Progress and Opportunities of In Planta and Topical RNAi for the Biotechnological Control of Agricultural Pests

Abstract

In planta RNAi or host-induced gene silencing (HIGS) has undergone significant advancements that have rendered it efficient and stable at the transgenerational level in plants for regulating host genes and targeting genes of insect pests and plant pathogens. Similarly, topical RNAi or spray-induced gene silencing (SIGS) has garnered considerable attention as an environmentally sustainable, selective, and alternative approach to chemical control of insect pests and plant pathogens. Several biotechnology companies and startups have focused their efforts on RNAi-based solutions for topical application in agriculture. Nevertheless, further technological advancements are required to enhance the efficacy of topical RNAi in agriculture, including improved dsRNA delivery systems, better target gene selection, and addressing biosafety regulatory issues. Herein, this review discusses key advances and bottlenecks in RNAi, and summarizes successful applications of these RNAi-based technologies in agriculture focusing on in planta and topical RNAi to control insect pests and plant pathogens. Furthermore, this review delves into the patenting landscape, biosafety considerations, risk evaluations, and the current regulatory status of RNAi in Latin America. Finally, it explores the contributions of RNAi to plant science, food production, and fostering a more sustainable form of agriculture.

1. Introduction

The RNA interference (RNAi) pathway has been explored in plants for proof-of-concept and biotechnological purposes (Figure 1). Initially, in transgenic plants (in planta or host-induced gene silencing, HIGS), the dsRNA expression was exploited to validate the biological function of genes and subsequently used for down-regulation of endogenous genes to achieve desired agronomic traits [1]. Virus-induced gene silencing (VIGS) has emerged as an alternative to transgenesis for proof-of-concept purposes [2]. Both in planta RNAi and VIGS have enabled the development of virus-resistant plants by expressing virus-specific dsRNAs to target viral genomes or their mRNAs [3,4]. Later, in planta RNAi was employed to confer resistance to other plant pathogens [5], insect pests [6], plant-parasitic nematodes [7,8], fungi [9], and oomycetes [10] (Figure 2). From this consolidation, transgene-free RNAi (topical or spray-induced gene silencing, SIGS) was established [11]. The first study showcasing the efficacy of topical RNAi delivery used Tobacco mosaic virus (TMV) RNA encapsulated in liposomes to deliver dsRNA molecules to tobacco protoplasts [12]. Despite these advancements, topical RNAi still encounters significant challenges; however, this approach aligns with the current pursuit of eco-sustainable and more selective agriculture technologies [13,14]. Notably, economic losses attributed to insect pests and plant pathogens in different crops have increased over the years, resulting in heightened agrochemical usage [15,16]. While considerable advancements have been achieved with in planta and topical RNAi, there are still substantial obstacles to overcome before making RNAi-based products more accessible to farmers [17]. The biosafety regulation of these RNAi-based technologies for commercial use remains a topic of discussion in Latin America, the USA, and Europe [18,19]. Herein, this review study presents advancements in (i) in planta and (ii) topical RNAi, (iii) intellectual property related to RNAi, (iv) biosafety and risk assessment of RNAi-based technologies, and (v) the major contributions of RNAi to plant science.

