Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

MAIN CONCLUSION: The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against the broad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

Limited dsRNA editing impedes leukemia stem cells.

Understanding the mechanisms underlying the generation and maintenance of leukemia stem cells (LSCs) is crucial for the development of effective therapies against T cell acute lymphoblastic leukemia (T-ALL). In a recent study, Rivera et al. discovered that elevated adenosine deaminase acting on RNA (ADAR)-1-mediated RNA editing is a distinguishing feature of T-ALL relapse, and that ADAR1 suppresses apoptosis triggered by the double-stranded (ds)RNA-sensing pathway.

RNA is a pro-apoptotic target of cisplatin in cancer cell lines and C. elegans.

Cisplatin not only targets DNA but also RNA. However, it is largely unknown whether platinated RNA (Pt-RNA) causes apoptosis and thus contributes to the cytotoxic effects of cisplatin. Consequently, cellular RNA was isolated from HepG2 and LS180 cells, exposed to cisplatin, and the resulting Pt-RNA (20 ng Pt/µg RNA) was transfected into these cancer cell lines or used to treat an apoptosis reporter Caenorhabditis elegans (C. elegans) strain (MD701, expressing CED-1::GFP). Cellular and molecular effects of Pt-RNA were evaluated by luminogenic caspase 3/7 assays, PCR array analysis, and fluorescence microscopy-based quantification of apoptosis in C. elegans gonads. Assuming RNA cross-linking (pseudo double-stranded RNA), the contribution of the Toll-like receptor 3 (TLR3, a sensor of double-stranded RNA) to apoptosis induction in cancer cell lines was investigated by pharmacological TLR3 inhibition and overexpression. In contrast to controls, Pt-RNA significantly enhanced apoptosis in C. elegans (2-fold) and in the cancer cell lines (2-fold to 4-fold). TLR3 overexpression significantly enhanced the pro-apoptotic effects of Pt-RNA in HepG2 cells. TLR3 inhibition reduced the pro-apoptotic effects of Pt-RNA and cisplatin, but not of paclitaxel (off-target control). Gene expression analysis showed that Pt-RNA (but not RNA) significantly enhanced the mRNA levels of nuclear factor kappa B subunit 2 and interleukin-8 in HepG2 cells, suggesting that Pt-RNA is a damage-associated molecular pattern that additionally causes pro-inflammatory responses. Together, this data suggests that not only DNA but also cellular RNA is a functionally relevant target of cisplatin, leading to pro-apoptotic and immunogenic effects.

Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells.

Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The IFN-inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for adenosine deaminase acting on RNA 1 (ADAR1) in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110-interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways. SIGNIFICANCE: These findings implicate DHX9 as a suppressor of dsRNA sensing. In some cell lines, loss of DHX9 alone is sufficient to cause activation of dsRNA sensing pathways, while in other cell lines DHX9 functions redundantly with ADAR1 to suppress pathway activation.

Multifaceted roles of RNA editing enzyme ADAR1 in innate immunity.

Innate immunity must be tightly regulated to enable sensitive pathogen detection while averting autoimmunity triggered by pathogen-like host molecules. A hallmark of viral infection, double-stranded RNAs (dsRNAs) are also abundantly encoded in mammalian genomes, necessitating surveillance mechanisms to distinguish "self" from "nonself." ADAR1, an RNA editing enzyme, has emerged as an essential safeguard against dsRNA-induced autoimmunity. By converting adenosines to inosines (A-to-I) in long dsRNAs, ADAR1 covalently marks endogenous dsRNAs, thereby blocking the activation of the cytoplasmic dsRNA sensor MDA5. Moreover, beyond its editing function, ADAR1 binding to dsRNA impedes the activation of innate immune sensors PKR and ZBP1. Recent landmark studies underscore the utility of silencing ADAR1 for cancer immunotherapy, by exploiting the ADAR1-dependence developed by certain tumors to unleash an antitumor immune response. In this perspective, we summarize the genetic and mechanistic evidence for ADAR1's multipronged role in suppressing dsRNA-mediated autoimmunity and explore the evolving roles of ADAR1 as an immuno-oncology target.

