Feasibility,limitation and possible solutions of RNAi‐based technology for insect pest control

Abstract Numerous studies indicate that target gene silencing by RNA interference (RNAi) could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi‐mediated crop protection. However, there are many limitations using RNAi‐based technology for pest control, with the effectiveness target gene selection and reliable double‐strand RNA (dsRNA) delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome‐scale high‐throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro‐injection or by feeding as a dietary component. However, micro‐injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi‐mediated crop protection has been considered as a potential new‐generation technology for pest control, or as a complementary method of existing pest control strategies; however, further development to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi‐based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.     

Spatial and temporal 'knock down' of gene expression by electroporation of double-stranded RNA and morpholinos into early postimplantation mouse embryos

Here we report the use of double-stranded RNA (dsRNA) and morpholino technologies to specifically 'knock down' gene expression in early postimplantation mouse embryos. Sequence specific interference mediated by either dsRNA or by morpholino has been a useful tool for studying gene function in several organisms. However, specifically for the dsRNA, doubts have been raised about whether it could successfully be applied on vertebrate embryos. We demonstrate that electroporation of dsRNA directed against Otx2 or Foxa2 into postimplantation mouse embryos results in specific knock down of the expression of the respective endogenous genes in a region- and germ-layer specific manner. We also show that electroporation of morpholino directed against Foxa2 into the node of mouse embryos leads to a specific down regulation of Foxa2 expression in the floor plate. Our results demonstrate for the first time that dsRNA and morpholino technologies can be successfully applied in early postimplantation mouse embryos to specifically knock down gene expression.     

Suppression of gene expression by RNA interference in cultured plant cells

Suppression by double-stranded RNA (dsRNA) of the expression of a target gene is known as RNA interference (RNAi). No quantitative analysis of the effects of RNAi on the expression of specific genes in cultured plant cells has been reported. However, as it is possible to produce populations of cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest, we performed a quantitative study of the effects of RNAi in such cells. We constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner. An RNAi effect was observed 24 hours after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.     

Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double‐stranded RNA

The induction of the naturally occurring phenomenon of RNA interference (RNAi) to study gene function in insects is now common practice. With appropriately chosen targets, the RNAi pathway has also been exploited for insect control, typically through oral delivery of dsRNA. Adapting current methods to deliver foreign compounds, such as amino acids and pesticides, to mosquitoes through sucrose solutions, we tested whether such an approach could be used in the yellow fever mosquito, Aedes aegypti. Using a non‐specific dsRNA construct, we found that adult Ae. aegypti ingested dsRNA through this method and that the ingested dsRNA can be recovered from the mosquitoes post‐feeding. Through the feeding of a species‐specific dsRNA construct against vacuolar ATPase, subunit A, we found that significant gene knockdown could be achieved at 12, 24 and 48 h post‐feeding.     

灰飞虱海藻糖酶基因的克隆及RNA干扰效应

RNA干扰（RNAi）不但可以用于研究基因的功能, 还可以通过沉默靶标基因干扰特定的生命过程。因此, 通过深入研究, 发掘高效专一性靶基因和RNAi技术, 有可能开辟针对性的害虫RNAi防控新途径。本研究通过灰飞虱Laodelphax striatellus转录组数据分析并结合RACE技术, 克隆了灰飞虱两种海藻糖酶的全长基因, 分别命名为LSTre-1和LSTre-2, 其GenBank登录号分别为JQ027050和JQ027051。它们均具有海藻糖酶基因的典型特征, 与已报道的其他昆虫的海藻糖酶基因具有很高的相似性, 并表现出一定的虫种亲缘关系。其中LSTre-1为水溶性海藻糖酶基因, 全长2 042 bp, 开放阅读框编码602个氨基酸, 前端有25个氨基酸的信号肽, 但无跨膜结构域; LSTre-2为膜结合型海藻糖酶基因, 全长2 619 bp, 开放阅读框编码618个氨基酸, 前端有26个氨基酸的信号肽, 有2个疏水性跨膜结构域。利用喂食法研究2种海藻糖酶基因dsRNA对灰飞虱的致死效应, 发现靶向水溶性酶基因的干扰效应略高于靶向膜结合型的, 但两种海藻糖酶基因的dsRNA都可以显著抑制灰飞虱海藻糖酶基因的表达, 降低其活力, 还能显著抑制试虫的生长, 大幅增加试虫死亡率。 结果提示, 通过适宜途径干扰海藻糖酶基因可以开发防治灰飞虱的新途径。     

Transient silencing of the grapevine gene VvPGIP1 by agroinfiltration with a construct for RNA interference

