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1. Target-specific requirements for RNA interference can arise through restricted RNA amplification despite the lack of specialized pathways

   https://doi.org/10.7554/eLife.97487.3  

   Knudsen-Palmer, Daphne R; Raman, Pravrutha; Ettefa, Farida; De_Ravin, Laura; Jose, Antony M (August 2024, eLife)

   Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematodeCaenorhabditis eleganscan selectively impair the silencing of some genes. Here, we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changingcis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells, and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, are enriched within ‘pUG zones’ matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs. 

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2. Evidence for multiple forms of heritable RNA silencing

   https://doi.org/10.1101/2024.04.28.591487  

   Chey, Mary S; Raman, Pravrutha; Ettefa, Farida; Jose, Antony M (April 2024, bioRxiv)

   Abstract Heritable gene silencing has been proposed to rely on DNA methylation, histone modifications, and/or non-coding RNAs in different organisms. Here we demonstrate that multiple RNA-mediated mechanisms with distinct and easily detectable molecular signatures can underlie heritable silencing of the same open-reading frame in the nematodeC. elegans. Using two-gene operons, we reveal three cases of gene-selective silencing that provide support for the transmission of heritable epigenetic changes through different mechanisms of RNA silencing independent of changes in chromatin that would affect all genes of an operon equally. Different heritable epigenetic states of a gene were associated with distinct populations of stabilized mRNA fragments with untemplated poly-UG (pUG) tails, which are known intermediates of RNA silencing. These ‘pUG signatures’ provide a way to distinguish the multiple mechanisms that can drive heritable RNA silencing of a single gene. 

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

   https://doi.org/10.1111/pbi.13589  

   Qiao, Lulu; Lan, Chi; Capriotti, Luca; Ah‐Fong, Audrey; Nino Sanchez, Jonatan; Hamby, Rachael; Heller, Jens; Zhao, Hongwei; Glass, N. Louise; Judelson, Howard S.; et al (May 2021, Plant Biotechnology Journal)

   Summary 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 pathogensBotrytis cinerea,Sclerotinia sclerotiorum,Rhizoctonia solani,Aspergillus nigerandVerticillium dahliae, but no uptake inColletotrichum 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. 

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4. A Tale of Two Lobsters—Transcriptomic Analysis Reveals a Potential Gap in the RNA Interference Pathway in the Tropical Rock Lobster Panulirus ornatus

   https://doi.org/10.3390/ijms231911752  

   Banks, Thomas M.; Wang, Tianfang; Fitzgibbon, Quinn P.; Smith, Gregory G.; Ventura, Tomer (October 2022, International Journal of Molecular Sciences)

   RNA interference (RNAi) has been widely utilised in many invertebrate models since its discovery, and in a majority of instances presents as a highly efficient and potent gene silencing mechanism. This is emphasized in crustaceans with almost all taxa having the capacity to trigger effective silencing, with a notable exception in the spiny lobsters where repeated attempts at dsRNA induced RNAi have demonstrated extremely ineffective gene knockdown. A comparison of the core RNAi machinery in transcriptomic data from spiny lobsters (Panulirus ornatus) and the closely related slipper lobsters (Thenus australiensis, where silencing is highly effective) revealed that both lobsters possess all proteins involved in the small interfering and microRNA pathways, and that there was little difference at both the sequence and domain architecture level. Comparing the expression of these genes however demonstrated that T. australiensis had significantly higher expression in the transcripts encoding proteins which directly interact with dsRNA when compared to P. ornatus, validated via qPCR. These results suggest that low expression of the core RNAi genes may be hindering the silencing response in P. ornatus, and suggest that it may be critical to enhance the expression of these genes to induce efficient silencing in spiny lobsters. 

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5. RNAi Strategies Against Downy Mildews: Insights Into dsRNA Uptake and Silencing

   https://doi.org/10.1111/mpp.70140  

   Göl, Deniz; Okechukwu, Emeka; Ünal, Gizem; Webb, Anne; Wood, Tom; Hong, Yiguo; Sherif, Sherif_M; Wacker, Theresa; Studholme, David_J; McDowell, John_M; et al (August 2025, Molecular Plant Pathology)

   Downy mildew (DM) diseases are caused by destructive obligate pathogens with limited control options, posing a significant threat to global agriculture. RNA interference (RNAi) has emerged as a promising, environmentally sustainable strategy for disease management. We evaluated the efficacy of dsRNA-mediated RNAi in suppressing key biological functions in DM pathogens of Arabidopsis thaliana, pea and lettuce: Hyaloperonospora arabidopsidis (Hpa), Peronospora viciae f. sp. pisi (Pvp) and Bremia lactucae (Bl), respectively. Conserved genes, cellulose synthase 3 (CesA3) and beta-tubulin (BTUB), were targeted. Silencing these genes significantly impaired spore germination and infection across species and reduced gene expression correlated with suppressed sporulation, confirming silencing efficacy. We tested dsRNAs from chemical synthesis, in vitro transcription, and Escherichia coli expression. Uptake and silencing efficiency varied with dsRNA length and concentration. In Hpa, short dsRNAs (21–25 bp) produced a variable spore germination rate, with 25 bp dsRNA causing a 247.10% increase, whereas longer dsRNAs (≥ 30 bp) completely inhibited germination. Similarly, in Pvp, dsRNAs of 21–25 bp resulted in a 73.05%–77.46% germination rate, while 30–75 bp dsRNAs abolished germination. Confocal microscopy using Cy-5-labelled short-synthesised dsRNA (SS-dsRNA) confirmed uptake by spores. Sequence specificity influenced efficacy, highlighting the need for precise target design. Multiplexed RNAi impacted silencing synergistically, further reducing germination and sporulation in Hpa. Importantly, SS-dsRNA-mediated silencing was durable, with reduced gene expression sustained at 4, 7, 10 and 11 days post-inoculation. Taken together, our findings demonstrate the potential of dsRNA-mediated gene silencing as a precise, sustainable tool for managing DM pathogens in multiple crop species 

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