2. In Planta RNAi

2.1. Establishment of In Planta RNAi

In planta RNAi is based on the transgenic overexpression of a dsRNA with high sequence homology to the endogenous or exogenous mRNAs, triggering post-transcriptional gene expression (PTGS) [8,20]. This overexpression must be engineered under the control of constitutive, stress-induced, and tissue-specific promoters, making this choice fundamental for regulating the activation of the RNAi machinery depending on the type of expected outcome [21,22]. This overexpression results in a primary transcript that is further processed by Dicer-like enzymes (DCLs) to generate small interfering RNA (siRNA) [23]. Initially, the overexpression of a full-length gene sequence was utilized to induce RNAi [24]. Subsequently, it was discovered that overexpression of a shorter antisense RNA is adequate to trigger RNAi [25]. Therefore, antisense RNA expression has undergone considerable optimization to enhance PTGS and its transgenerational stability in transgenic plants [23]. The key advancement was determining the minimum length of an antisense RNA (200–400 nucleotides) capable of triggering RNAi, effectively down-regulating the target gene without causing off-target effects. Subsequently, it was shown that simultaneous overexpression of sense and antisense RNA results in increased siRNA production and more efficient PTGS [26]. However, superior RNAi responses have been achieved by overexpression of a sense and antisense RNA separated by a spacer sequence [27,28]. While these in planta RNAi proof-of-concept studies have led to high RNAi efficiency, concerns regarding the transgenerational stability of this technology still exist [6,29,30]. The transgene methylation induced by transcriptional gene silencing (TGS), the reduced transcription of RNAi-inducing RNA, and the decline in PTGS efficiency over successive generations in transgenic plants have been reported [31,32]. The overexpression of sense and antisense RNA driven by a constitutive promoter results in continuous siRNA production, which can eventually lead to TGS through transgene methylation across generations [23]. Hence, it is advisable to utilize tissue-specific promoters with targeted activity [22,33], cognate promoters [34], and stress-induced promoters [35]. The RNA structure has also been refined to ensure enhanced stability of the dsRNA, minimizing premature degradation by nucleases, promoting transgenerational stability, and augmenting PTGS efficiency [6,30,36].

Fitch et al. [37] developed transgenic papayas resistant to the Papaya ringspot virus by overexpressing the CP gene, resulting in high resistance against this virus [38]. This study served as a key reference for the biotechnological purposes of in planta RNAi. Likewise, Bonfim et al. [3] developed transgenic common beans by overexpression of sense and antisense RNAs, spaced by a PDK intron, capable of down-regulating the AC1 gene of Bean golden mosaic virus (BGMV). These transgenic beans exhibited high resistance to this virus and demonstrated transgenerational stability under field conditions [39]. This sustained transgenerational stability can be attributed to the RNAi mechanism being activated during plant infection by BGMV. Head et al. [40] further showed that transgenic maize, constitutively overexpressing a sense and antisense RNA separated by an HSP70 intron, effectively down-regulates the DvSnf7 gene of Diabrotica virgifera virgifera. This transgenic maize (event MON-87411-9) is commercially available in various countries and has proven transgenerational stability [41]. Recently, Zhang et al. [30] introduced nucleotide mismatches between the sense and antisense RNA to prevent the self-TGS. Specifically, it has been shown that replacing all cytosines with thymines maintains the hairpin RNA (hpRNA)structure and enhances the transgenerational stability of dsRNA [30]. Additionally, Hunter and Wintermantel [36], enhanced dsRNA stability and increased insect mortality by modifying the 2′-F pyrimidine nucleotides (2′-F-Uridine and 2′-F-Cytosine).

In addition to transgenerational instability, premature dsRNA processing by plant DCLs also reduces the efficiency of RNAi for pest control [42]. This premature dsRNA processing can diminish RNAi efficacy particularly when pests are unable or inefficient in uptaking siRNAs rather than dsRNA [43]. One strategy to address this challenge involves modifying the secondary structure of dsRNA to render these molecules inaccessible to plant machinery [44,45]. Abbasi et al. [46] showed that “paperclip”-structured RNAs enter cells through a clathrin-independent pathway, enhancing PTGS, while the uptake of siRNAs, short hpRNAs, and long dsRNA was suppressed. Similarly, CSIRO researchers have developed the LedRNA strategy, which facilitates high siRNA accumulation and diminishes transgene self-silencing, thereby enhancing RNAi effectiveness (WO2019051563; WO2021022325A1). Furthermore, specific structured dsRNAs may also remain inaccessible to the RNAi machinery of pests. Ribeiro et al. [6] showed that overexpression of a dsRNA resembling a viroid-like architecture in the chloroplast increased dsRNA stability in transgenic plants and enhanced its accessibility to insects. Therefore, structured dsRNA can enhance stability within the plant and make it more accessible to pests [6].

