Foliar application of clay-delivered RNA interference for whitefly control.

Whitefly (Bemisia tabaci) is a phloem-feeding global agricultural pest belonging to the order Hemiptera. Foliar application of double-stranded RNA (dsRNA) represents an attractive avenue for pest control; however, limited uptake and phloem availability of the dsRNA has restricted the development of RNA interference (RNAi)-based biopesticides against sap-sucking insects. Following high-throughput single and combinational target gene identification for additive effects, we report here that foliar application of dsRNA loaded onto layered double hydroxide (LDH), termed BioClay, can effectively disrupt multiple whitefly developmental stages in planta. Adjuvants were shown to enhance uptake and movement of foliar-applied dsRNA to vascular bundles and into the whitefly. Notably, delivering the dsRNA as a BioClay spray instead of as naked dsRNA improved protection against immature insect stages, demonstrating the platform's potential to extend the benefits offered by RNA insecticides towards complete life cycle control of whitefly and potentially other pests.

Crowdsourced Identification of Potential Target Genes for CTV Induced Gene Silencing for Controlling the Citrus Greening Vector Diaphorina citri.

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.

Current scenario of RNAi-based hemipteran control.

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.

RNAi-Based Functional Genomics in Hemiptera.

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.

Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses.

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.

Diet-delivered RNAi in Helicoverpa armigera--Progresses and challenges.

Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.