RNAi-mediated crop protection against insects

Downregulation of the expression of specific genes through RNA interference (RNAi), has been widely used for genetic research in insects. The method has relied on the injection of double-stranded RNA (dsRNA), which is not possible for practical applications in crop protection. By contrast, specific suppression of gene expression in nematodes is possible through feeding with dsRNA. This approach was thought to be unfeasible in insects, but recent results have shown that dsRNA fed as a diet component can be effective in downregulating targeted genes. More significantly, expression of dsRNA directed against suitable insect target genes in transgenic plants has been shown to give protection against pests, opening the way for a new generation of insect-resistant crops.     

Delivery of dsRNA for RNAi in insects: an overview and future directions

Abstract RNA interference (RNAi) refers to the process of exogenous double‐stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi‐based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.     

RNAi在害虫防治中应用的重要进展及存在问题

RNAi是目前最有可能应用于害虫绿色防控的新技术。2017年6月,美国环境署(EPA)批准了国际上第一例表达昆虫双链RNA(dsRNA)的抗虫转基因玉米MON87411,掀起了利用RNAi技术进行害虫防治研究新的热潮。但是,目前RNAi在害虫防治中的应用还存在一些问题,例如有效靶标基因筛选和应用策略,鳞翅目昆虫对RNAi的敏感性以及双链RNA在环境中的稳定性等等。本文系统总结了RNA干扰现象发现20年来,该技术在害虫防治领域的研究及应用概况,并对RNAi技术应用的可行性、应用方法、存在问题和目前的一些解决办法进行了比较详细的综述。通过对近期研究结果的综合分析发现,dsRNA进入某些鳞翅目昆虫中肠或血淋巴后,被相关核酸酶降解可能是其RNAi效率较低的首要原因。通过对dsRNA进行脂质体修饰,纳米粒子包埋可以在一定程度上解决dsRNA降解的问题,进而提高RNAi效率。     

RNA干扰技术在昆虫学领域研究进展

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,广泛存在于生物体内。RNAi主要由小干扰RNA诱发阻碍目的基因的翻译或转录,造成目标信使RNA沉默。RNAi具有高效、特异性强等优点,被广泛应用于昆虫基因功能研究,并显示出了开发新型病虫害管理策略的巨大潜力。主要阐述了RNAi的沉默机制,双链RNA转入昆虫体内的几种方式,以及RNAi技术在不同目昆虫中研究的最新进展。最后,对RNAi技术存在的不足之处进行了简单总结,还对RNAi技术在害虫防治中的应用进行了展望,以期为该技术广泛应用于农业害虫防治提供理论支持。     

Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review

RNA interference already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. For this purpose, the insect should be able to autonomously take up the dsRNA, for example through feeding and digestion in its midgut. In this review we bring together current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to affect pest insects. At least two pathways for dsRNA uptake in insects are described: the transmembrane channel-mediated uptake mechanism based on Caenorhabditis elegans’ SID-1 protein and an ‘alternative’ endocytosis-mediated uptake mechanism. In the second part of the review dsRNA feeding experiments on insects are brought together for the first time, highlighting the achievement of implementing RNAi in insect control with the first successful experiments in transgenic plants and the diversity of successfully tested insect orders/species and target genes. We conclude with points of discussion and concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in insect control.     

病虫害RNAi技术中的外源RNA递送策略

病虫害严重威胁着作物安全生产。近年来,在RNA干扰(RNA interference,RNAi)基础上开发病虫害防控策略的研究得到越来越多的关注。RNAi是真核生物体内的一种基因调控过程,如何将外源RNA有效地递送到靶标生物体内,是病虫害RNAi技术能否成功的关键之一。国内外学者进行了大量研究和实践,探究影响病虫害吸收和传递外源双链RNA(double-stranded RNA,dsRNA)的因素,探索提高dsRNA递送效率的方法,取得了重要的进展。本文对相关研究进行了梳理,简述了影响病虫害对dsRNA吸收和递送的因素,对外源RNA的递送策略进行了综述,讨论了纳米颗粒复合物在dsRNA递送中的应用前景,以期为相关研究提供参考。     