2.2. In Planta RNAi for Insect Pest Control

In addition to the stability of dsRNA in plants, the expression of sufficient amounts of dsRNA to achieve efficient uptake and PTGS in insect pests remains a major challenge [6]. Nucleases present in the insect gut degrade dsRNAs even before they enter the cells [47]. By targeting the genes encoding nucleases with the dsRNA, it was possible to enhance the RNAi efficiency in insect pests [48]. Moreover, the co-overexpression of dsRNAs and chimeric PTD-DRBD proteins containing binding domains to this dsRNA improved the efficiency of ribonucleoprotein internalization across the insect cell membrane [49]. The chloroplast-specific transgenic overexpression of dsRNAs triggered by chloroplast-specific promoters also provides greater stability to the dsRNA, as PTGS does not occur in this organelle [50,51,52]. Additionally, chloroplast-triggered overexpression of dsRNA can bypass these obstacles by accumulating high levels of protected dsRNA [53,54]. Although this strategy is not yet commonly employed due to the challenges associated with plastid transformation, transplastomic plants are being developed to combat both chewing and/or sap-sucking insect pests, such as Leptinotarsa decemlineata [42], Helicoverpa zea [55], Myzus persicae [56], Bemisia tabaci [57], Frankliniella occidentalis [51], and Manduca sexta [58]. The length and stability of the dsRNA directly impact its accumulation in chloroplasts and the efficacy of insect control [57,59]. For example, 60-nucleotide dsRNA induced a weak RNAi response in L. decemlineata, whereas 200-nucleotide dsRNAs resulted in higher insect mortality rates [50,60]. The target gene selection in insect pests also influences the effectiveness of RNAi [61], while various factors combined can contribute to the ineffectiveness of transplastomic plants in controlling insect pests [57,59]. Furthermore, the evaluation of transplastomic plants overexpressing self-replicating dsRNAs for enhanced accumulation is still pending [62].

Conversely, nuclear transgenic plants have been successfully developed to control insect pests, including D. virgifera virgifera [63], Helicoverpa armigera [64], Adelphocoris suturalis [65], Sitobion avenae [66], and Chilo suppressalis [67]. SmartStax PRO maize overexpressing a dsRNA to target the DvSnf7 gene of D. virgifera virgifera, in addition to Cry toxin, was the first commercially available RNAi-based crop to control insect pests [40]. As stated previously, the primary bottleneck impeding the efficiency of nuclear transgenic plants is the early processing of dsRNA within plant cells [42]. This issue is particularly troublesome as most species possess mechanisms for the uptake of long dsRNAs and are unable to spread siRNAs systemically [68,69]. The assertive choice of appropriate target genes in insect pests is crucial for the effectiveness of RNAi. Efficient insect control is usually achieved by targeting genes linked to a lethal phenotype or disrupting insect viability [70,71]. For instance, some potential targets for RNAi in insect pests include ATPases, proteases, kinases, ABC transporters, vitellogenin, AgraRelish, chitin synthase, laccase, Distal-less, and ecdysis-triggering hormone receptors [6,70,72,73]. Integrated approaches using multiple target genes, or strategies, such as RNAi combined with entomotoxic proteins, can reduce plant susceptibility to insect pests [74,75]. Moreover, when choosing the target gene region, it is crucial to ensure both the efficacy of RNAi in the target insect and the absence of off-target effects in non-target organisms [76].

Another challenge of in planta RNAi for controlling insect pests is the necessity to adhere to stringent regulatory frameworks [77]. In turn, in planta RNAi offers the advantage of continuous dsRNA expression, providing constant exposure to the target insect pest [78]. Consequently, this approach might be favored for its practicality in field applications. In contrast, developing transgenic plants could be more time-consuming, labor-intensive, and costly [79]. Examples of in planta RNAi applications for insect pest management and an overview of dsRNA delivery methods, RNAi strategies, targeted insect species, genes, crops, and resulting plant phenotypes are detailed in Table S1.

