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At least 19 recordsLinked to original sources
PubMed2025

Argonaute 2 targets viral transcripts but not genomes of RNA viruses during antiviral RNA interference in Drosophila.

RNA interference (RNAi) mediated by the small interfering RNA (siRNA) pathway is a major antiviral mechanism in insects. This pathway is triggered when double-stranded RNA (dsRNA) produced during virus replication is recognized by Dicer-2, leading to the formation of virus-derived siRNA duplexes. These siRNAs are loaded onto the programmable nuclease Argonaute-2 (AGO2), with one strand serving as a guide to target and cleave fully complementary sequences of viral RNAs. While siRNAs are generated from viral dsRNA, the specific viral RNA species targeted for silencing during RNA virus replication remains unclear. In this study, we characterized the primary viral RNA targets of the Drosophila siRNA pathway during infections caused by negative and positive RNA viruses, namely Vesicular stomatitis virus (VSV) and Sindbis virus (SINV). Our findings reveal that polyadenylated transcripts of VSV and SINV are the major targets of silencing by the siRNA pathway during infection, likely when they are poised for translation. Consistent with earlier findings, we show that AGO2 is associated with ribosomes in control and virus infected cells. Therefore, we propose that the inhibition of the replication of RNA viruses in Drosophila results from the silencing of incoming viral transcripts, facilitated by the association of AGO2 with ribosomes.

Animals↗
PubMed2026

Development of a Droplet-Based RNA Interference Feeding Assay for Neonates of the Citrus Root Weevil Diaprepes abbreviatus.

The citrus root weevil, Diaprepes abbreviatus, is an economically important pest of citrus and ornamental crops whose subterranean larval feeding damages roots and predisposes plants to secondary pathogen infection. Development of efficient RNA interference (RNAi) delivery methods for early larval stages is essential for functional genomics studies and the evaluation of RNAi-based pest management strategies. In this study, we developed a droplet-based feeding assay for oral delivery of double-stranded RNA (dsRNA) to neonates of D. abbreviatus using chitin synthase 2 (DaCHS2) as a model RNAi target to validate the assay. Feeding solutions containing dsRNA were supplemented with sucrose and bromophenol blue dye, with bromophenol blue used to visually confirm ingestion. Across three independent biological replicates, all neonates exposed to DaCHS2-dsRNA, GFP-dsRNA, and water control droplets were confirmed to have ingested the feeding solution (45/45 neonates per treatment; 100% feeding success). Oral delivery of dsRNA targeting DaCHS2 reduced transcript abundance and was associated with developmental abnormalities and mortality, including incomplete molting, abnormal pigmentation, cuticular deformities, defective pupation, and malformed adults. Regression analysis demonstrated moderate and significant relationship between dsRNA concentration and neonate mortality and developmental abnormalities. RT-qPCR further confirmed reduced DaCHS2 transcript abundance following oral dsRNA exposure. The developed assay provides a simple, reproducible, and minimally invasive proof-of-concept platform for oral dsRNA delivery to D. abbreviatus neonates. The assay requires only small dsRNA volumes, provides visual confirmation of ingestion, and may facilitate laboratory-based screening of additional RNAi target genes in D. abbreviatus and other coleopteran pests.

Animals↗
PubMed2026

Exploring the potential of RNA interference (RNAi) in mosquito control: from mechanisms to molecular insights.

Mosquito-borne diseases represent a growing global health crisis, exacerbated by climate change and insecticide resistance. RNA interference (RNAi), a natural mechanism of gene silencing, offers a promising, target-specific alternative for mosquito control. This review explores the potential of RNAi to disrupt critical physiological processes, such as reproduction and disease transmission, thereby reducing vector populations and competence. We examine the mechanisms of RNAi, its application in combatting insecticide resistance, and recent advancements in delivery systems, including nanobody- and chitosan-based nanoparticles, which enhance the stability and uptake of double-stranded RNA (dsRNA) molecules. However, significant challenges remain, such as optimizing field-effective delivery methods and assessing potential off-target effects on non-target organisms. Continued innovation in RNAi technology is pivotal for developing sustainable and environmentally sound vector control strategies. This review synthesizes current research, highlighting the molecular insights, practical applications, and future directions for integrating RNAi into modern public health initiatives.

RNA Interference↗
PubMed2024

Antiviral RNA interference inhibits virus vertical transmission in plants.

