Overview of the recently published studies on the use of plant-RNAi against different insect pests
Abstract
The fast-growing human population requires the development of new agricultural technologies to meet consumers´ demand, while minimizing environmental impacts. Insect pests are one of the main causes for losses in agriculture production, and current control technologies based on pesticide application or the use of transgenic crops expressing Bacillus thuringiensis toxin proteins are facing efficacy challenges. Novel approaches to control pests are urgently necessary. RNA interference (RNAi) is a gene silencing mechanism triggered by providing double-stranded RNA (dsRNA), that when ingested into insects can lead to death or affect the viability of the target pest. Transgenic plants expressing dsRNA version of insect specific target genes are the new generation of resistant plants. However, the RNAi mechanism is not conserved among insect orders, and its elucidation is the key to develop commercial RNAi crops. In this chapter, we review the core RNAi pathway in insects and the dsRNA uptake, amplification, and spread of systemic silencing signals in some key insect species. We also highlight some of the experimental steps before developing an insect-pest-resistant “RNAi plant”. Lastly, we review some of the most recent development studies to control agricultural insect pests by RNAi transgenic plants.
Keywords
- Biotechnology
- dsRNA
- Entomology
- Gene silencing
- Insect control
- RNA interference
1. Introduction
Agriculture has to continually adapt to rising environmental concerns in conjunction with meeting the increasing consumers´ demand. The fast-growing human population creates the need for the sustainable intensification of agriculture throughout the world which can be acomplished by adopting mechanization and new technologies to close yield gaps while minimizing environmental impacts. In the past few decades, insect pest control has been mainly conducted by the application of chemical pesticides because of the low cost and efficacy; but their indiscriminate use has caused escalating problems with the evolution of insect resistance to the pesticides together with secondary pest outbreaks. The development of new biotechnological approaches, with the introduction of transgenic crops expressing
RNA interference (RNAi) is a gene silencing mechanism at the cellular level triggered by double-stranded RNA (dsRNA) and is likely to be the new approach underlying the next generation of insect-resistant transgenic plants. In some studies, successful delivery of dsRNA molecules to insects by ingestion resulted in the expected essential gene target silencing [5, 6], which led to death or affected the viability of the target insect, resulting in control of the pest.
In general, long dsRNAs are processed by species-specific RNase-III-like enzymes, resulting in smaller double-stranded molecules. These shorter RNAs are loaded into RNA complexes as a guide for finding target mRNAs that are either cleaved or blocked for translation in posttranscriptional silencing, or inducing histone modifications when involved in transcriptional silencing response [7, 8]. However, the RNAi systemic spreading mechanism is not conserved across organisms, and its elucidation is an essential step in developing an efficient method to control agricultural pests by RNAi technology.
In this chapter, we review how the RNAi mechanism occurs in insects, highlighting the core RNAi pathway and components, and new developments regarding dsRNA uptake, amplification, and the spread of systemic silencing signals in some key insect species. We also discuss the critical experimental steps before developing an “RNAi plant” protected against a specific insect pest, with consideration to application. Lastly, we review some of the most recent published studies to control agriculturally important insect pests based on RNAi transgenic plants.
2. RNAi mechanism
RNAi is an important and natural antiviral defense mechanism, protecting organisms from RNA viruses, or even avoiding the random integration of transposons [9]. Over time, with the discovery of some aspects of the mechanism, RNAi has become a widely used tool to knock down and analyze the function of genes. Most of the RNAi pathways have dsRNA as the precursor triggering molecule that vary in length and origin [7, 8]. In addition, the RNAi pathways differ not only in the RNA precursor molecule, but also in genes, enzymes, and effector complexes involved throughout the process. However, some key steps are conserved. Briefly, RNA duplexes are processed into short RNA duplexes, which are then used to guide the recognition of their target, either to cleave a complementary mRNA, or to repress their target translation at a posttranscriptional silencing level, or to modify the chromatin structure at the transcriptional level [7, 8].