2.3. In Planta RNAi to Control Plant-Parasitic Nematodes

In planta RNAi studies with nematodes have provided promising results, particularly when targeting genes encoding effector proteins [8,80,81,82]. Targeting a single effector gene was sufficient to decrease plant susceptibility by 70–80% [8]. Comparatively, in planta RNAi directed at other functional genes proved effective in managing plant-parasitic nematodes but it demonstrated lower efficacy compared to effector genes [7]. Therefore, several validated genes in model plants have emerged as targets for RNAi in crops [83]. These studies suggest that pyramiding dsRNAs to target different essential genes could be an alternative for durable plant resistance under field conditions [84]. However, the main barrier to its commercial use in main crops is the fact that it is regulated as a transgenic. In planta RNAi on rootstocks could be a good strategy for fruit and vegetables propagated by grafting to control nematodes and reduce public concern regarding to transgenic products [85]. Furthermore, the additional cost of intellectual property is another factor that hampers the adoption of in planta RNAi to control nematodes in crops [86]. Examples of in planta RNAi applications for nematode control, along with an overview of dsRNA delivery methods, RNAi strategies, nematode species, target genes, crops, and plant phenotypes, are detailed in Table S2.

2.4. In Planta RNAi to Control Other Important Plant Pathogens

In planta RNAi has shown significant potential in managing viruses, subviral agents, fungi, and oomycetes (Tables S3 and S4). Notably, viral agents, which depend entirely on the plant cell, display high sensitivity to the plant’s RNAi machinery [87]. Numerous studies employing RNAi targeting different or multiple genes and genomes of RNA and DNA viruses have underscored the effectiveness of this technology in crops [88,89]. Similarly, several target genes have been identified in fungi and oomycetes for in planta RNAi targeting, resulting in pathogen suppression and reduced plant susceptibility [11,90,91]. In this context, the selection of the target gene in the specific pathogen is decisive for the effectiveness of RNAi. Therefore, in planta RNAi has proven to significantly decrease crop susceptibility to plant pathogens. However, the utilization of in planta RNAi in crops has been limited primarily due to transgenic regulations and the current practicality of implementing topical RNA. Nonetheless, both RNAi approaches have been deemed highly promising and environmentally sustainable alternatives to synthetic chemical fungicides [14,92]. The primary advantage of in planta RNAi lies is the steady and continual availability of dsRNA in crops compared to topical RNAi, which requires regular applications that may face challenges due to the instability and limited uptake of dsRNAs by these pathogens [93]. Conversely, in planta RNAi-based technologies for controlling bacteria and phytoplasmas remain limited, as these pathogens lack the RNAi machinery [94]. Nevertheless, in planta RNAi has been effectively utilized to down-regulate plant genes associated with host susceptibility, enhancing plant resistance to these pathogens [5]. Examples of in planta RNAi for control of plant viruses, subviral agents, fungi, and oomycetes, as well as an overview of the RNA delivery methods, RNAi strategies, pathogens targeted by RNAi, target genes, crops, and plant phenotypes are outlined in Tables S3 and S4.

2.5. Alternative Methods for In Planta RNAi

Symbiont microorganisms engineered to overexpress dsRNAs to cross-act on target genes of insect pests have been developed, such as Saccharomyces cerevisiae and Bacillus thuringiensis to target Drosophila suzukii, Spodoptera littoralis, and Plutella xylostella [95,96,97]. Similarly, engineered viruses have been utilized as viral vectors (virus-induced gene silencing) to suppress plant genes for proofs of concept or pest genes for biocontrol [98,99,100]. Concerns regarding transgenesis are still under regulatory consideration for the commercial deployment of these approaches. Artificial miRNAs (amiRNAs), which provide enhanced efficacy and specificity for the down-regulation of target genes offer a valuable alternative to control insect pests and pathogens but also require plant genetic engineering [20,101]. Furthermore, the stable knockout of plant genes using genome editing through CRISPR/Cas9 technology by Non-Homologous End-Joining (NHEJ or SDN1) or equivalent approaches has been reported to be relevant for commercial purposes [102,103,104]. This relevance occurs because it allows overcoming transgenic restrictions in certain countries [102,105]. In this context, both in planta RNAi and NHEJ/SDN1-mediated genome editing have been employed to down-regulate or knockout genes linked to plant susceptibility to multiple stresses, or genes encoding negative regulators of other proteins associated with increased plant resistance [106,107]. Meanwhile, genome editing of pests to knockout target genes is not yet available as an open field control measure in agriculture.

