Common strategies* for mass production of dsRNA with pros and cons.
RNAi in crop protection can be achieved not only by plant-incorporated protectants through plant transformation (transgenic) but also by nontransformative strategies such as formulations of sprayable dsRNAs used as direct control agents, resistance factor repressors, or developmental disruptors. Therefore, the RNAi-based biopesticides are expected to reach the market also in the form of nontransgenic strategies such as sprayable products, stem injection, root drenching, seed treatment, or powder/granule. While the delivery of dsRNA by transgenic expression is well established, it requires generations of crop plants and is costly, which may take years and delays for practical application, depending on the regulatory rules, plant transformability, genetic stability, and public acceptance of genetically modified crop species. DsRNA delivery as a nontransgenic approach was already published as a proof-of-concept work, so it is time to point out some directions on how the real potential for agriculture and crop protection is.
- nontransgenic approaches
- nontransformative plant protection
1. Crop protection and RNAi
The beginning of human civilization can be traced back to the ability of cultivating crops. This has allowed that a higher number of people could be supported in the same environment; however, it also brought several crop protection challenges that mankind has been facing continuously. To ensure sufficient food production, since the earliest days of agriculture, farmers have had a history of using agrochemicals to protect their crops against yield loss from a vast range of organisms including pest insects, mites, fungi, weeds, and others.
The modern era of synthetic pesticides began in the 1930s, and with insects, fungal pathogens, and weeds, destroying each one more than 13% before harvest and about 10% in postharvest , the pesticide use has become fundamental to modern crop protection technology. The increasing resistance  of weeds, pest insects, and fungi to established agrochemical compound classes, stringent regulatory environment rules, and market growth has stimulated demand for more selective, safer, and cost-effective pest control methods. Crop protection scientists have allocated a great deal of intellectual energy into seeking of more refined strategies to reduce crop losses such as transgenic crops expressing
RNAi is a natural process present in eukaryotic cells for gene regulation and antiviral defense. Although, from a crop protection perspective, RNAi refers to double-stranded RNA (dsRNA)-mediated gene silencing that involves the blocking of the expression of specific target genes by destroying the corresponding mRNA molecules affecting only the translation process. Due to its sequence-dependent mode of action, the RNAi technology, as referred nowadays by industry, has a vast range of potential crop protection application, including genetic studies and pest control research in insects [6, 7, 8, 9, 10, 11, 12], mites [13, 14, 15] and ticks , plant pathogens [17, 18, 19, 20, 21, 22, 23], termites, nematodes, and weeds [2, 24, 25] in a range of crops. These RNAi practical applications have been pursuing over the last decade for the development of novel crop protection methods.
The application of this technology did not go unnoticed in agriculture; hence, since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in opening a new vista for crop protection. Nevertheless, one of the biggest challenges for the RNAi technology is to make possible that target organisms (i.e., pest insects, plant pathogens, nematodes, viruses) uptake intact and active molecules that will trigger an RNAi pathway. Delivery of dsRNA to a target organism is the easiest through transformative RNAi approach (i.e., transgenic plants) [26, 27], but it is not practical to every target and crop. Therefore, the development of nontransformative approach (i.e., sprayable dsRNA) [9, 11, 28] for RNAi delivery will boost up its use in the field.
2. RNAi mechanism in brief
The cellular mechanics of gene silencing by RNAi was largely misunderstood or even unknown until the work of Andrew Fire and Craig Mello with the nematode
3. Transformative versus nontransformative RNAi
As aforementioned for field applications, the transformative RNAi includes the plant-incorporated protectants (PIPs; i.e., transgenic plants/RNAi-based plant traits), whereas non-PIP dsRNA-containing end-use products (dsRNA-EPs) might be formulated as dsRNA active ingredient such as a raw material for insecticide, antifungal, or antiviral with variable delivery modes (see Section 4).
The RNAi mechanism works at mRNA level exploring a sequence-dependent mode of action, which makes it unique in potency and selectivity compared to any regular agrochemicals. Therefore, one advantage of RNAi either by transformative or by nontransformative approach is that it would allow farmers to target pests more specifically. The technology can be designed by using sequences of RNA that match very specific gene sequences in a target pest; hence, RNAi should leave other species unharmed. The careful selection of unique regions of pest genes results in effects highly targeted, avoiding unintended effects.
The transformative RNAi strategy through transgenic plants known as host-induced gene silencing (HIGS) has proved to be successful in the protection of crops against their specific pest insects [26, 36, 37, 38], plant pathogens [18, 19], viruses [22, 39, 40, 41], and nematodes [42, 43], recently reviewed .