Known for over a century, seed transmission of plant viruses promotes trans-continental virus dissemination and provides the source of infection to trigger devastating disease epidemics in crops. However, it remains unknown whether there is a genetically defined immune pathway to suppress virus vertical transmission in plants. Here, we demonstrate potent immunosuppression of cucumber mosaic virus (CMV) seed transmission in its natural host Arabidopsis thaliana by antiviral RNA interference (RNAi) pathway. Immunofluorescence microscopy reveals predominant embryo infection at four stages of embryo development. We show that antiviral RNAi confers resistance to seed infection with different genetic requirements and drastically enhanced potency compared with the inhibition of systemic infection of whole plants. Moreover, we detect efficient seed transmission of a mutant CMV lacking its RNAi suppressor gene in mutant plants defective in antiviral RNAi, providing further support for the immunosuppression of seed transmission by antiviral RNAi.

Plant Diseases↗
PubMed2026

A small viral protein suppresses immune amplification by two distinct mechanisms.

Diverse viral suppressors of RNA interference (RNAi) and RNA silencing (VSRs) interact directly with core protein and/or RNA components of the host RNAi pathway. However, the specific counter-defense function of any VSR biochemical activity is fully validated only when it is shown as essential for viral infection in the wild-type but not mutant hosts defective in antiviral RNAi. Here, we investigated the role of VSR activities for direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1) during plant infection by wild-type and mutant cucumber mosaic virus (CMV), a positive-strand RNA virus expressing the 110-residue 2b protein as its VSR. We demonstrate that a C-terminally truncated 2b mutant (2b1-93) active in direct binding to siRNA and dsRNA, but not RDR1, was able to suppress the amplification of virus-derived siRNAs (vsiRNA) and antiviral RNAi mediated by RDR6, but not RDR1. By contrast, an N-terminally truncated 2b mutant (2b18-110) inactive in direct binding to siRNA or dsRNA was able to suppress vsiRNA amplification and antiviral RNAi mediated by RDR1, but not RDR6, and was less effective to promote systemic CMV infection and disease development than 2b1-93. Together, our results show that whereas RDR1 suppression requires direct binding of VSR-2b to RDR1, but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Therefore, CMV, through its VSR-2b, suppresses two parallel vsiRNA amplification pathways by distinct molecular mechanisms, and this unique property may account for the unusually wide host range of CMV.IMPORTANCEHost amplification of antiviral immunity is essential for robust control of viral infections. However, little is known about the mechanisms that viruses have evolved to suppress immune amplification in plants. Here, we characterized whole plant infection by cucumber mosaic virus (CMV) with its viral suppressor of RNA interference (RNAi) mutated to become inactive in direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1). We demonstrate maximal suppression of both RDR1- and RDR6-mediated antiviral RNAi amplification by the CMV 2b protein, a viral suppressor of RNAi (VSR). Notably, whereas RDR1 suppression requires direct binding of 2b to RDR1 but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Our findings reveal a novel counter-defense strategy evolved by a wide host range positive-strand RNA virus to suppress two pathways of immune amplification by distinct mechanisms.

Cucumovirus↗
PubMed2025

Microbe-induced gene silencing of fungal gene confers efficient resistance against Fusarium graminearum in maize.

UNLABELLED: Small RNAs (sRNAs), the main effectors of RNA interference (or RNA silencing, RNAi), mediate cell-autonomous and non-cell-autonomous gene silencing. The discoveries of trans-kingdom RNAi and interspecies RNAi have accelerated the development of RNAi-based crop protection technologies. Recently, based on interspecies RNAi, a practical technology termed microbe-induced gene silencing (MIGS) without the need of host genetic modification is developed for crop protection against Verticillium dahliae and Fusarium oxysporum in cotton and rice plants. In this study, we utilized MIGS technology to protect maize against Fusarium graminearum, which is responsible for maize stalk rot. An RNAi-engineered Trichoderma harzianum strain, Th-FgPmt2i, was exploited to generate double-stranded RNAs (dsRNAs) to trigger the silencing of the FgPTM2 gene. Our data verify that sRNAs generated from Th-FgPmt2i can silence the FgPMT2 gene via translational inhibition in F. graminearum. We further demonstrated that Th-FgPmt2i has a stronger capacity than does the T. harzianum chassis for protection of maize against F. graminearum. Coupled with our studies on crop protection against V. dahliae and F. oxysporum, our findings reveal that MIGS can be exploited to protect various crops against distinct fungal pathogens and has extensive applicability. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-025-00212-9.