The RNA precursor molecules from the RNAi pathways, all of which are already identified in insects, are small RNAs, categorized into three classes [8, 10]: the first two classes are small interfering RNAs (siRNAs; 20–25 nucleotides) and microRNAs (miRNAs; 21–24 nucleotides) [7] (Figure 1). Both miRNAs and siRNAs share a common RNase-III processing enzyme, Dicer, and closely related effector complexes and both can regulate gene expression at the posttranscriptional level [8, 10, 11]. Conversely, the third class of small RNAs, the PIWI-interacting RNAs (piRNAs; 24–30 nucleotides), are generated independent of the Dicer activity [12]. piRNAs have been reported to play an essential role in germ-line development, stem cell renewal, transposon silencing, and epigenetic regulation [13-16]. These piRNAs originate from a diversity of sequences, including repetitive DNA and transposons, and they seem to act both at the posttranscriptional and chromatin levels [10]. The mechanism that generates and amplifies piRNAs is not well-understood, but involves slicer activities (Argonaute proteins associated with cleaving activity) [8, 10, 17]. Considered a specialized subclass of piRNAs,
The most recognized RNAi pathways are the siRNA and miRNA; despite being triggered by different molecules, both precursors are long double-stranded RNAs (dsRNAs). Naturally in a cell, long dsRNAs can derive from RNA virus replication, from the transcription of convergent cellular genes or mobile genetic elements, or from self-annealing cellular transcripts [8]. In the siRNA pathway, these long dsRNA are processed by Dicer into siRNA duplexes. By contrast, in the miRNA pathway, miRNAs are generated from endogenous transcripts (primary miRNAs; pri-miRNAs) that form stem–loop structures [19, 20]. In the nucleus, these hairpin regions are recognized and cleaved into precursor mirRNAs (pre-miRNAs) by Drosha, another RNase-III-family enzyme, and the pre-miRNAs are transported to the cytoplasm through the nuclear export receptor Exportin-5 (Expo-5) [19, 20]. Subsequently, the pre-miRNA undergoes another endonucleolytic cleavage, now catalyzed by Dicer, generating an miRNA duplex [19, 20] (Figure 1).
The siRNA and miRNAs duplex containing ribonucleoprotein particles (RNPs) are subsequently rearranged into effector complexes. Although it is difficult to assign distinct functional labels, an siRNA-containing effector complex is referred to as an “RNA-induced silencing complex” (RISC), and an miRNA-containing effector complex is referred to as an miRNP [7]. In these complexes, the regulation is at a posttranscriptional level and every RISC or miRNP contains a member of the Argonaute (Ago) protein family [7]. For the regulation at the transcriptional level as guided by rasiRNAs, a specialized nuclear Argonaute-containing complex, known as the RNA-Induced Transcriptional Silencing complex (RITS) mediates gene silencing [10]. In general, one strand of the short-RNA duplex (the guide strand) is loaded onto an Argonaute protein at the core of the effector complexes. During loading, the nonguide strand is cleaved by an Argonaute protein and ejected. The Argonaute protein then uses the guide RNA to associate with target RNAs that contain a perfectly complementary sequence and then catalyzes the slicing of these targets, either to be cleaved by RISC, to be blocked for translation in miRNP or by inducing histone modifications in RITS [7] (Figure 1). The mechanism of miRNA-guided translational regulation is not as well-understood in the case of target-RNA cleavage, and to make things more complicated, miRNAs can act as siRNAs, and siRNAs can act as miRNAs [7].