A proof-of-concept milestone paper of Baum et al.  demonstrated that a dsRNA construct in a genetically engineered plant could provoke larval mortality in western corn rootworm (WCR),
The development of a transgenic plant expressing dsRNA as a strategy for plant protection is straightforward, but it is not practical to every pest organism and crop [9, 10, 12]. Although the delivery of dsRNA by transgenic expression is well established, it requires generations of crop plants, which may take years and delays for practical application, depending on the regulatory rules, plant transformability, genetic stability, and public acceptance of genetically modified (GM) crop species .
While RNAi-based crops are expensive to produce and have a high risk of resistance breakdown, topical application is underway as a nontransformative approach that might enable RNAi-based pest management. Therefore, triggering an RNAi pathway in a pest organism may also be possible through a spray-induced gene silencing (SIGS) approach, without changing the plant DNA. The SIGS as a nontransgenic approach for pest control was already published  as a proof-of-concept work and recently reviewed [9, 11]. Because of using this approach to silence genes without introducing heritable changes into the genome, it may not be regulated as a GM product. A dsRNA spray can be used almost immediately as a regular pesticide without having to go through several years involved in the development of a GM or conventionally bread crop. Besides, in several countries due to the slow regulatory procedure to approve transgenic crops, nontransformative RNAi strategies with similar results such as some of those demonstrated above could be applied. Still, the main drawback of nontransformative RNAi strategy is that as a plant grows, new leaves have to be sprayed to guarantee protection, so this implies in possible higher costs to farmers, whereas transgenic plants will produce dsRNA continuously. However, the vascular system of plants naturally translocates RNAs . Therefore, sprays on leaves, injection in trunks, or soil application of dsRNA can travel long distances through plant vessels; hence, this can be exploited for the development of pest control strategies [11, 28].
The idea to use sprayable dsRNA was followed by an underlying supposition that this type of molecule would have a short half-life for an effective crop protection agent , and the short half-life of dsRNA in soil and by UV light has been confirmed . This apparent challenge posed by SIGS approach is that the effects on plants last only a few days because unprotected RNAs break down soon. Farmers may not want to apply extensive sprays to keep plants protected; however, there are some positive issues because the sprays of dsRNA can be quickly tailored toward a pest organism, much faster than a GM crop, and last only a few days or weeks different from most regular pesticides. Crop protectors should bear in mind that there is no need for a pest control agent persist active for months to become an efficient pest control agent.
Regardless of the target species, for a successful nontransformative RNAi strategy, it is of paramount importance to identify unique regions in very essential target genes, so that brief changes in the level of expression can provoke severe consequences as well as delivery of sufficient amount of intact dsRNA. Alternatively to transgenic plants, the delivery of dsRNA can be through other routes including dsRNA sprays, dsRNA expression in bacteria, trunk injection, and engineered viruses, among others. For example, to control plant viruses, farmers are obligated to either grow varieties with resistance to viruses or try to kill the organism that spread, such as aphids or hemipterans. Sprays with dsRNA might be rapidly tailored against existing or new type of virus, and the gene-silencing effects of RNAi will last only a few days, enough to suppress virus replication. Overall, the SIGS approach opens up a range of possibilities for several pest insects that are difficult to control such as root-feeding and sap-feeding insects, plant viruses, and plant pathogens, especially in perennial crops (e.g., fruits such as grapes, apples, and citrus), where plant transformation takes years to develop and is costly.
4. Successes of RNAi through nontransformative approaches
The delivery of dsRNA through nontransformative approaches is likely to hit the market in four categories: (i) direct control agents; (ii) resistance factor repressors; (iii) developmental disruptors; and (iv) growth enhancers [9, 10, 11, 49, 50, 51, 52]. As a direct control agent, nontransgenic approaches were successfully managed to achieve long-lasting gene silencing [9, 10, 18, 19, 41].
In some experiments, full-sized citrus and grapevine trees were treated with dsRNA using foliar sprays, root drenching, or trunk injections. Two hemipteran insects, a xylem- and a phloem-feeding, and a coleopteran chewing insect took up the dsRNA after feeding on plants previously treated with dsRNA [11, 28]. Similarly, rice plants were able to take up dsRNAs when their roots were soaked in dsRNA solution showing resistance against piercing-sucking and stem-borer pest insects  and also mites . Altogether, these experiments are clear demonstrations that drench/soak roots, trunk injections, and sprays on leaves are success strategies for delivery of dsRNA molecules without any modification on plants DNA.
