Topical application of pathogen-specific double-stranded RNA (dsRNA) for virus resistance in plants represents an attractive alternative to transgenic RNA interference (RNAi). However, the instability of naked dsRNA sprayed on plants has been a major challenge towards its practical application. We demonstrate that dsRNA can be loaded on designer, non-toxic, degradable, layered double hydroxide (LDH) clay nanosheets. Once loaded on LDH, the dsRNA does not wash off, shows sustained release and can be detected on sprayed leaves even 30 days after application. We provide evidence for the degradation of LDH, dsRNA uptake in plant cells and silencing of homologous RNA on topical application. Significantly, a single spray of dsRNA loaded on LDH (BioClay) afforded virus protection for at least 20 days when challenged on sprayed and newly emerged unsprayed leaves. This innovation translates nanotechnology developed for delivery of RNAi for human therapeutics to use in crop protection as an environmentally sustainable and easy to adopt topical spray.

RNA interference (RNAi) is an homology‐dependent gene silencing mechanism that is a feasible and sustainable avenue for the management of hemipteran pests. Commercial implementation of RNAi‐based control strategies is impeded by limited knowledge about the mechanism of double‐stranded RNA (dsRNA) uptake, the function of core RNAi genes and systemic RNAi mechanisms in hemipteran insects. This review briefly summarizes recent progress in RNAi‐based studies aimed to reduce insect populations, viral transmission and insecticide resistance focusing on hemipteran pests. This review explores RNAi‐mediated management of hemipteran insects and offers potential solutions, including in silico approaches coupled with laboratory‐based toxicity assays to circumvent potential off‐target effects against beneficial organisms. We further explore ways to mitigate degradation of dsRNA in the environment and the insect such as stacking and formulation of dsRNA effectors. Finally, we conclude by considering nontransformative RNAi approaches, concatomerization of RNAi sequences and pyramiding RNAi with active constituents to reduce dsRNA production and application cost, and to improve broad‐spectrum hemipteran pest control. © 2020 Society of Chemical Industry

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RNA interference (RNAi) is a powerful approach for sequence-specific gene silencing, displaying tremendous potential for functional genomics studies in hemipteran insects. Exploiting RNAi allows the biological roles of critical genes to be defined and aids the development of RNAi-based biopesticides. In this review, we provide context to the rapidly expanding field of RNAi-based functional genomics studies in hemipteran insects. We highlight the most widely used RNAi delivery strategies, including microinjection, oral ingestion and topical application. Additionally, we discuss the key variables affecting RNAi efficacy in hemipteran insects, including insect life-stage, gene selection, the presence of nucleases, and the role of core RNAi machinery. In conclusion, we summarise the application of RNAi in functional genomics studies in Hemiptera, focusing on genes involved in reproduction, behaviour, metabolism, immunity and chemical resistance across 33 species belonging to 14 families.

Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama vectoring a phloem-limited bacterium Candidatus Liberibacter sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in D. citri to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to D. citri using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH4) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (Citrus tristeza virus, CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated “para-transgenic” citrus plant system caused higher mortality to adult D. citri than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult D. citri. Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding D. citri CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS.

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) is a significant global pest of economically important vegetable, fibre, and ornamental crops. Whiteflies directly damage the plants by piercing and sucking essential nutrients, indirectly through honeydew secretion and by transmitting more than 200 plant viruses that cause millions of dollars in produce losses per year. Whitefly management is mostly reliant on the heavy use of chemical insecticides. However, this ultimately leads to increasing resistance development, detrimental effects on beneficial insects and biomagnification of ecologically harmful chemicals in the environment. Responding to consumer demands for more selective, less toxic, non-GM insect control strategies, RNA interference (RNAi) has emerged as a potential game-changing solution. The RNA interference (RNAi) is a homology-dependent mechanism of gene silencing that represents a feasible and sustainable technology for the management of insect pests. In the present study, twenty-two whitefly genes were selected based on their essential function in the insect and tested in artificial diet bioassays for mortality and gene silencing efficacy. The nine most effective dsRNA constructs showed moderate-to-high whitefly mortality as compared to negative controls six days post-feeding. qPCR analysis further demonstrated significant knockdown of target gene mRNA expression. Additionally, uptake and spread of fluorescently labelled dsRNA was evident beyond the midgut of the whitefly supporting the systemic spreading of RNAi effectors. Taken together, the oral delivery of dsRNA shows effective RNAi mediated gene silencing of target genes and offers a viable approach for the development of dsRNA biopesticides against hemipteran pest.

Animal health measures mainly rely on vaccination or chemical control for major pests and pathogens, causing issues of residue, toxicity and development of resistance. For example, control of Sheep flystrike and lice-infestation affecting the Australia's sheep/wool industry (>3.5 B) have developed resistance to nearly all control chemicals used in the past. Topicals RNAi provides an innovative clean-green, non-toxic, environmentally sustainable biological control solution. Biodegradable clay particles as carriers can be used to deliver double stranded RNA (dsRNA), the key trigger molecule of RNA interference pathway. As an early proof of concept, we investigated the stability dsRNA loaded on two types of Clay particles: Clay 1 (releases dsRNA under acidic conditions) and Clay 2 (releases dsRNA under alkaline conditions) on cattle hide. Cattle skin was treated with Cy3 labelled dsRNA alone and Cy3 labelled dsRNA loaded on Clay1 or Clay2. The skin samples treated with the Cy3 formulations were imaged using confocal microscopy. Once imaged, the skin samples were washed and stored at room temperature for 5 days, later the samples were re-imaged to detect the fluorescent signal (Figure 1). The dsRNA loaded on clay particles was stable unlike naked Cy3-dsRNA which degraded and was not visible after washing. This increased inherent stability of the dsRNA molecules, combined with the environmental stability afforded by the Clay particles, offers promise to provide a sustainable solution for animal health. Topical RNAi can reduce reliance on trade withholding periods of meat/wool without chemical residues, enhance animal welfare and increase production of premium quality meat/wool, improve export potential, competitiveness and long-term profitability of livestock industry.