3. Topical RNAi

3.1. Establishment of Topical RNAi

Topical RNAi is a promising approach to down-regulate genes in plants, insect pests, and plant pathogens. Induction of RNAi might be achieved by ingestion of dsRNA sprayed on artificial diets or leaves and topical spray of dsRNA directly on the target organisms [17,71]. Previous studies have identified several target genes associated with lethal or deleterious effects in these organisms that can be used in topical RNAi (Tables S1–S4) [108,109,110]. The primary challenges include dsRNA instability when exposed to adverse environmental conditions, high costs for dsRNA production, low dsRNA uptake by plants or pests, and the substantial dsRNA dosages required to induce knockdown in some species [7,111]. Furthermore, factors such as leaf wettability, hydrophobic cuticle, wax layers, trichomes, cell wall barriers, and stomatal aperture and closure variations, which can all impede the efficiency of dsRNA uptake by plants, need to be considered as barriers [112,113]. Therefore, the selection of target genes and optimization of dsRNA delivery systems represent crucial steps towards achieving high control rates using minimal doses of dsRNA [114]. In contrast, topical RNAi can be applied to a wide range of organisms and can be associated with biological control and synthetic agrochemicals for crop protection [115]. It has also gained prominence in the last five years as an alternative to address the current demands for eco-sustainable technologies and the need to reduce excessive applications of non-selective agrochemicals to control pests [14]. Furthermore, it offers an important regulatory advantage by not relying on transgenic-based delivery and allowing high target specificity [17,77]. However, transforming proof-of-concept results using topical RNAi into commercial products has encountered significant challenges [116]. In this context, biotechnology companies have focused on the research and development of these topical RNAi-based products to increase efficacy, reduce costs, and streamline biosafety regulatory issues [117].

3.2. Large-Scale dsRNA Production

Topical RNAi initially involved the in vitro production of RNA in sense or antisense orientation and its delivery to target organisms through spraying, ingestion, or injection at the laboratory scale [113,118]. However, the low dose and rapid degradation of RNA highlighted the need for advancements in the stability of these molecules [119]. Consequently, dsRNA molecules have emerged as RNAi triggers because they are more effective and stable compared to sense or antisense RNA [120]. Therefore, topical RNAi depends on the large-scale production of dsRNA, either by cell-free synthesis or cell-dependent production [118]. The dsRNA production in microorganism systems such as yeast, bacteriophage, Bacillus, Pseudomonas, Corynebacterium, and Escherichia coli has been demonstrated as the most cost-effective method [118,121]. E. coli co-expressing a viral capsid protein has proven to be effective by forming complexes between the dsRNA and viral protein, protecting against degradation by nucleases [122]. However, this alternative had limited commercial application due to concerns regarding the potential viral hetero-encapsidation [123]. Given the challenges in obtaining systems that provide stability and protection for dsRNA, this approach has again been revisited [124]. Furthermore, compartmentalized dsRNA in E. coli itself has been successfully tested, facilitated by the use of an RNase III-deficient strain [119]. Even so, the cost of large-scale production, the amount of dsRNA required to be delivered to RNAi target cells, and co-contamination with other bacterial RNA/dsRNA that overload the RNAi pathway are still factors to be overcome [118]. Therefore, producing sufficient quantities of highly intact and pure dsRNA at a lower cost remains a challenge that persists to date [115].

3.3. Nanocarriers for Topical Delivery of dsRNA

The highly variable sensitivity to the RNAi and ability to access exogenous dsRNAs (such as sap-sucking and chewing insects) among target organisms pose a constraint that hampers the widespread application of topical RNAi-based products [125,126,127]. Therefore, topical RNAi often requires the formulation of dsRNAs and adjuvant molecules, ensuring their functional stability until delivered to the target cell [17]. The use of nanocarriers is considered one of the most promising approaches nowadays. Nanomaterials act not only as stabilizers, protecting dsRNA molecules against degrading agents but also as transporters, facilitating the delivery of dsRNA into target cells. Several nanocarriers improve the RNAi efficiency enhacing dsRNA stability, delivery, uptake, and endosomal escape in target organisms, [17,113,128,129]. Furthermore, nanocarriers that exhibit no harmful effects on RNA and non-target organisms, biodegradability, cellular compatibility to improve dsRNA uptake and processing into siRNAs, and low production costs have been preferred [113,130].