Plant diseases caused by virus have a tremendous impact in food production and quality, being responsible for loses in several crops, fruits, and vegetables worldwide. Coherent with an ancient role to protect genome from invasive viruses, the RNAi mechanism can be reprogrammed to work by destroying any virus RNA. Without viral RNA, no viral proteins are made, thus preventing virus replication and plant diseases. Some studies have already been conducted on topical application of dsRNA to control plant viruses [41, 54]. However, a major limitation in the practical application of dsRNA to control viruses is that RNAs face a hostile environment where it is rapidly degraded with not only low uptake into plants, but also the short virus protection window of few days postspray. There are some rumors that the initiated pipeline branded “BioDirect” by Monsanto controls pest insects and plant viruses with sprays of dsRNA, but details on this probably are not publically available. To address some of these limitations, a layered double hydroxide (LDH) clay nanosheet, called “BioClay,” was developed  and combined with dsRNA molecules. The clay nanoparticles are positively charged and so bind and protect the negatively charged RNAs; delivery occurs when atmospheric carbon dioxide and moisture react with clay nanoparticles breaking down LDH, gradually releasing RNAs. Using this dsRNA-LDH complex was possible to achieve long-lasting gene silencing, protecting tobacco plants from a virus for 20 days with a single spray [55, 56], thus extending the period of 5–7 days using naked dsRNA. The complex dsRNA-LDH protected plants in both local lesions and systematically. Also using RNAi to control plant viruses, the mechanism of dsRNA uptake into the leaf was investigated. It was reported a rapid systemic spread of dsRNA when leaves of
Direct spray of dsRNA was also used experimentally to control the Colorado potato beetle (CPB),
The fungi kingdom consists of a large and diverse group of eukaryotes, and plant diseases caused by fungi exert particular and agronomic impact on global grain and food production. Generally, the proteins dicer, argonaute, and RdRP, which are some of the major components of RNAi pathways, are present in most fungi species . Therefore, the RNAi pathways can be harnessed to control plant diseases . Sprays of CYP3-dsRNAs, targeting simultaneously three fungal ergosterol biosynthesis genes (P450 lanosterol C-14α-demethylases—
The study with full-sized citrus trees (2.5 m tall) was performed with 2 g of dsRNA in 15 l of water applied by root drench and injections . The dsRNA was detected in psyllids and leafhoppers 5–8 days postingestion from plants and for at least for 57 days in the citrus trees; this allows the development of an area-wide pest suppression approach. Similarly, Koch et al.  showed that the CYP3-dsRNA labeled with the green fluorescent dye (ATTO 488) was detected in the vascular tissue 24 h after spraying leaves. Also, the leaf sections demonstrated that the fluorescence was detected in the xylem, in the apoplast and symplast of phloem parenchyma cells, companion cells, mesophyll cells, as well as in trichomes and stomata. The labeled dsRNA was detected also inside fungal conidia and germ tubes as well as in the fungal mycelium. These experiments conducted by Koch et al.  using sprays on barley leaf surface are the first examples of active dsRNA uptake by plant cells.
The uptake of RNAs from the environment, a phenomenon known as environmental RNAi , has not yet been observed in mammals. This phenomenon was observed in
One obstacle, if not the biggest, is the cost for the mass production of dsRNA. While the issues of environmental stability and delivery are being addressed with creative innovations such as BioClay, making mass amounts of RNA is still expansive. Indeed, cost-effective methods will allow real-world applications of exogenous dsRNA for RNAi-mediated crop protection. To our knowledge, currently, there are no commercial RNAi-based products that utilize dsRNA as a spray for crop protection. Since the discovery of dsRNA and its potential for crop protection, some companies and academic scientists are seeking to develop more cost-efficient methods for large production of dsRNA. Similarly, RNAi to control devastating pests such as the Colorado potato beetle has obviously attracted attention in private research and development. As mentioned before, Monsanto (currently Bayer) and Syngenta (current ChemChina) have allocated major investments toward SIGS technology. Already in mid-2015, Monsanto launched its technology BioDirect, and although the principle was the same as we had seen in academia, these products work differently because they are not expressed in the leaves, but applied exogenously to the plants. Syngenta scientists also are developing lines of biocontrol products based on RNAi (https://www.youtube.com/embed/BiVZbAy4NHw?ecver=1). For example, these dsRNA-based products when sprayed onto the potato plants (field trials) or soy plants targeting genes of Colorado potato beetle and stink bug,
|Approach||Cost||Purification||Labor demand||Mass production|
In the last few years, we have experienced an ever-growing interest in the market for dsRNA that has pushed long-established companies and startups toward better production, cost-efficient, and stable delivery systems. In instance, the cost to produce 1 g of dsRNA (100 up to 800 pb) has dropped from $12,500 USD in 2008 to $100 USD in 2016, and to less than $60 USD today (July 2018) (http://www.agrorna.com/sub_05.html). The agroRNA  produces bulk amounts of dsRNA that could be used in agriculture; however, it is worth noting that naked dsRNA as sold by agroRNA needs to be formulated if the objective is a long-lasting crop protection; otherwise, the dsRNAs will last only a few days. For crop protection, dsRNA does not need to be as pure as for medical application; however, at least for gene silencing in insects, the efficacy of dsRNA increased using purified RNA.