Blockade of pan-viral propagation by inhibition of host cell PNPT1.

For successful viral propagation within infected cells, the virus needs to overcome the cellular integrated stress response (ISR), triggered during viral infection, which, in turn, inhibits general protein translation. This paper reports a tactic employed by viruses to suppress the ISR by upregulating host cell polyribonucleotide nucleotidyltransferase 1 (PNPT1). The propagation of adenovirus, murine cytomegalovirus and hepatovirus within their respective host cells induces PNPT1 expression. Notably, when PNPT1 is knocked down, the propagation of all three viruses is prevented. Mechanistically, the inhibition of PNPT1 facilitates the relocation of mitochondrial double-stranded RNAs (mt-dsRNAs) to the cytoplasm, where they activate RNA-activated protein kinase (PKR). This activation leads to eukaryotic initiation factor 2α (eIF2α) phosphorylation, resulting in the suppression of translation. Furthermore, by scrutinizing the PNPT1 recognition element and screening 17,728 drugs and bioactive compounds approved by the US Food and Drug Administration, lanatoside C was identified as a potent PNPT1 inhibitor. This compound impedes the propagation of adenovirus, murine cytomegalovirus and hepatovirus, and suppresses production of the severe acute respiratory syndrome coronavirus-2 spike protein. These discoveries shed light on a novel strategy to impede pan-viral propagation by activating the host cell mt-dsRNA-PKR-eIF2α signalling axis.

DsRNA-based pesticides: Considerations for efficiency and risk assessment.

In view of the ongoing climate change and the ever-growing world population, novel agricultural solutions are required to ensure sustainable food supply. Microbials, natural substances, semiochemicals and double stranded RNAs (dsRNAs) are all considered potential low risk pesticides. DsRNAs function at the molecular level, targeting specific regions of specific genes of specific organisms, provided that they share a minimal sequence complementarity of approximately 20 nucleotides. Thus, dsRNAs may offer a great alternative to conventional chemicals in environmentally friendly pest control strategies. Any low-risk pesticide needs to be efficient and exhibit low toxicological potential and low environmental persistence. Having said that, in the current review, the mode of dsRNA action is explored and the parameters that need to be taken into consideration for the development of efficient dsRNA-based pesticides are highlighted. Moreover, since dsRNAs mode of action differs from those of synthetic pesticides, custom-made risk assessment schemes may be required and thus, critical issues related to the risk assessment of dsRNA pesticides are discussed here.

Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

Mechanistic exploration of autophagy and aging by RNA interference.

Cellular senescence is a cellular process with organismal impact that is mechanistically counterbalanced to a certain extent by frequent episodes of autophagy. Here we describe a detailed, automation-compatible method for the use of RNA-interference (RNAi; also called post-transcriptional gene silencing (PTGS))-mediated silencing of autophagy related protein-coding gene expression. RNAi is a conserved biological response to double-stranded RNA that mediates resistance to endogenous parasites and exogenous pathogenic nucleic acids. RNAi mediated by short interfering RNA (siRNA) is widely used for gene function analysis. The accurate use of RNAi for the inference of gene function necessitates that both specificity and efficacy of the siRNA-mediated knockdown are monitored. In this manuscript, we exemplify these crucial steps employing siRNAs targeting the autophagy and lysosomal biogenesis associated transcription factor TFE3 and validate their specificity on protein and mRNA level.

RNA interference-based exogenous double-stranded RNAs confer resistance to Rhizoctonia solani AG-3 on Nicotiana tabacum.

