Search Results (17)

In planta RNAi or host-induced gene silencing (HIGS) has undergone significant advancements that have rendered it efficient and stable at the transgenerational level in plants for regulating host genes and targeting genes of insect pests and plant pathogens. Similarly, topical RNAi or spray-induced gene silencing (SIGS) has garnered considerable attention as an environmentally sustainable, selective, and alternative approach to chemical control of insect pests and plant pathogens. Several biotechnology companies and startups have focused their efforts on RNAi-based solutions for topical application in agriculture. Nevertheless, further technological advancements are required to enhance the efficacy of topical RNAi in agriculture, including improved dsRNA delivery systems, better target gene selection, and addressing biosafety regulatory issues. Herein, this review discusses key advances and bottlenecks in RNAi, and summarizes successful applications of these RNAi-based technologies in agriculture focusing on in planta and topical RNAi to control insect pests and plant pathogens. Furthermore, this review delves into the patenting landscape, biosafety considerations, risk evaluations, and the current regulatory status of RNAi in Latin America. Finally, it explores the contributions of RNAi to plant science, food production, and fostering a more sustainable form of agriculture. Full article

Double-stranded RNA (dsRNA) biopesticides offer the potential for highly targeted pest control with minimal off-target impacts. Varroa mites (Varroa destructor) are an important pest of honey bees (Apis mellifera) that are primarily managed by synthetic pesticides. A next-generation treatment using a varroa-active dsRNA, vadescana, has been developed to target calmodulin expression in varroa. We evaluated the potential exposure of non-target species to vadescana. First, we assessed potential gene silencing effects on 39 arthropods with known genomes via bioinformatics. Three mite species, monarch butterflies (Danaus plexippus), fruit flies (Drosophila melanogaster), and European earwigs (Forficula auricularia) showed theoretical potential for off-target effects. These in silico results could be used to help inform risk assessments. Second, we conducted vadescana feeding trials on the greater wax moth (Galleria mellonella), a common beehive associate. There were no significant differences in wax moth reproduction, survival, or adult F2 wing length between vadescana-fed and control groups. Male F2 body weight was slightly but significantly lower in wax moths exposed to the highest vadescana dose, with no such effect observed in female moths. Calmodulin gene expression was unaffected in wax moths. Our hazard assessment of vadescana’s lethal and sublethal effects on wax moths indicates minimal impact following continuous dietary exposure far greater than any exposure that might be expected in the field, in line with the bioinformatics findings. This biopesticide appears highly varroa-specific and likely has fewer non-target effects than many current varroa control methods. Full article

Ceratitis capitata, known as the Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on pollinators. A more sustainable and species-specific alternative strategy may be based on double-stranded RNA (dsRNA) delivery through feeding to disrupt essential functions in pest insects, which is poorly reported in dipteran species. Previous reports in Orthoptera and Coleoptera species suggested that dsRNA degradation by specific nucleases in the intestinal lumen is among the major obstacles to feeding-mediated RNAi in insects. In our study, we experimented with three-day adult feeding using a combination of dsRNA molecules that target the expression of the ATPase vital gene and two intestinal dsRNA nucleases. These dsRNA molecules were recently tested separately in two Tephritidae species, showing limited effectiveness. In contrast, by simultaneously feeding dsRNA against the CcVha68-1, CcdsRNase1, and CcdsRNase2 genes, we observed 79% mortality over seven days, which was associated with a decrease in mRNA levels of the three targeted genes. As expected, we also observed a reduction in dsRNA degradation following RNAi against nucleases. This research illustrates the potential of utilizing molecules as pesticides to achieve mortality rates in Medfly adults by targeting crucial genes and intestinal nucleases. Furthermore, it underscores the importance of exploring RNAi-based approaches for pest management. Full article