Considering the rapid half-life of dsRNA mainly regulated due to action of RNases and sunlight in the hostile environment, a biotechnology company RNAagri (former APSE) developed a technology “Apse RNA Containers” (ARCs) that allows the mass production of encapsulated ready-to-spray dsRNA with costs near $1 USD per 1 g . In brief, this technology is based on plasmids engineered to produce naturally occurring proteins such as capsids that are cotransformed with another plasmid coding for the target dsRNA with a sequence called the “packing site.” The proteins produced by bacteria self-assemble around RNAs, resulting in RNA protected and resistant to environmental hostile conditions. For long-lasting crop protection with exogenous applications, the dsRNAs should be protected with coating of nanoparticles, liposomes, or polymers, which will increase the efficacy by reducing dsRNA degradation .
Alternatively to pure dsRNA, the
The virus-induced gene silencing (VIGS) has also a great potential [70, 71, 72] to transiently silence target genes of insects or pathogens on host plants. Therefore, if an insect or pathogen-specific RNAi inducer sequence is introduced into an engineered plant virus, siRNAs specific for insect/pathogen targets will be produced upon plant infection [18, 73].
5. Postharvest protection using nontransformative RNAi
Yearly, vegetables, grains, flowers, and fresh fruits are damaged by microbial pathogens and insects. Sprays of dsRNA may also be efficient on postharvest products  to protect them during processing, transportation, and storage. Indeed, spraying
6. Other applications
RNAi naturally protects the cell from invasive viruses. Therefore, beyond the application of dsRNA sprays for pest and pathogen control, there is also a potential for the protection of beneficial insects such as bees from viral diseases. For example, the Israeli acute paralysis virus (IAPV)  is a single-stranded RNA virus in the family Dicistroviridae that increases bee mortality. The ingestion of dsRNAs from two regions of the IAPV genome protected bees from subsequent IAPV infection. The success of this experiment has encouraged field trials . Large-scale field trials tested the efficiency of a dsRNA product, Rembee™ (Beeologics, LLC, Miami, FL, USA), in protecting honeybees from IAPV infection. The result was twice as many bees in the dsRNA-treated hives when compared to untreated hives. Additionally, dsRNA-treated hives produced threefold more honey than the untreated hives infected with IAPV. Similar results are also observed. A similar result was observed in bumblebees (
Crop protection against pathogens and pest insects relies mostly on the widespread use of chemical pesticides that are applied to the environment in large amounts yearly; some of these chemicals are in use for almost half a century. Therefore, there is a need for novel tools more sustainable and less detrimental to the environment. Therefore, scientists have harnessed RNAi to turn off genes that they are studying. RNAi through nontransformative strategies will demand mass production of dsRNA, efficient delivery methods, and methods to validate its environmental stability.
A large number of studies have demonstrated the feasibility and efficacy of RNAi-based approaches, and some transgenic plants have been approved for commercialization and release [38, 39, 79, 80]. However, unlike these strategies, which depend on plant transformation, the spray of dsRNA externally realizes crop protection without changing the plant DNA. The dsRNA-containing end-use products, nevertheless, will be differently regulated when compared to transgenic plants such as
It is worth to remind that a specific dsRNA exerts its mode of action throughout entire sequence length by generating a large pool of target-specific siRNAs [29, 30, 32]. This large pool of siRNAs for a single target increases target specificity and largely reduces evolution of mutations and resistance in the targeted organism. Indeed, the dsRNA is designed to match a long nucleotide sequences in the target organism (i.e., insects, pathogens, or viruses). The effectiveness of a long dsRNA will remain even when parts of this sequence mutate. So that it is believed that it is unlike to face resistance evolution that commonly makes a chemical pesticide ineffective. Resistance development toward RNAi has not been documented in insects and fungi, but as a famous artist says, “
The sprayed dsRNAs, different from regular pesticides, are biocompatible compounds as they occur naturally in the nature as well as inside/outside body of organisms and in food. The dsRNA ultimately is a regular RNA molecule that enters naturally within plants and other organisms. These molecules are subject of pathways from RNAi silencing mechanism, converted into siRNA and finally degraded by natural cell processes. In water and soil, dsRNAs are rapidly degraded as regular RNA molecules do , so unlike to left considerably novel residues in food products.
New genomic tools will allow the development of technologies such as dsRNA sprays that increase crop resistance against insects, pathogens, and viruses; these could even replace chemical pesticides in some applications.
The authors acknowledge support over the years for their RNAi-related research from the National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES) in Brazil and from the Foundation Research-Flanders (FWO-Vlaanderen) and the Special Research Fund of Ghent University (BOF-UGent) in Belgium.