植物介导的害虫RNA干扰

摘要: 应用植物介导的昆虫RNAi进行害虫防治近10年来受到了广泛的关注,其作用机理包括两个阶段,首先是害虫靶标基因dsRNA在植物体内的表达、运输和贮存,然后是害虫取食该植物后,dsRNA特异性抑制害虫体内靶标基因的表达。目前,植物介导的昆虫RNAi主要针对鳞翅目、鞘翅目和同翅目害虫,可以引起害虫生长发育的异常,导致死亡/繁殖力下降,甚至影响到其子代的生长。影响植物介导昆虫RNAi效率的因素主要包括害虫靶标基因的选择、dsRNA靶定位点及长度、植物表达dsRNA载体的结构和转基因植物的遗传转化方式等。植物介导昆虫RNAi防治害虫的策略也面临着潜在的安全性问题,如转基因植物安全性和RNAi潜在脱靶性等。随着植物介导昆虫RNAi技术的成熟,该方法有望成为害虫防治的新策略。     

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.     

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。     

The Insect Ecdysone Receptor is a Good Potential Target for RNAi-based Pest Control

RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.     

GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER,Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE)

RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double‐strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase‐2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12–14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high‐efficiency determination of gene functions in this species.     

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.     

Transport of orally delivered dsRNA in southern green stink bug,Nezara viridula

The southern green stink bug (SGSB, Nezara viridula) is an emerging polyphagous pest in many regions of the world. RNA interference (RNAi) is a valuable method for understanding gene function and holds great potential for pest management. However, RNAi efficiency is variable among insects and the differences in transport of double-stranded RNA (dsRNA) are one of the major factors that contribute to this variability. In this study, Cy3 labeled dsRNA was used to track the transport of dsRNA in SGSB tissues. Cy3_dsRNA was detected in the hemocytes, fat body (FB), epidermis, and midgut tissues at 24–72 hr after injection. Orally delivered Cy3_dsRNA or Cypher-5E labeled dsRNA was mostly detected in the midgut and a few signals were detected in parts of the FB and epidermis. Both injected and fed Cy3_dsRNA showed stronger signals in SGSB tissues when compared to Cy3_siRNA (small interfering RNA) or Cy3_shRNA (short hairpin RNA). dsRNA targeting the gene for a vacuolar-sorting protein, SNF7, induced higher knockdown of the target gene and greater SGSB mortality compared to siRNA or shRNA targeting this gene. ³²P-labeled dsRNA injected into SGSB was processed into siRNA, but fed ³²P-labeled dsRNA was not efficiently processed into siRNA. These data suggest that transport of orally delivered dsRNA across the midgut epithelium is not efficient in SGSB which may contribute to variable RNAi efficiency. Targeting genes expressed in the midgut rather than other tissues and using dsRNA instead of siRNA or shRNA would be more effective for RNAi-mediated control of this pest.     

Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control

The key of RNAi approach success for potential insect pest control is mainly dependent on careful target selection and a convenient delivery system. We adopted second-generation sequencing technology to screen RNAi targets. Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies were used to screen optimal RNAi targets from Ostrinia furnalalis. Total 14690 stage specific genes were obtained which can be considered as potential targets, and 47 were confirmed by qRT-PCR. Ten larval stage specific expression genes were selected for RNAi test. When 50 ng/μl dsRNAs of the genes DS10 and DS28 were directly sprayed on the newly hatched larvae which placed on the filter paper, the larval mortalities were around 40～50%, while the dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities reached 73% to 100% at 5 d after treatment. The qRT-PCR analysis verified the correlation between larval mortality and the down-regulation of the target gene expression. Topically applied fluorescent dsRNA confirmed that dsRNA did penetrate the body wall and circulate in the body cavity. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost less and an easy way to select the candidate target genes for RNAi. More importantly, it demonstrated that dsRNAs are able to penetrate the integument and cause larval developmental stunt and/or death in a lepidopteron insect. This finding largely broadens the target selection for RNAi from just gut-specific genes to the targets in whole insects and may lead to new strategies for designing RNAi-based technology against insect damage.