Fusarium graminearum↗
PubMed2021

Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake.

Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen's RNA uptake efficiency.

Ascomycota↗
PubMed2004

Expression of a major surface protein of Trypanosoma brucei insect forms is controlled by the activity of mitochondrial enzymes.

In cycling between the mammalian host and the tsetse fly vector, trypanosomes undergo major changes in energy metabolism and surface coat composition. Early procyclic (insect) forms in the tsetse fly midgut are coated by glycoproteins known as EP and GPEET procyclins. EP expression continues in late procyclic forms, whereas GPEET is down-regulated. In culture, expression of GPEET is modulated by glycerol or glucose. Here, we demonstrate that a glycerol-responsive element of 25 nucleotides within the 3' untranslated region of GPEET mRNA also controls expression by glucose and during development in the fly. In trypanosomes, mitochondrial ATP is produced mainly by the acetate: succinate-CoA transferase/succinyl-CoA synthetase (ASCT) cycle, the citric acid cycle, and the cytochromes. Silencing of the pyruvate dehydrogenase or succinyl-CoA synthetase from the ASCT cycle by RNA interference induces reexpression of GPEET in late procyclic forms, whereas inhibition of the citric acid cycle or the cytochromes has no effect. In contrast, inhibition of the alternative oxidase, the second branch of the electron transport chain, with salicylhydroxamic acid overrides the effect of glucose or glycerol and causes a reduction in the level of GPEET mRNA. Our results reveal a new mechanism by which expression of a surface glycoprotein is controlled by the activity of mitochondrial enzymes.

3' Untranslated Regions↗
PubMed2026

Comparative evaluation of chitosan-based and star polycation nanocarriers for enhanced RNAi efficacy targeting CmFibL in Cnaphalocrocis medinalis.

BACKGROUND: The rice leaf folder, Cnaphalocrocis medinalis, causes substantial rice yield losses through larval leaf-rolling behavior. RNA interference (RNAi) offers a sustainable alternative, but its application in Lepidoptera is hindered by dsRNA degradation and poor cellular uptake. This study developed nanocarrier-mediated dsRNA delivery to overcome these limitations. RESULTS: Three nanocarriers - chitosan (CS), chitosan-tripolyphosphate (CS-TPP), and star polycation (SPc) - were compared for enhancing RNAi efficiency targeting the C. medinalis fibroin light chain gene (CmFibL). CS-TPP and SPc achieved 61% and 55% silencing efficiency, respectively, representing 2.7-fold improvement over naked dsRNA (23%). All nanocarriers protected dsRNA from RNase A (30 min) and midgut fluid (6 h) degradation. CmFibL knockdown caused severe silk defects, prolonged pupal duration by 23%, reduced pupal weight by 33%, and decreased leaf-rolling damage by 31% in glasshouse cage trials. Transcriptomics revealed down-regulation of amino acid metabolism and activation of endoplasmic reticulum (ER) stress and immune responses. No off-target effects were detected in human genome, nor in any predators or parasitoids sharing the same ecological niche. CONCLUSION: CS-TPP and SPc nanocarriers effectively enhance RNAi efficiency in a Lepidopteran pest. Targeting CmFibL disrupts silk-mediated feeding shelters with minimal ecological risk, providing a practical framework for field application of RNAi-based biopesticides against leaf-rolling rice pests. © 2026 Society of Chemical Industry.

Animals↗
PubMed2026

De novo transcriptome meta-analysis reveals candidate genes involved in life-stage transitions for RNAi-mediated management of the citrus root weevil (Diaprepes abbreviatus).

BACKGROUND: The citrus root weevil, Diaprepes abbreviatus, is a destructive agricultural pest for which molecular control options remain limited due to historically sparse genomic resources. Leveraging a comprehensive de novo transcriptome, we investigated developmental gene regulation across larval, pupal, and adult stages and identified essential targets for RNA interference (RNAi)-based intervention. RESULTS: Stage-resolved transcriptomic analyses revealed extensive transcriptional reprogramming associated with metabolism, detoxification, cuticle biosynthesis, endocrine signaling, and sensory perception. Among these, chitin synthase (DaCHS) emerged as a critical developmental gene, exhibiting pronounced up-regulation during late larval and pupal stages corresponding to intensive cuticle synthesis. Phylogenetic and structural analyses demonstrated that DaCHS is highly conserved among insects and retains canonical catalytic domains and transmembrane topology. Alpha Fold-based structural modeling and molecular docking confirmed stable interaction of DaCHS with its substrate, N-acetylglucosamine, supporting functional conservation of enzymatic activity. Oral delivery of DaCHS double-stranded RNA induced robust transcript suppression, leading to significant mortality and severe developmental defects, including larval and pupal abnormalities, and adults with disrupted wing and abdominal morphogenesis. CONCLUSION: These findings establish DaCHS as an indispensable gene for D. abbreviates development and validate transcriptome-guided RNAi as a powerful framework for target discovery. This work provides a strong molecular foundation for developing RNAi-based strategies that can be integrated into sustainable management programs for citrus root weevil control. © 2026 Society of Chemical Industry.