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. Full article

The Asian citrus psyllid (ACP) is a citrus pest and insect vector of “Candidatus Liberibacter asiaticus”, the causal agent of citrus greening disease. Double-stranded RNA (dsRNA) biopesticides that trigger RNA interference (RNAi) offer an alternative to traditional insecticides. Standardized laboratory screening of dsRNA requires establishing the minimal effective concentration(s) that result in effective RNAi “penetrance” and trigger RNAi, resulting in one or more measurable phenotypes, herein, significant gene knockdown and the potential for mortality. In this study, knockdown was evaluated for a range of dsRNA concentrations of three ACP candidate genes, clathrin heavy chain (CHC), vacuolar ATPase subunit A (vATPase-A), and sucrose non-fermenting protein 7 (Snf7). Gene knockdown was quantified for ACP teneral adults and 3rd instar nymphs allowed a 48 h ingestion-access period (IAP) on 10, 50,100, 200, and 500 ng/µL dsRNA dissolved in 20% sucrose followed by a 5-day post-IAP on orange jasmine shoots. Significant gene knockdown (p < 0.05) in ACP third instar nymphs and adults ranged from 12–34% and 18–39%, 5 days post-IAP on dsRNA at 10–500 and 100–500 ng/µL, respectively. The threshold concentration beyond which no significant gene knockdown and adult mortality was observed post-48 h IAP and 10-day IAP, respectively, was determined as 200 ng/µL, a concentration indicative of optimal RNAi penetrance. Full article

The Varroa destructor mite is a devastating parasite of honey bees; however the negative effects of varroa parasitism are exacerbated by its role as an efficient vector of the honey bee pathogen, Deformed wing virus (DWV). While no direct treatment for DWV infection is available for beekeepers to use on their hives, RNA interference (RNAi) has been widely explored as a possible biopesticide approach for a range of pests and pathogens. This study tested the effectiveness of three DWV-specific dsRNA sequences to lower DWV loads and symptoms in honey bees reared from larvae in laboratory mini-hives containing bees and varroa. The effects of DWV-dsRNA treatment on bees parasitised and non-parasitised by varroa mites during development were investigated. Additionally, the impact of DWV-dsRNA on viral loads and gene expression in brood-parasitising mites was assessed using RNA-sequencing. Bees parasitised during development had significantly higher DWV levels compared to non-parasitised bees. However, DWV-dsRNA did not significantly reduce DWV loads or symptoms in mini-hive reared bees, possibly due to sequence divergence between the DWV variants present in bees and varroa and the specific DWV-dsRNA sequences used. Varroa mites from DWV-dsRNA treated mini-hives did not show evidence of an elevated RNAi response or significant difference in DWV levels. Overall, our findings show that RNAi is not always successful, and multiple factors including pathogen diversity and transmission route may impact its efficiency. Full article

Double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) and lead to directed silencing of specific genes. This natural defense mechanism and RNA-based products have been explored for their potential as a sustainable and ecofriendly alternative for pest control of species of agricultural importance and disease vectors. Yet, further research, development of new products and possible applications require a cost-efficient production of dsRNA. In vivo transcription of dsRNA in bacterial cells has been widely used as a versatile and inducible system for production of dsRNA combined with a purification step required to extract the dsRNA. Here, we optimized an acidic phenol-based protocol for extraction of bacterially produced dsRNA at low cost and good yield. In this protocol, bacterial cells are efficiently lysed, with no viable bacterial cells present in the downstream steps of the purification. Furthermore, we performed a comparative dsRNA quality and yield assessment of our optimized protocol and other protocols available in the literature and confirmed the cost-efficiency of our optimized protocol by comparing the cost of extraction and yields of each extraction method. Full article

Food losses, defined as a reduction in the quantity and quality of food during production and storage, impact food safety and security. Losses caused by plant pathogens are among the most significant. Chemical pesticides have been extensively used to prevent microbial diseases. Their toxicity and reduced efficacy, however, have encouraged investigators to develop alternatives. Alternatives based on microbial biopesticides tend to be safer and more environmentally benign than conventional pesticides. In recent years, formulations based on biopesticides have progressively increased in number and diversity and have attracted commercial interest. Understanding the mechanisms by which biopesticides control the disease is fundamental to achieving optimal disease control. Biocontrol mechanisms can be divided into two main categories: those related to the ability to inhibit pathogens or their virulence factors, and those that enhance host plant fitness and induce disease resistance. Here, the first type of strategy is reviewed, which is directly mediated by physical contact between biocontrol agents and pathogens or indirectly by exposure of a pathogen to antimicrobial or microbial-inhibiting compounds produced by the microbial antagonist. Mechanisms involving physical contact include mycophagy, destruction of pathogenic bacteria by bacteriophages or predation, and disease inhibition by topical applications of specific dsRNA. Indirect mechanisms that do not involve direct contact with a pathogen include the production of antimicrobial compounds, competition, and virulence factor suppression by quorum quenching. These topics are reviewed and discussed. Full article