BACKGROUND: Rhizoctonia solani Kühn is a pathogenic fungus causing tobacco target spot disease, and leads to great losses worldwide. At present, resistant varieties and effective control strategy on tobacco target spot disease are very limited. Host-induced gene silencing (HIGS) as well as the exogenous dsRNA can be used to suppress disease progression, and reveal the function of crucial genes involved in the growth and pathogenesis of the fungus. RESULTS: The silencing of endoPGs or RPMK1 in host plants by TRV-based HIGS resulted in a significant reduction in disease development in Nicotiana benthamiana. In vitro analysis validated that red fluorescence signals were consistently observed in the hyphae treated with Cy3-fluorescein-labeled dsRNA at 12, 24, 48 and 72 h postinoculation (hpi). Additionally, application of dsRNA-endoPGs, dsRNA-RPMK1 and dsRNA-PGMK (fusion of partial endoPGs and RPMK1 sequences) effectively inhibited the hyphal growth of R. solani YC-9 in vitro and suppressed disease progression in the leaves, and quantitative real-time PCR confirmed that the application of dsRNAs significantly reduced the expression levels of endoPGs and RPMK1. CONCLUSION: These results provide theoretical basis and new direction for RNAi approaches on the prevention and control of disease caused by R. solani. © 2024 Society of Chemical Industry.

In Vivo Production of dsRNA Using Bacteriophage ϕ6 in Pseudomonas syringae Cit7 Cells.

RNA interference (RNAi), also known as post-transcriptional gene silencing (PTGS), is one of the emerging genetic engineering techniques to effectively silence or inhibit the expression of target genes. This chapter describes a method for in vivo production of dsRNA in non-pathogenic Pseudomonas syringae strains using phage ϕ6 RNA-dependent RNA polymerase, extraction and purification of dsRNA from bacterial solution, and the use of dsRNA to induce silencing of green fluorescent protein (GFP) in transgenic Nicotiana benthamiana.

Subcellular Colocalization Assay of Host Factors with Viral Replication Complex in the dsRNA Reporter Nicotiana benthamiana.

As obligate pathogens, plant viruses co-opt several host factors for viral replication. Double-stranded RNA (dsRNA) plays important roles in plants, including eliciting innate immune responses and RNA interference. dsRNA also represents the genetic entities of a number of viruses and is a marker of infection by positive-sense single-stranded RNA viruses. Previous detection methods for RNA viruses basically relied on immunological methods, but later researchers discovered that the dsRNA-binding domain of the Flock house virus B2 protein is a perfect alternative to the J2 mAb for sensitive and rapid detection of long dsRNA in vitro and in vivo, and developed B2:GFP transgenic Nicotiana benthamiana line. This method describes in detail how to visualize host factors in the viral replication complex in time and space with the help of B2:GFP transgenic plants, exemplified by Turnip mosaic virus (TuMV), a representative virus member of the Potyviruses.

Production of Double-Stranded RNA in Planta by a Potato Mop-Top Virus (PMTV)-Based Vector for Inducing Gene Silencing.

RNA silencing (also known as gene silencing) is an evolutionary conserved mechanism that is involved in regulating gene expression, suppressing mobile elements, and defensing virus infection. RNA silencing is triggered by double-stranded RNA via Dicer or Dicer-like riboendonucleases. DsRNAs are also the replication intermediates of all RNA viruses; as a result, plant RNA viruses are ideal candidates to induce RNA silencing. A large body of plant viruses have been modified into vectors for RNA silencing in varied plant species. Here, we described a simple, time-saving, and operable system for gene function and genetic breeding study of potato and Nicotiana benthamiana using a potato mop-top (MPTV)-based vector.

Transiently Induce RNA Silencing in Plants Using a Tobacco Necrosis Virus A (TNV-A)-Based dsRNA Production System.

Transgenic expression of hairpin RNA or artificial microRNA is widely used for genetic studies in plant science. However, induction of RNA silencing by transgenic method may have a problem when studying essential genes. Here, we provide an in planta transient double-stranded RNA (dsRNA) producing system using a tobacco necrosis virus A (TNV-A)-based replicon for efficiently inducing RNA silencing in plants. In this system, the target sequence is placed between the cauliflower mosaic virus 35S promoter and the 3'-terminal part of viral genomic RNA, while the C-terminal part of TNV-A RNA-dependent RNA polymerase (p82C) is expressed by a different promoter. The endogenous RNA polymerase-synthesized target sequence is recruited by p82C to produce dsRNA to induce RNA silencing.