Animals↗
PubMed2027

Lentiviral Transduction of Embryonic Stem Cells.

Lentiviral vectors provide an efficient and reliable method for stable gene knockdown in embryonic stem cells (ESCs) through RNA interference. Here, we describe a detailed protocol for lentiviral transduction of mouse ESCs using lentiviral shRNA expression vectors. The protocol encompasses lentiviral particle production in HEK-293T packaging cells, determination of viral titer, transduction of ESCs cultured under feeder-free conditions, and selection of stably transduced cells. Additionally, we describe methods for evaluating transduction efficiency using fluorescence microscopy and flow cytometry, as well as for assessing gene knockdown efficacy by quantitative real-time PCR (Q-RT-PCR). This protocol is suitable for functional genomic studies in pluripotent stem cells and can be adapted for other difficult-to-transfect cell types.

Lentivirus↗
PubMed1979

[Stimulation of induction or inhibition of crown-gall tumor development by RNA-fragments U2. Interference by auxin].

RNA-fragments U2 obtained by mild degradation with RNase U2 of ribosomal RNA containing A and G nucleotides in excess are capable of exhibiting either a stimulatory effect on the induction of Crown-gall tumors or an inhibitory action on their subsequent development. These different effects are dependent on the moment at which RNA-fragments were introduced into wounded Pea seedlings infected by Agrobacterium tumefaciens B6. The results obtained in vitro and in vivo suggest that an interaction between auxin and RNA-fragments U2 may take place, either increasing the tumor induction or inhibiting the proliferation of tumourous cells.

Cell Transformation, Neoplastic↗
PubMed2026

Transcriptomic and RNAi analyses reveal chloride channel 3-associated osmoregulation in Litopenaeus vannamei under low-salinity stress.

Chloride channels and transporters are important for cellular volume regulation and salinity adaptation in euryhaline crustaceans, yet the intestinal transcriptional relationship between plasma-membrane and intracellular chloride pathways remains unclear in Litopenaeus vannamei. In this study, RNA interference of anoctamin 1 (ANO1) was combined with intestinal transcriptome sequencing under the production-relevant low-salinity condition of salinity 3. ANO1 silencing produced a focused transcriptional response, with 16 differentially expressed genes (DEGs) identified (11 upregulated and 5 downregulated). Functional enrichment indicated that these genes were associated with transporter activity, cytoskeletal organization, extracellular matrix-receptor interaction, membrane lipid metabolism, and vesicular processes. Notably, a transcript encoding chloride channel protein 3 (CLC-3) was significantly upregulated following ANO1 knockdown, suggesting a potential transcriptional relationship between ANO1 and CLC-3 in chloride homeostasis. Based on this finding, CLC-3 was selected for full-length cDNA cloning, sequence characterization, salinity-gradient expression analysis, and RNAi-based functional assessment. The cloned CLC-3 cDNA was 2883 bp in length and encoded an 850 amino acid protein containing a conserved voltage-gated chloride channel (Voltage-CLC) domain and two cystathionine β-synthase domains. Phylogenetic analysis placed LvCLC-3 within the intracellular CLC-c clade, and tissue distribution analysis showed the highest CLC-3 expression in the intestine. Intestinal CLC-3 expression responded nonlinearly to salinity variation, peaking at salinity 20. Under salinity 3, CLC-3 knockdown reduced ANO1, Na+/K+-ATPase alpha subunit, and Na+-K+-2Cl- cotransporter transcript levels, whereas glutamate-gated chloride channel expression increased. Mild hepatopancreatic structural alterations were also observed after CLC-3 knockdown. These findings suggest that CLC-3 is a salinity-responsive intracellular chloride-transporter candidate associated with intestinal ion-transport-related transcriptional responses after ANO1 suppression in L. vannamei, although the underlying physiological mechanism requires further validation.