To fulfil the growing needs of the global population, sustainability in food production must be ensured. Insect pests and pathogens are primarily responsible for one-third of food losses and harmful synthetic pesticides have been applied to protect crops from these pests and other pathogens such as viruses and fungi. An alternative pathogen control mechanism that is more “friendly” to the environment can be developed by externally applying double-stranded RNAs (dsRNAs) to suppress gene expression. However, the use of dsRNA sprays in open fields is complicated with respect to variable efficiencies in the dsRNA delivery, and the stability of the dsRNA on and in the plants, and because the mechanisms of gene silencing may differ between plants and between different pathogen targets. Thus, nanocarrier delivery systems have been especially used with the goal of improving the efficacy of dsRNAs. Here, we highlight recent developments in nanoparticle-mediated nanocarriers to deliver dsRNA, including layered double hydroxide, carbon dots, carbon nanotubes, gold nanoparticles, chitosan nanoparticles, silica nanoparticles, liposomes, and cell-penetrating peptides, by review of the literature and patent landscape. The effects of nanoparticle size and surface modification on the dsRNA uptake efficiency in plants are also discussed. Finally, we emphasize the overall limitation of dsRNA sprays, the risks associated, and the potential safety concerns for spraying dsRNAs on crops. Full article

Reticulitermes chinensis Snyder is a serious pest in China, and the odorant receptor co-receptor gene RcOrco plays a crucial role in olfaction. However, the function of RcOrco in the resistance of termites to entomopathogens has not been reported. We constructed dsRcOrco-HT115 engineered bacteria based on the RcOrco sequence from the full-length transcriptome data of R. chinensis. The engineered bacteria expressed dsRNA of RcOrco. Sonication was used to inactivate the dsRNA-HT115 strain and obtain a large amount of dsRcOrco. The dsRcOrco produced using this method overcame the problem that genetically engineered bacteria could not be applied directly and improved its effectiveness against termites. Bioassays using the dsRcOrco generated using this method showed that dsRcOrco significantly increased the toxicity of the bacterial and fungal pathogens to R. chinensis. The present study showed, for the first time, the function of Orco in termite resistance to pathogens, and the results provide a theoretical basis for the development and application of termite RNA biopesticides. Full article

Invasive insects cost the global economy around USD 70 billion per year. Moreover, increasing agricultural insect pests raise concerns about global food security constraining and infestation rising after climate changes. Current agricultural pest management largely relies on plant breeding—with or without transgenes—and chemical pesticides. Both approaches face serious technological obsolescence in the field due to plant resistance breakdown or development of insecticide resistance. The need for new modes of action (MoA) for managing crop health is growing each year, driven by market demands to reduce economic losses and by consumer demand for phytosanitary measures. The disabling of pest genes through sequence-specific expression silencing is a promising tool in the development of environmentally-friendly and safe biopesticides. The specificity conferred by long dsRNA-base solutions helps minimize effects on off-target genes in the insect pest genome and the target gene in non-target organisms (NTOs). In this review, we summarize the status of gene silencing by RNA interference (RNAi) for agricultural control. More specifically, we focus on the engineering, development and application of gene silencing to control Lepidoptera through non-transforming dsRNA technologies. Despite some delivery and stability drawbacks of topical applications, we reviewed works showing convincing proof-of-concept results that point to innovative solutions. Considerations about the regulation of the ongoing research on dsRNA-based pesticides to produce commercialized products for exogenous application are discussed. Academic and industry initiatives have revealed a worthy effort to control Lepidoptera pests with this new mode of action, which provides more sustainable and reliable technologies for field management. New data on the genomics of this taxon may contribute to a future customized target gene portfolio. As a case study, we illustrate how dsRNA and associated methodologies could be applied to control an important lepidopteran coffee pest. Full article

