RNAi‐mediated plant protection against aphids

Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double‐stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant‐mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant‐mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant‐mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid‐resistant plants through plant‐mediated RNAi strategy. © 2016 Society of Chemical Industry     

RNA interference technology in crop protection against arthropod pests,pathogens and nematodes

Scientists have made significant progress in understanding and unraveling several aspects of double‐stranded RNA (dsRNA)‐mediated gene silencing during the last two decades. Now that the RNA interference (RNAi) mechanism is well understood, it is time to consider how to apply the acquired knowledge to agriculture and crop protection. Some RNAi‐based products are already available for farmers and more are expected to reach the market soon. Tailor‐made dsRNA as an active ingredient for biopesticide formulations is considered a raw material that can be used for diverse purposes, from pest control and bee protection against viruses to pesticide resistance management. The RNAi mechanism works at the messenger RNA (mRNA) level, exploiting a sequence‐dependent mode of action, which makes it unique in potency and selectivity compared with conventional agrochemicals. Furthermore, the use of RNAi in crop protection can be achieved by employing plant‐incorporated protectants through plant transformation, but also by non‐transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. In this review, RNAi is presented in an agricultural context (discussing products that have been launched on the market or will soon be available), and we go beyond the classical presentation of successful examples of RNAi in pest‐insect control and comprehensively explore its potential for the control of plant pathogens, nematodes and mites, and to fight against diseases and parasites in beneficial insects. Moreover, we also discuss its use as a repressor for the management of pesticide‐resistant weeds and insects. Finally, this review reports on the advances in non‐transformative dsRNA delivery and the production costs of dsRNA, and discusses environmental considerations. © 2017 Society of Chemical Industry     

Validation of RNA interference in western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) adults

BACKGROUND: RNA interference (RNAi) is commonly used in insect functional genomics studies and usually involves direct injection of double‐stranded RNA (dsRNA). Only a few studies have involved exposure to dsRNAs through feeding. For western corn rootworm (Diabrotica virgifera virgifera) larvae, ingestion of dsRNA designed from the housekeeping gene, vacuolar ATPase (vATPase) triggers RNAi causing growth inhibition and mortality; however, the effect of dsRNA feeding on adults has not been examined. In this research, WCR adults were fed with vATPase‐dsRNA‐treated artificial diet containing a cucurbitacin bait, which is a proven feeding stimulant for chrysomelid beetles of the subtribe Diabroticina to which rootworms belong. RESULTS: Real‐time PCR confirmed suppression of vATPase expression and western blot analysis indicated reduced signal of a protein that cross‐reacted with a vATPase polyclonal antiserum in WCR adults exposed to artificial diet treated with dsRNA and cucurbitacin bait. Continuous feeding on cucurbitacin and dsRNA‐treated artificial diet resulted in more than 95% adult mortality within 2 weeks while mortality in control treatments never exceeded 20%. CONCLUSIONS: This research clearly demonstrates the effect of RNAi on WCR adults that have been exposed to dsRNA by feeding and establishes a tool to screen dsRNAs of potential target genes in adults. This technique may serve as an alternative to target screening of larvae which are difficult to maintain on artificial diets. Copyright © 2011 Society of Chemical Industry     

Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata

BACKGROUND: RNA interference (RNAi) is a breakthrough technology for conducting functional genomics studies and also as a potential tool for crop protection against insect pests. The major challenge for efficient pest control using RNAi in the field is the development of efficient and reliable methods for production and delivery of double‐stranded RNA (dsRNA). In this paper, the potential of feeding dsRNA expressed in bacteria or synthesized in vitro to manage populations of Colorado potato beetle, Leptinotarsa decemlineata (Say) (CPB), was investigated. RESULTS: Feeding RNAi successfully triggered the silencing of all five target genes tested and caused significant mortality and reduced body weight gain in the treated beetles. This study provides the first example of an effective RNAi response in insects after feeding dsRNA produced in bacteria. CONCLUSION: These results suggest that the efficient induction of RNAi using bacteria to deliver dsRNA is a possible method for management of CPB. This could be also a promising bioassay approach for genome‐wide screens to identify effective target genes for use as novel RNAi‐based insecticides. Copyright © 2010 Society of Chemical Industry     

Molecular mechanisms influencing efficiency of RNA interference in insects

RNA interference (RNAi) is an endogenous, sequence‐specific gene‐silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi‐based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double‐stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small‐interfering RNA/double‐stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. © 2018 Society of Chemical Industry     