Co-immunoprecipitation-Based Isolation of Double-Stranded RNA-Associated Protein Complexes in Nicotiana benthamiana.

Double-stranded RNA (dsRNA) is associated with most viral infections, and is generated in host cells during viral replication. Viral RNA replication occurs within the viral factories called the viral replication complexes (VRCs). In addition to viral genome, viral-derived dsRNA and replicase, the VRCs composition remains largely unexplored. The dsRNA binding domain of the B2 protein from Flock house virus has been reported to be used for detecting viral-derived long dsRNA in plants efficiently. Nicotiana benthamiana is widely used as a model plant for plant-microbe interactions owing to its susceptibility to diverse plant diseases, especially viral diseases. Here, we describe the use of Nicotiana benthamiana stably expressing GFP-tagged dsRNA binding protein (B2: GFP) to pull down dsRNA and associated host and viral proteins from turnip mosaic virus-infected plants. The obtained protein complexes are compatible with functional assays, Western blotting, and mass spectrometry. This system provides a valuable and robust tool to study VRC proteome in N. benthamiana upon plant viral infections.

Utilization of Bacteriophage phi6 for the Production of High-Quality Double-Stranded RNA Molecules.

Double-stranded RNA (dsRNA) molecules are mediators of RNA interference (RNAi) in eukaryotic cells. RNAi is a conserved mechanism of post-transcriptional silencing of genes cognate to the sequences of the applied dsRNA. RNAi-based therapeutics for the treatment of rare hereditary diseases have recently emerged, and the first sprayable dsRNA biopesticide has been proposed for registration. The range of applications of dsRNA molecules will likely expand in the future. Therefore, cost-effective methods for the efficient large-scale production of high-quality dsRNA are in demand. Conventional approaches to dsRNA production rely on the chemical or enzymatic synthesis of single-stranded (ss)RNA molecules with a subsequent hybridization of complementary strands. However, the yield of properly annealed biologically active dsRNA molecules is low. As an alternative approach, we have developed methods based on components derived from bacteriophage phi6, a dsRNA virus encoding RNA-dependent RNA polymerase (RdRp). Phi6 RdRp can be harnessed for the enzymatic production of high-quality dsRNA molecules. The isolated RdRp efficiently synthesizes dsRNA in vitro on a heterologous ssRNA template of any length and sequence. To scale up dsRNA production, we have developed an in vivo system where phi6 polymerase complexes produce target dsRNA molecules inside Pseudomonas cells.

Effective delivery and selective insecticidal activity of double-stranded RNA via complexation with diblock copolymer varies with polymer block composition.

BACKGROUND: Chemical insecticides are an important tool to control damaging pest infestations. However, lack of species specificity, the rise of resistance and the demand for biological alternatives with improved ecotoxicity profiles means that chemicals with new modes of action are required. RNA interference (RNAi)-based strategies using double-stranded RNA (dsRNA) as a species-specific bio-insecticide offer an exquisite solution that addresses these issues. Many species, such as the fruit pest Drosophila suzukii, do not exhibit RNAi when dsRNA is orally administered due to degradation by gut nucleases and slow cellular uptake pathways. Thus, delivery vehicles that protect and deliver dsRNA are highly desirable. RESULTS: In this work, we demonstrate the complexation of D. suzukii-specific dsRNA for degradation of vha26 mRNA with bespoke diblock copolymers. We study the ex vivo protection of dsRNA against enzymatic degradation by gut enzymes, which demonstrates the efficiency of this system. Flow cytometry then investigates the cellular uptake of Cy3-labelled dsRNA, showing a 10-fold increase in the mean fluorescence intensity of cells treated with polyplexes. The polymer/dsRNA polyplexes induced a significant 87% decrease in the odds of survival of D. suzukii larvae following oral feeding only when formed with a diblock copolymer containing a long neutral block length (1:2 cationic block/neutral block). However, there was no toxicity when fed to the closely related Drosophila melanogaster. CONCLUSION: We provide evidence that dsRNA complexation with diblock copolymers is a promising strategy for RNAi-based species-specific pest control, but optimisation of polymer composition is essential for RNAi success. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Larvae of Colorado potato beetle (Leptinotarsa decemlineata Say) resistant to double-stranded RNA (dsRNA) remain susceptible to small-molecule pesticides.