Insects and ectoparasites are causes for major concern throughout the world due to their economic and welfare impacts on livestock agriculture. Current control measures involve chemicals such as acaricides which pose challenges like chemical resistance and longer withholding periods. To enable more sustainable agriculture practices, it is important to develop technologies that combine targeted effectiveness with minimal environmental footprint. RNA interference (RNAi) is a eukaryotic process in which transcript expression is reduced in a sequence-specific manner. This makes it a perfect tool for developing efficient and effective biological control against pests and pathogens. Double-stranded RNA (dsRNA) is the key trigger molecule for inducing RNAi; this concept is widely studied for development of RNA-based biopesticides as an alternative to chemical controls in crop protection for targeting pests and pathogens with accuracy and specificity. In this review, we discuss key advances made using RNAi technology and how they can be applied to improve health in livestock industries. This includes research focused on different delivery mechanisms of dsRNA, important developments in regulatory frameworks, and risk identification, that will enable the future adoption of RNAi technologies to improve animal health. Full article

The advantages from exogenously applied RNAi biopesticides have yet to be realized in through commercialization due to inconsistent activity of the dsRNA trigger, and the activity level of RNAi suppression. This has prompted research on improving delivery methods for applying exogenous dsRNA into plants and insects for the management of pests and pathogens. Another aspect to improve RNAi activity is the incorporation of modified 2′-F pyrimidine nucleotides into the dsRNA trigger. Modified dsRNA incorporating 32–55% of the 2′-F- nucleotides produced improved RNAi activity that increased insect mortality by 12–35% greater than non-modified dsRNA triggers of the same sequence. These results were repeatable across multiple Hemiptera: the Asian citrus psyllid (Diaphorina citri, Liviidae); whitefly (Bemisia tabaci, Aleyroididae); and the glassy-winged sharpshooter (Homalodisca vitripennis, Cicadellidae). Studies using siRNA with modified 2′-F- pyrimidines in mammalian cells show they improved resistance to degradation from nucleases, plus result in greater RNAi activity, due to increase concentrations and improved binding affinity to the mRNA target. Successful RNAi biopesticides of the future will be able to increase RNAi repeatability in the field, by incorporating modifications of the dsRNA, such as 2′-F- pyrimidines, that will improve delivery after applied to fruit trees or crop plants, with increased activity after ingestion by insects. Costs of RNA modification have decreased significantly over the past few years such that biopesticides can now compete on pricing with commercial chemical products. Full article

RNA interference (RNAi) is a naturally occurring process inhibiting gene expression, and recent advances in our understanding of the mechanism have allowed its development as a tool against insect pests. A major challenge for deployment in the field is the development of convenient and efficient methods for production of double stranded RNA (dsRNA). We assessed the potential for deploying bacterially produced dsRNA as a bio-pesticide against an invasive forest pest, the emerald ash borer (EAB). EAB feeds on the cambial tissue of ash trees (Fraxinus spp.), causing rapid death. EAB has killed millions of trees in North America since its discovery in 2002, prompting the need for innovative management strategies. In our study, bacterial expression and synthesis of dsRNA were performed with E. coli strain HT115 using the L4440 expression vector. EAB-specific dsRNAs (shi and hsp) over-expressed in E. coli were toxic to neonate EAB after oral administration, successfully triggering gene silencing and subsequent mortality; however, a non-specific dsRNA control was not included. Our results suggest that ingestion of transformed E. coli expressing dsRNAs can induce an RNAi response in EAB. To our knowledge, this is the first example of an effective RNAi response induced by feeding dsRNA-expressing bacteria in a forest pest. Full article

Helicoverpa armigera, the cotton bollworm, is a major insect pest for a wide range of agricultural crops. It causes huge yield losses through feeding damage and increasing the crop’s vulnerability to bacterial and fungal infection. H. armigera has evolved substantial resistance to many different chemical insecticides, prompting the development of transgenic crop plants with alternative insect-resistance-conferring mechanisms. For example, transgenic crops producing Bacillus thuringiensis (Bt) toxins have been very successful. However, there is still a concern about insect populations emerging with resistance to these biopesticides. Novel strategies that give effective protection, without affecting the environment, need to be continuously developed and implemented. Such a strategy is Trans-kingdom RNAi, which is based on making plants express double-stranded (ds) or hairpin (hp) RNA for ingestion by herbivorous pests. The RNA triggers silencing of specific genes within the pest leading to its death or impaired growth. However, the efficacy of the approach appears to depend on the means of delivering the RNA. We will describe new approaches and delivery strategies, including chloroplast-based expression, which greatly enhance the potency of insect protection. Full article