Prospects,challenges and current status of RNAi through insect feeding

RNA interference is a phenomenon in which the introduction of double‐stranded RNA (dsRNA) into cells triggers the degradation of the complementary messenger RNA in a sequence‐specific manner. Suppressing expression of vital genes could lead to insect death, therefore this technology has been considered as a potential strategy for insect pest control. There are three main routes of dsRNA administration into insects: (i) injections to the hemolymph, (ii) topical, and (iii) feeding. In this review, we focus on dsRNA administration through feeding. We summarize novel strategies that have been developed to improve the efficacy of this method, such as the use of nano‐based formulations, engineered microorganisms, and transgenic plants. We also expose the hurdles that have to be overcome in order to use this technique as a reliable pest management method. © 2019 Society of Chemical Industry     

RNAi基因沉默技术及其在植物抗病性改良中应用的策略

RNAi (RNA interference) 是一种由dsRNA参与、对靶基因表达进行干扰或沉默的现象。由此发展起来的RNAi基因沉默技术已成为当今植物基因功能研究和遗传改良的一个重要手段。该技术已经在靶向病原物(真菌、细菌、病毒和线虫)基因沉默方面得到了广泛的应用,并且产生了一批抗病性增强的转基因植物。人工设计和合成的amiRNAs和ata siRNAs的成功研发加快了RNAi技术的应用。本文对RNAi基因沉默机制、RNAi技术研发进展及其在植物抗病性遗传改良中的应用进行综述,并对其应用策略进行探讨。     

RNA interference in Nilaparvata lugens (Homoptera: Delphacidae) based on dsRNA ingestion

BACKGROUND: An efficient and convenient RNA interference (RNAi) technique involving double‐stranded RNA (dsRNA) ingestion is useful for gene function studies of non‐model insects. RESULTS: Three dsRNAs targeting different sites within a gene encoding vacuolar ATP synthase subunit E (V‐ATPase‐E, 21E01) were synthesised for RNAi in Nilaparvata lugens. dsRNA was found to be stable in 0.1 g mL^?1 sucrose solution, but unstable in artificial fodder. Therefore, dsRNAs were orally delivered into N. lugens in 0.1 g mL^?1 sucrose solution. RNAi was induced by all three of the dsRNAs at 0.05 µg µL^?1 in N. lugens. Time dynamics analysis of gene silencing indicated that significant suppression of the target gene began as early as 2 days after ingestion of ds2‐21E01 and ds3‐21E01. However, significant repressive effects were recorded up to 10 days after exposure to ds1‐21E01. The maximum reduction in target gene mRNA was observed after 10 days of treatment, with suppression ratios induced by ds1‐21E01, ds2‐21E01 and ds3‐21E01 of 41, 55 and 48% respectively. CONCLUSION: An efficient and convenient RNAi technique involving dsRNA ingestion has been successfully developed for N. lugens. This will be a useful tool for further functional genomic investigation in this organism. Copyright © 2011 Society of Chemical Industry     

Combined use of lipopolysaccharide-binding protein dsRNA and Gram-negative bacteria for pest management of Coptotermes formosanus

BACKGROUND

RNA interference (RNAi) technology is an environmentally friendly strategy for controlling insect pests. Lipopolysaccharide-binding protein (LBP) recognizes lipopolysaccharides, which are a major outer membrane constituent of Gram-negative bacteria. We propose that the LBP gene is a potential target for termite management; however, to date, no studies have examined this gene in termites.

RESULTS

In this study, we cloned the LBP gene of Coptotermes formosanus (Cf) and found that the mortality rate of termite workers significantly increased, and the repellence of these workers to Gram-negative bacteria was suppressed after knockdown of CfLBP using double-stranded RNA (dsRNA) injection and feeding. Moreover, the mortality rate of termite workers fed with CfLBP dsRNA and three Gram-negative bacteria (provided separately) was over 50%, which was much higher than that of termites treated with either CfLBP dsRNA or Gram-negative bacteria. Finally, we found that CfLBP impacts the IMD pathway to regulate the immune response of C. formosanus to Gram-negative bacteria.

CONCLUSION

CfLBP plays a important role in the immune defense of termites against Gram-negative bacteria. It can be used as an immunosuppressant for RNAi-based termite management and is an ideal target for termite control based on the combined use of RNAi and pathogenic bacteria. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.     

Arabidopsis thaliana resistance to insects, mediated by an earthworm-produced organic soil amendment