Grapevine is an economically important crop, and the recent completion of its genome makes it possible to study the function of specific genes through reverse genetics. However, the analysis of gene function by RNA interference (RNAi) in grapevine is difficult, because the generation of stable transgenic plants has low efficiency and is time consuming. Recently, transient expression of genes in grapevine leaves has been obtained by Agrobacterium tumefaciens infiltration (agroinfiltration). We therefore tested the possibility to silence grapevine genes by agroinfiltration of RNAi constructs. A construct to express a double strand RNA (dsRNA) corresponding to the defense-related gene VvPGIP1, encoding a polygalacturonase-inhibiting protein (PGIP), was obtained and transiently expressed by agroinfiltration in leaves of grapevine plants grown in vitro. Expression of VvPGIP1 and accumulation of PGIP activity were strongly induced by infiltration with control bacteria, but not with bacteria carrying the dsRNA construct, indicating that the gene was efficiently silenced. In contrast, expression of another defense-related gene, VST1, encoding a stilbene synthase, was unaffected by the dsRNA construct. We have therefore demonstrated the possibility of transient down-regulation of grapevine genes by agroinfiltration of constructs for the expression of dsRNA. This system can be employed to evaluate the effectiveness of constructs that can be subsequently used to generate stable RNAi transgenic plants.     

RNA干扰在昆虫中的作用机理及应用进展

RNA干扰（RNAi）是生物体内源基因发生转录后特异性降解的一种生理现象,作为抵抗病毒的免疫机制,广泛存在于生物体内。RNAi在秀丽隐杆线虫中的发生机制已明确,但昆虫的系统性RNAi不同于线虫,在昆虫中尚未发现线虫跨膜蛋白SID．2的同源蛋白,且果蝇中不存在依赖于RNA的RNA聚合酶（RdRP）,但存在具有相似活性的物质。昆虫发生RNAi的效率不仅与靶标基因自身及双链RNA的选择有关,而且与虫体的发育状态及摄入双链RNA的剂量相关。随着RNAi在昆虫中作用特点的阐明,RNAi的应用价值也逐渐体现。近年来,通过RNAi沉默靶标基因,不但促进了昆虫基因功能研究的发展,而且被广泛用于重要农业害虫抗药性基因的研究。最新研究表明,RNAi结合第2代测序技术,针对非模式昆虫,能迅速找到具有致死效应的靶标序列,加快了利用RNAi技术生产生物农药的步伐。     

Knocking down the sex peptide receptor by dsRNA feeding results in reduced oviposition rate in olive fruit flies

Insect pests can cause crop damage in yield or quality, resulting in profit losses for farmers. The primary approach to control them is still the use of chemical pesticides resulting in significant hazards to the environment and human health. Biological control and the sterile insect technique are alternative strategies to improve agriculture protection. However, both strategies have significant limitations. A newly introduced approach that could be both effective and species-specific is the RNA interference mechanism. One key point for the success of this strategy is the delivery method of double-strand RNA (dsRNA) to the insects. A method of dsRNA delivery to insects with potential use in the field is the oral delivery, feeding the insects engineered microorganisms that produce dsRNA. Here, we present the first protocol for dsRNA feeding using modified bacteria, in the olive fruit fly, the most important insect pest of cultivated olives. We chose to target the sex peptide receptor gene. The sex peptide receptor interacts with the sex peptide, a peptide that is responsible for the postmating behavior in the model organism, Drosophila melanogaster. Feeding the female olive fruit fly with bacteria that produced dsRNA for the sex peptide receptor gene resulted in the development of female insects with significantly lower oviposition rates. Administration of dsRNA producing bacteria in insect diet against target genes that lead to genetic sexing or female-specific lethality could be added in the armory of control methods.     

RNA干涉在纤毛虫中的研究进展

RNA干涉是dsRNA介导的基因沉默现象,本文简要介绍了其作用的机制和生物学意义,重点阐述了RNA干涉在原生动物纤毛虫中的发现与应用,比较了RNA干涉与纤毛虫大核基因组重排机理的异同,并对RNA干涉在纤毛虫中传输的技术途径-RNAi喂饲法的原理也做了详细的介绍。     

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.     

BioClay™ prolongs RNA interference-mediated crop protection against Botrytis cinerea

One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.     

RNAi技术在昆虫功能基因研究中的应用进展

RNA干扰（RNA interference,RNAi）是指外源或内源的双链RNA（dsRNA）特异性地引起基因表达沉默的现象,它作为一种有效的工具用来产生转录后沉默,从而抑制特定基因的表达,成为基因功能研究的一种新方法,除了在模式昆虫如果蝇Drosophila中广泛应用之外,也在非模式昆虫中得到成功应用。近年来,RNAi技术在导入方法和基因功能分析方面都取得了飞速发展,且与转基因技术相结合成功应用于害虫防治领域。本文综述了RNAi技术在导入方法、昆虫功能基因组功能分析及害虫防治等领域新近的研究成果,并展望了该技术的应用前景。     

Nanoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management

RNA interference (RNAi) targeting lethal genes in insects has great potential for sustainable crop protection. Compared with traditional double-stranded (ds)RNA delivery systems, nanoparticles such as chitosan, liposomes, and cationic dendrimers offer advantages in delivering dsRNA/small interfering (si)RNA to improve RNAi efficiency, thus promoting the development and practice of RNAi-based pest management strategies. Here, we illustrate the limitations of traditional dsRNA delivery systems, reveal the mechanism of nanoparticle-mediated RNAi, summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management, and finally address the prospects of nanoparticle-based RNA pesticides.