BACKGROUND: Implementation of resistance management tools is crucial for the continued efficacy of insect control technologies. An important aspect of insect resistance management (IRM) is the combined or sequential use of different modes-of-action to reduce selection pressure and delay evolution of resistance. This is especially important for insect pests with established ability to develop resistance to insecticides, such as the Colorado potato beetle (Leptinotarsa decemlineata, CPB). A new class of insecticides, based on double-stranded RNA (dsRNA) activating the gene silencing RNA-interference (RNAi) pathway, are currently under review for regulatory approval and commercial use in the USA against CPB. However, there is no information available on the potential for cross-resistance between RNAi insecticides and other classes of insecticides used against CPB. Herein, we aim to fill this knowledge gap by capitalizing on the availability of a CPB strain highly resistant to dsRNAs and test its susceptibility to diverse small-molecule insecticide classes compared to reference dsRNA-susceptible CPB strains. RESULTS: Differences in activity were observed among the four insecticides tested, with abamectin demonstrating highest activity against all three strains of CPB. However, no differences were observed among the dsRNA-resistant and susceptible CPB strains for any of the tested compounds. Overall, these results demonstrate lack of cross-resistance to commonly used chemical insecticides in the dsRNA-resistant strain of CPB. CONCLUSION: These data support the use of these different insecticide classes along with RNAi-based insecticides as part of an effective insect resistance management framework aimed at delaying resistance in CPB. © 2023 Society of Chemical Industry.

The nanovirus U2 protein suppresses RNA silencing via three conserved cysteine residues.

Nanoviruses have multipartite, circular, single-stranded DNA genomes and cause huge production losses in legumes and other crops. No viral suppressor of RNA silencing (VSR) has yet been reported from a member of the genus Nanovirus. Here, we demonstrate that the nanovirus U2 protein is a VSR. The U2 protein of milk vetch dwarf virus (MDV) suppressed the silencing of the green fluorescent protein (GFP) gene induced by single-stranded and double-stranded RNA, and the systemic spread of the GFP silencing signal. An electrophoretic mobility shift assay showed that the U2 protein was able to bind double-stranded 21-nucleotide small interfering RNA (siRNA). The cysteine residues at positions 43, 79 and 82 in the MDV U2 protein are critical to its nuclear localization, self-interaction and siRNA-binding ability, and were essential for its VSR activity. In addition, expression of the U2 protein via a potato virus X vector induced more severe necrosis symptoms in Nicotiana benthamiana leaves. The U2 proteins of other nanoviruses also acted as VSRs, and the three conserved cysteine residues were indispensable for their VSR activity.

In search of critical dsRNA targets of ADAR1.

Recent studies have underscored the pivotal role of adenosine-to-inosine RNA editing, catalyzed by ADAR1, in suppressing innate immune interferon responses triggered by cellular double-stranded RNA (dsRNA). However, the specific ADAR1 editing targets crucial for this regulatory function remain elusive. We review analyses of transcriptome-wide ADAR1 editing patterns and their evolutionary dynamics, which offer valuable insights into this unresolved query. The growing appreciation of the significance of immunogenic dsRNAs and their editing in inflammatory and autoimmune diseases and cancer calls for a more comprehensive understanding of dsRNA immunogenicity, which may promote our understanding of these diseases and open doors to therapeutic avenues.