BACKGROUND: Vermicompost is an organic soil amendment produced by earthworm digestion of organic waste. Studies show that plants grown in soil amended with vermicompost grow faster, are more productive and are less susceptible to a number of arthropod pests. In light of these studies, the present study was designed to determine the type of insect resistance (antixenosis or antibiosis) present in plants grown in vermicompost‐amended potting soil. Additionally, the potential role of microarthropods, entomopathogenic organisms and non‐pathogenic microbial flora found in vermicompost on insect resistance induction was investigated. RESULTS: Findings show that vermicompost from two different sources (Raleigh, North Carolina, and Portland, Oregon) were both effective in causing Arabidopsis plants to be resistant to the generalist herbivore Helicoverpa zea (Boddie). However, while the Raleigh (Ral) vermicompost plant resistance was expressed as both non‐preference (antixenosis) and milder (lower weight and slower development) toxic effect (antibiosis) resistance, Oregon (OSC) vermicompost plant resistance was expressed as acute antibiosis, resulting in lower weights and higher mortality rates. CONCLUSION: Vermicompost causes plants to have non‐preference (antixenosis) and toxic (antibiosis) effects on insects. This resistance affects insect development and survival on plants grown in vermicompost‐amended soil. Microarthropods and entomopathogens do not appear to have a role in the resistance, but it is likely that resistance is due to interactions between the microbial communities in vermicompost with plant roots, as is evident from vermicompost sterilization assays conducted in this study. Copyright © 2010 Society of Chemical Industry     

Food safety assessment of crops engineered with RNA interference and other methods to modulate expression of endogenous and plant pest genes

Genetically modified crops have been grown commercially for more than two decades. Some of these crops have been modified with genetic constructs that induce gene silencing through RNA interference (RNAi). The targets for this silencing action are genes, either specific endogenous ones of the host plant or those of particular pests or pathogens infesting these plants. Recently emerging new genetic tools enable precise DNA edits with the same silencing effect and have also increased our knowledge and insights into the mechanisms of RNAi. For the assessment of the safety of foodstuffs from crops modified with RNAi, internationally harmonized principles for risk assessment of foods derived from genetically modified crops can be followed. Special considerations may apply to the newly expressed silencing RNA molecules, such as their possible uptake by consumers and interference with expression of host genes, which, however, would need to overcome many barriers. Bioinformatics tools aid the prediction of possible interference by a given RNA molecule with the expression of genes with homologous sequences in the host crop and in other organisms, or possible off‐target edits in gene‐edited crops. © 2020 The Author. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Host-delivered RNAi-mediated root-knot nematode resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis> by targeting <Emphasis Type="Italic">splicing factor</Emphasis> and <Emphasis Type="Italic">integrase</Emphasis> genes

Root-knot nematodes (RKNs) are one of the most important biotic factors limiting crop productivity in many crop plants. The major RKN control strategies include development of resistant cultivars, application of nematicides and crop rotation, but each has its own limitations. In recent years, RNA interference (RNAi) has become a powerful approach for developing nematode resistance. The two housekeeping genes, splicing factor and integrase, of Meloidogyne incognita were targeted for engineering nematode resistance using a host-delivered RNAi (HD-RNAi) approach. Splicing factor and integrase genes are essential for nematode development as they are involved in RNA metabolism. Stable homozygous transgenic Arabidopsis lines expressing dsRNA for both genes were generated. In RNAi lines of splicing factor gene, the number of galls, females and egg masses was reduced by 71.4, 74.5 and 86.6%, respectively, as compared with the empty vector controls. Similarly, in RNAi lines of the integrase gene, the number of galls, females and egg masses was reduced up to 59.5, 66.8 and 63.4%, respectively, compared with the empty vector controls. Expression analysis revealed a reduction in mRNA abundance of both targeted genes in female nematodes feeding on transgenic plants expressing dsRNA constructs. The silencing of housekeeping genes in the nematodes through HD-RNAi significantly reduced root-knot nematode infectivity and suggests that they will be useful in developing RKN resistance in crop plants.     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

Outbreak of a new alien invasive plant Salvia reflexa in north‐east China

The establishment of invasive species is widely recognised as a pivotal issue in the preservation of biodiversity. Salvia reflexa, a species native to the south‐central United States and Mexico, has been widely introduced in Argentina, Australia, New Zealand and Japan. In China, the first population of this plant was found growing adjacent to a grain depot in Shahai village, Jianping County, Liaoning Province, on 25 July 2007. Since the grain depot imported foodstuffs from regions where the plant is native, we infer that S. reflexa was introduced into China via imported foodstuff in the early to mid‐2000s. Based on field observations, at least seven populations of this plant were observed in north‐east China. The plants displayed vigorous growth in midsummer and produced prolific seeds to overcome the cold environment in winter. Salvia reflexa occurred in both dense monocultures and in mixed stands with native plants. In order to validate a system for recognising and categorising non‐native plants in China, the Australian Weed Risk Assessment system was used to assess the invasiveness status of 19 exotic and 16 native plants in north‐east China. Salvia reflexa exhibited a high score of 10, suggesting it is a potentially pernicious alien invasive plant. Although the current distribution of S. reflexa is restricted to Liaoning province and thus far has limited impact on local environments, local regulatory authorities should pay close attention to this plant and take measures to stop its expansion. This is the first time that an invasive plant from the Lamiaceae (mint family) has been documented from cold environments in China.