Dicer is one of the enzymes involved in RNAi mechanism that is encoded by a variable number of genes and presents distinct specificity among organisms [21]. For instance, mammals and
Another gene family involved in RNAi pathways is the Argonaute proteins (Ago). Ago is a central protein component of silencing complexes (RISC, RITS, miRNP) that acts in mediating target recognition and silencing [24]. Argonaute proteins contain two domains: a PAZ domain involved in dsRNA binding, and a PIWI-domain responsible for RNase activity [23]. In
2.1. Systemic RNAi
Systemic RNAi is described as a silencing signal transmitted widely throughout a treated organism [5, 31]. The knowledge about the systemic RNAi mechanism in insects is important as it may affect the approaches adopted to develop “RNAi-mediated pest control” because the systemic mechanism is not conserved among those organisms. Systemic RNAi has two important steps to be considered: the uptake of dsRNA by the cells and the systemic spreading of the signals. Some of the main genes involved in systemic RNAi are presented below and discussed for the model organisms.
2.1.1. dsRNA uptake
In insects, two types of dsRNA uptake mechanisms have been identified [32]. The first one involves a multi-transmembrane domain protein, Systemic Interference Defective (Sid). In
Other important proteins for systemic RNAi were identified in
2.1.2. RNAi systemic spreading: amplification and maintenance of dsRNA
Once the dsRNA overcomes all the uptake barriers, the silencing signal should be transported from treated cell to other cells, and spread to other tissues. Further, dsRNA should be constantly produced, e.g., either by the amplification of dsRNA by an RNA-dependent RNA polymerase (RdRP), and/or constantly acquired for the maintenance of the silencing responses.
In
The presence of the main genes involved in systemic RNAi and amplification in
Another component that might affect RNAi efficiency in different insects are the proteins containing the dsRNA-binding motif (dsRBM), which help small molecules to properly load inside of the silencing complexes [23]. These proteins act together with Dicer, and seem to be responsible for determining Dicer specificity in
3. Factors affecting the silencing effect and RNAi efficiency as an insect control method
The RNAi approach to control insect pests had been considered for many years, but application of this technology was just realized after it was shown that ingestion of dsRNA would trigger RNAi. The concept of RNAi-plant mediated pest control was demonstrated in 2007 by the development of transgenic plants producing dsRNAs against specific insect genes, with the consequent effect on the target species [43, 44]. The main prerequisites to generate successful RNAi insect-resistant transgenic plants are: (i) identification of a specific gene with an essential function in the insect that can cause developmental deformities and/or larval lethality when knocked down or knocked out; and (ii) dsRNA delivery by oral ingestion that must be uptaken by the insect cells, and spread systemically.
The insect must uptake the dsRNA version of a target gene region by feeding. To silence the target gene, this specific dsRNA must be taken up from the gut lumen into the gut cells as what is considered as “environmental RNAi.” If the target gene is expressed in a tissue distinct from the digestive system, the silencing signal should successfully spread via cells and tissues as a systemic RNAi. Both environmental and systemic RNAi are considered noncell-autonomous RNAi, which means that the interfering effect takes places in tissues/cells different from the location of application or production of the dsRNA. Conversely, in the cell-autonomous RNAi, the silencing process is limited to the cell in which the dsRNA is introduced [5]. However, the mechanism of ingested dsRNA uptake and systemic spreading of the silencing signal in the insect have yet to be fully characterized and understood.
Some factors can affect the efficiency of the dsRNA uptake and systemic silencing spread in different insects. Here, we highlight important points that must be considered in developing an RNAi approach against insect pests.
3.1. Target gene
The choice of the target gene should be carefully considered. Each gene requires particular effort to be silenced. Terenius and colleagues [28] reviewed more than 150 RNAi experimental results from RNAi of lepidopterans involving 130 genes, from which only 38% were silenced at a satisfactory level, while 48% failed to be silenced, and 14% were silenced at insufficient levels. Among the target genes, those involved in immunity were more effectively silenced, and, in contrast, genes expressed in epidermal tissues seem to be most difficult. Differences for RNAi sensitivity among genes in the same tissue was described in [28].
