. Drosophila DL1 cells were treated with dsRNA for 3 days to deplete factors involved in the antiviral RNAi pathway and miRNA pathway, then were challenged with one of four viruses for 4 days. Total RNA was collected, and the small RNA populations from 15-29 nt were cloned and sequenced.
Project description:RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl−/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.
Project description:RNA interference (RNAi) functions as an antiviral immune response in plants and invertebrates, whereas mammalian RNAi response has been found so far only in undifferentiated cells and in differentiated cells inactive in interferon (IFN) system or in infections with viruses disabling viral suppressors of RNAi (VSRs), thereby leading to question the physiological importance of the RNAi pathway in mammals. Here, we identified that wild-type Semliki Forest virus (SFV), a prototypic alphavirus, triggered the Dicer-dependent production of abundant viral (v)siRNAs in different mammalian somatic cells in the presence of VSR. These vsiRNAs were produced from viral dsRNA replicative intermediates, almost exclusively located at the 5’ termini of the viral genome, and loaded into AGO, and they were fully active in slicing cognate viral RNAs. Besides, Sindbis virus, another alphavirus, also induced vsiRNA generation in mammalian somatic cells. AGO2 deficiency increased SFV and SINV replication, while enoxacin, a known RNAi enhancer that functions at post steps of siRNA production, efficiently reduced viral replication. The nucleotide sequence at the 5’ termini of SFV and SINV genome is conserved among the Old World alphaviruses, and mutating the conserved sequences resulted in the recombinant SFV being deficient in vsiRNA production and irresponsive to antiviral RNAi. SFV infection also enabled the production of abundant vsiRNAs and antiviral RNAi in IFN-competent adult mice, and importantly, enhanced RNAi by enoxacin protected adult mice from lethal SFV challenge and reduced the virus-induced neuropathogenesis in the central neuron system. Overall, our findings provide evidence that mammalian antiviral RNAi is active in differentiated cells and adult mice with intact IFN response even in the presence of VSR and present a therapeutic strategy against alphaviruses that include many important emerging and reemerging human pathogens.
Project description:Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI) are well-defined modes of plant immunity triggered by recognition of pathogen effector proteins and microbe-associated molecular patterns, respectively. While ETI and PTI network extensively share signaling components, the shared components are used in different ways, resulting in distinct network properties in the model plant Arabidopsis: immunity is highly robust against network perturbations in ETI but relatively sensitive in PTI. However, the molecular mechanism how the shared network leads to the different properties is not known. Here we show that sustained MAPK activation compensate salicylic acid (SA) signaling. A 12 DNA microarray study using total RNA from Arabidopsis transgenic plants carrying DEX-inducible MKK4DD in Col or sid2-2 background treated with DEX or control.
Project description:In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG-I like receptor (RLR) family , which signal to induce production of type I interferons (IFN). These key anti-viral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon-stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG-I, MDA5 and the least-studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus-derived double stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We identify Dicer as an LGP2-associated protein and show that LGP2 inhibits Dicer cleavage of dsRNA into siRNAs both in vitro and in vivo. Further, we show that in cells lacking an IFN response, ectopic expression of LGP2 interferes with RNAi-dependent suppression of gene expression. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs.
Project description:The function and mechanism of the interferon-regulated antiviral responses have been extensively characterized. Recent studies have demonstrated induction of antiviral RNA interference (RNAi) in somatic cells against several mammalian RNA viruses rendered incapable of RNAi suppression. However, little is known about Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNAs) in these cells active in the type I interferon response. Here we show that the dsRNA precursors of influenza vsiRNAs were processed more efficiently than cellular precursor microRNA hairpins by wild-type human Dicer expressed de novo in Dicer-knockout somatic cells. We found that infection of two strains of suckling mice with wild-type Nodamura virus (NoV) was associated with production of silencing-active vsiRNAs and direct sequestration of duplex vsiRNAs by its RNAi suppressor protein B2. Our findings from in vivo infection with Sindbis virus recombinants expressing NoV B2 or carrying a vsiRNA-targeted insert provide evidence for an antiviral function of the induced RNAi response. Interestingly, NoV infection induces expression of RIG-I-like receptor LGP2 to inhibit vsiRNA biogenesis and promote virulent infection in suckling mice. Our findings together reveal efficient Dicer processing of vsiRNA precursors in interferon-competent somatic cells and suckling mice in contrast to synthetic long dsRNA examined previously by in vitro dicing.
Project description:Background: RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double stranded (ds) RNA precursors by Dicer and direct the RNA-induced silencing complex (RISC) to target transcripts. Recent efforts have uncovered important principles governing small RNA (smRNA) sorting into RISC, yet mechanisms defining substrate selection by Dicer proteins remain uncharacterized. Methodology: To better characterize Dicer-2 substrates in Drosophila, we examined the antiviral RNAi response, which generates virus-derived siRNAs from viral RNA. Using high-throughput sequencing, we found that diverse viruses were uniquely targeted; substrates included dsRNA replication intermediates and intramolecular RNA stem loops. smRNA distribution patterns from viral and synthetic dsRNA precursors were highly reproducible, and machine learning techniques identified characteristics of precursor molecules and smRNA duplexes important in determining relative smRNA abundance. Significance: To our knowledge, this study provides the first description of the rules governing Dicer-2 substrate selection, which has important implications for exogenous RNA silencing technologies and the development of smRNA-based antiviral therapeutics. virus-derived siRNA (vsiRNA) expression comparison between control and 4 different virus-infected cells in control as well as 5 different RNAi pathway protein knock-downs in Drosophila dl1 cells
Project description:In RNA interference (RNAi), long double-stranded RNA (dsRNA) is cleaved by Dicer endonuclease into small RNA interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates adopted sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi. Next, we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: Coxsackievirus B3 (CVB3) and encephalomyocarditis virus (ECMV) from Picornaviridae; tick-borne encephalitis virus (TBEV) from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice had no antiviral effect. This result supports insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also report that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.