Animals↗
PubMed2025

Generation of Cdc20 RNAi-Sensitive Cell Lines to Study Mitotic Exit.

Accurate mitotic progression ensures the fidelity of genome passage. Cdc20 is a key mitotic regulator. It promotes mitotic exit by activating the anaphase-promoting complex or cyclosome (APC/C) and monitors kinetochore-microtubule attachment through activating the spindle assembly checkpoint (SAC). Precise characterization of Cdc20 requires efficient depletion of endogenous Cdc20, which is extremely difficult to achieve by RNA interference (RNAi). This chapter describes the methodology to generate Cdc20 RNAi-sensitive cell lines with the help of CRISPR/Cas9 technology. These cell lines are highly sensitive to Cdc20 RNAi and provide a very useful tool for Cdc20 functionality investigation without the interference of endogenous Cdc20 protein. Similar strategy could be applied to other genes.

Cdc20 Proteins↗
PubMed2026

RNAi in the Rhizarian Phytopathogen Plasmodiophora brassicae: The Causal Agent of Clubroot Disease in Cruciferous Crops.

Although RNA interference (RNAi) is widespread and functionally important across eukaryotes, RNAi pathways are diverse or even lost in some lineages. Rhizaria represents a major and distinct eukaryotic supergroup that includes Plasmodiophora brassicae (Pb), the causal agent of cruciferous clubroot disease, yet RNAi in this lineage remains poorly understood. Here, we characterized an unusual RNAi pathway in Pb. Small RNA sequencing across five representative Pb life stages revealed abundant siRNAs and miRNAs characterized by a predominant 21-nt length, phased genomic distribution, 2-nt 3' overhangs, and a strong 5'-cytidine bias. Three Pb miRNAs were further validated by northern blotting and stem-loop RT-qPCR. Genome analysis identified two canonical AGO homologs, PbAGO1 and PbAGO2, but no Dicer homologs, except for an RNase III-containing Drosha-like protein, PbDRL. Functional analyses showed that PbAGO1 and PbAGO2 mediate gene silencing, whereas PbDRL is required for sRNA biogenesis. Further, the cell wall component chitin was identified from Pb zoosporangia during the early infection and RNAi interfering with its biosynthesis in transgenic plants of Arabidopsis and Brassica napus blocked Pb early infection and conferred broad-spectrum resistance. Our study uncovers an unusual RNAi pathway in Rhizaria and provides a promising strategy to control cruciferous clubroot disease.

Plasmodiophora brassicae↗
PubMed2004

Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene.

Genetic studies show that Msx2 and Dlx5 homeodomain (HD) proteins support skeletal development, but null mutation of the closely related Dlx3 gene results in early embryonic lethality. Here we find that expression of Dlx3 in the mouse embryo is associated with new bone formation and regulation of osteoblast differentiation. Dlx3 is expressed in osteoblasts, and overexpression of Dlx3 in osteoprogenitor cells promotes, while specific knock-down of Dlx3 by RNA interference inhibits, induction of osteogenic markers. We characterized gene regulation by Dlx3 in relation to that of Msx2 and Dlx5 during osteoblast differentiation. Chromatin immunoprecipitation assays revealed a molecular switch in HD protein association with the bone-specific osteocalcin (OC) gene. The transcriptionally repressed OC gene was occupied by Msx2 in proliferating osteoblasts, while Dlx3, Dlx5, and Runx2 were recruited postproliferatively to initiate transcription. Dlx5 occupancy increased over Dlx3 in mature osteoblasts at the mineralization stage of differentiation, coincident with increased RNA polymerase II occupancy. Dlx3 protein-DNA interactions stimulated OC promoter activity, while Dlx3-Runx2 protein-protein interaction reduced Runx2-mediated transcription. Deletion analysis showed that the Dlx3 interacting domain of Runx2 is from amino acids 376 to 432, which also include the transcriptionally active subnuclear targeting sequence (376 to 432). Thus, we provide cellular and molecular evidence for Dlx3 in regulating osteoprogenitor cell differentiation and for both positive and negative regulation of gene transcription. We propose that multiple HD proteins in osteoblasts constitute a regulatory network that mediates development of the bone phenotype through the sequential association of distinct HD proteins with promoter regulatory elements.

Amino Acid Sequence↗