3.2. dsRNA design
The design of the dsRNA determines the one particular target gene to be silenced, but off-target effects can occur if siRNAs have some sequence similarity with unintended genes. Tobacco plants expressing
3.3. dsRNA length
The length of the dsRNA fragments plays an essential role in the effectiveness of molecular uptake in insects, which is directly involved in the success of the target gene silencing. In most of the RNAi experiments, the insects are fed with long dsRNAs [5]. Some experiments showed that long dsRNAs are more efficiently uptaken than siRNAs [37, 46]. This may be due to the fact that a long dsRNA, with 100% match of the target mRNA, after processing into siRNA will provide a greater diversity of siRNAs available to cause specific suppression of target gene and increase the desired effect, and, additionally, reduce the likelihood of developing resistance [47]. In contrast, other studies reported suppression of genes in different insects via incorporation of siRNA in diet instead of dsRNA [48, 49].
3.4. dsRNA concentration
Optimal concentration of dsRNA delivered to the insect is required to induce sufficient gene target silencing. It is noteworthy to mention that exceeding the optimal dsRNA concentration may not result in more silencing [50, 51]. However, higher concentration of dsRNA decreased the duration of dsRNA exposure to reach 50% mortality of
3.5. Controls
Empty vector, empty cassette, buffer only, irrelevant or nonspecific control (such as dsGFP – Green Fluorescent Protein gene region), or any other kind of negative control are essential to discriminate specific gene silencing from the simple induction of siRNA processing machinery by exposure to a dsRNA. Mainly, a negative control should demonstrate the specificity of the dsRNA designed for a target, not interfering in specific target expression, and even unspecific effects. Also, any control should have similar size and concentration of the used dsRNA [53].
3.6. Molecular silencing confirmation
An efficient molecular confirmation of the RNAi silencing should be conducted, which includes target RNA expression, and analyses of protein amount and/or enzyme activity. In RNA analysis, additional care should be taken for expression analysis. The method of choice for RNA expression analysis is the quantitative amplification of reversed transcripts or RT-qPCR, considered a very sensitive and accurate method. To provide precision in RT-qPCR, some essential care is required, such as the choice of appropriate stable reference genes and primer pair design with sufficient amplification efficiency. The reference genes should exhibit stable expression among experimental conditions, providing reliable estimate of gene expression results [54]. Additionally, primers should be designed flanking the region used to design the dsRNA to ensure that the initial cleavage of the mRNA could be detected, thus avoiding false-positives [55]. Conventional care of RT–qPCR reactions defined by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines must be strictly followed [56].
3.7. Protein stability and phenotype analysis
Proteins can exhibit a long half-life and interfere with the phenotypic changes. However, phenotype changes are not totally related to small decrease of protein levels; haplo-sufficient genes produce proteins capable of performing the biological processes normally, even at half of the protein levels [53]. Phenotype changes could be more difficult to be observed in RNAi responses if the protein product of the target gene has a long half-life. For example, the reduction of
3.8. Insect issues, life stage, nucleases, and gut pH
Some insect characteristics should also be considered before starting an RNAi experiment including the developmental stage of insects. Although handling advanced developmental stages of insects is more efficient, silencing effects are more prominent in earlier stages. For instance, in second instar larvae of
Another consideration that can affect the RNAi silencing efficiency is the presence of insect nucleases and gut pH. For instance, feeding assays with
4. Overview on the use of RNAi to control insects by transgenic plants
Most of the current transgenic crops with specific control against insect pests are based on
In 2007, two studies demonstrated the concept of plants expressing dsRNAs derived from hairpin vectors that directed dsRNAs to target gene regions of economically important agricultural pests: the cotton bollworm (
To implement RNAi in agricultural pest control, the target insect should uptake the dsRNA autonomously, e.g., from transgenic plants expressing dsRNA. This feeding should be continuous, since insects lack an amplification mechanism based on RdRP, such as
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Coleoptera |
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Mortality | [67] |
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Coleoptera |
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Mortality | [68] |
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Lepidoptera |
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Nuclear receptor complex of 20-hydroxyecdysone ( |
Molting defect and larval lethality | [45] |
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Lepidoptera |
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Molt-regulating transcription factor gene ( |
Developmental deformities and larval lethality | [70] |
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Lepidoptera |
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Developmental Deformities and larval lethality | [71] |
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Hemiptera |
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Progeny reduced | [72] |
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Hemiptera |
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Progeny reduced | [73] |
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Hemiptera |
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Inhibited reproduction | [74] |
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Hemiptera |
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Mortality | [75] |
4.1. Coleoptera
The coleopterans are likely to be the first target to be controlled by the new generation of transgenics, the “RNAi-plants.”
Studies have also been performed in other species, such as the important potato pest
An explanation for this result is that since choroplasts do not have cellular RNAi machinery [69], the dsRNAs produced inside these organelles are not cleaved by a plant Dicer and the beetles ingest almost entirely long dsRNA. In contrast, beetles fed on nuclear-transformed plants consumed mostly siRNAs; previous dsRNA-feeding studies already indicated that ingested long dsRNAs were much more effective than ingested siRNAs. It should be highlighted that all the other studies have been based on an efficient cytoplasm-derived dsRNA in various crops, indicating that possibly potato plants process long dsRNA more effective than the plants from those other studies (Table 1 [69]).
4.2. Lepidoptera
Plants producing
The first successful RNAi plant protected against a lepidopteran (
The expression of dsRNA in both
4.3. Hemiptera
Hemipterans are characterized as piercing/sucking insects, representing major agricultural pests that inflict direct damages by sucking sap, or indirectly by acting as a vector of several viruses and bacterial infections. Since hemipterans feed through sucking the phloem, only systemic chemical insecticides are effective against these insects, resulting in high residual toxicity. The problem is further aggravated as no Bt toxin has been identified as exhibiting adequate insecticidal effects against hemipterans. Transgenic crops based on RNAi offer a large potential to control hemipteran, requiring expression of target gene dsRNAs on the phloem. One first report was published in 2011 about developing transgenic
The same phenotype was observed when
5. Conclusion and future perspectives
Since the concept of a transgenic plant expressing dsRNA targeted to a specific essential gene in the insect that affects its viability was first demonstrated in 2007, the technology has been extended to a large number of insect species from various orders. Elucidating the various mechanisms and components of the RNA interference pathway has progressed, but many aspects remain to be clarified. Many differences in components and mechanisms among insect orders and between insects and other organisms still need to be worked out. Some of these differences (e.g., genes involved, gene number, and level of expression) may explain variation in recalcitrance among insect species and need to be further investigated. Of particular interest are the mechanisms of dsRNA uptake, signal amplification, and systemic spread in the major pest species. Additional insect- or order-specific characteristics, such as gut pH, presence of dsRNA-degrading activity in digestive system, among others that could be associated with differences in recalcitrance to RNAi need to be dissected and clarified.
Due to the variety of RNAi response to RNAi in insects, no single protocol is suitable for all species. Issues related to the choice of effective target genes, including determining the size of optimal dsRNA length and ideal gene region. Assuming that the method of choice to deliver dsRNA is transgenic plants, a major question still to be addressed is the impact of plant dsRNA processing in the effective RNAi-induced silencing. There is still a need for investigation in this area. The choice of a suitable inducible promoter for expressing the dsRNA construct is another point barely explored.
Based on the recent publications reviewed in this chapter, the progress in developing “RNAi-plants” to control important insect pests widely demonstrated the potential of this technology to complement or replace Bt crops, providing resistance against a broad variety of insect pests. However, to be applied on a commercial level, several issues related to the RNAi mechanism and biosafety still need to be addressed. As a new technology, risk assessments and government regulations still have to be developed. However, RNAi transgenic crops are expected to have wider acceptance and reduced biosafety requirements for RNAi traits, in comparison to a protein incorporated into a plant, such as a Bt transgenic [39]. Thus, RNAi-mediated pest control will open a new paradigm in insect pest management.
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