Most nanocarriers used to protect and deliver RNAi molecules follow the same principle of complexation, which is based on the electrostatic interactions between the negatively charged phosphate backbone of RNA molecules and cationic groups in the carrier molecule [131]. These interactions are strong enough to shield RNA molecules from most enzymes and compounds that could interact with them and lead to their degradation, while also being weak enough, as compared to covalent interactions, to release the RNA within the cell to trigger the RNAi mechanism [132]. Interestingly, protection at the extracellular level is not the sole advantage of using nanocarriers. It has been recently demonstrated to light that nanocarriers are uptaken by cells through the clathrin-mediated endocytosis mechanism and facilitate endosomal escape, ultimately preventing the dsRNA/siRNA degradation by the lysosome [128,133]. MgAl-layered double hydroxides [134], chitosan [135], E. coli-derived anucleated minicells [136], microvesicles such as exosomes [137,138,139,140,141], star polycations [128,142], clay nanostructures [143], DNA nanostructures [144], silica [145], liposomes [146], carbon dots such as polyethyleneimine [147,148], gold nanocluster [149], single-walled carbon nanotubes [150], and cell-penetrating peptides [49,151] are among the main examples of nanocarriers.

One of the most studied nanocarriers is chitosan, which has been shown to effectively enhance the RNAi effect in pests [135,152,153]. In addition, chitosan is easily degraded in the environment and has low toxicity levels when accumulated in mammal cells [154,155]. Hence, it is considered one of the most suitable nanocarriers for dsRNA delivery in agricultural systems. Dendrimers, such as star polycations, are also cheap to produce with the advantage, compared to chitosan, that the flexibility of their architecture allows them to easily modify their size, branching density, and surface functionality [156]. This is particularly interesting for topical delivery, in which interaction with different types of plant surfaces requires nanocarriers with different characteristics to penetrate a certain tissue, facilitate adsorption, and endure weather conditions. The major challenge for the development of cationic polymers is their variable stability due to the capability of most polymers to interact with molecules present in the target pests or their hosts, which can lead to the premature disassembly of nanoformulations. On the other hand, molecules that naturally occur inside cells, such as lipids, peptides, and proteins, also have the potential to deliver dsRNAs due to their biodegradable composition and high stability [157,158,159,160,161,162,163,164]. However, the main limitations of these molecules are related to production costs and eventual cytotoxicity effects [165,166]. Furthermore, chemical modifications of nanocarriers [167,168], dsRNA/nanocarrier delivery in association with surfactants to increase uptake [169], and conjugation with other chemicals to increase stability [152,170] are some alternatives to improve certain nanoformulations. Therefore, each of these nanocarriers has particular characteristics and variable efficacy for each delivery system and target pest (Table S5), from enhancing the RNAi effect to eliciting the basal defense of plants [171,172,173,174].

Despite the wide variety of nanomaterials currently available, some parameters have become standard measurements of the potential efficacy of a given nanoformulation. Ideally, nanocarriers smaller than 50 nm have been preferred as they offer a high surface-to-volume ratio, facilitating effective dsRNA binding, and are small enough to be uptaken by the plant cell wall and membrane of target organisms [143,175]. In addition, water-soluble nanocarriers with a positively charged core and surface have been preferred by facilitating dsRNA assembly [143]. Likewise, nanocarriers with a zeta potential greater than +25 mV and a low polydispersity index have shown the ability to achieve uniform distribution in the solvent [176]. However, recent studies have indicated that even nanocarriers larger than 150 nm can efficiently deliver dsRNA and enhance the RNAi effect [162,177,178]. The dsRNA fragment size is a determinant of the size of the resulting nanocarrier. While longer fragments (>60 bp) are usually required for efficient gene silencing in insects [41,179], some researchers have opted for shorter dsRNA sequences (<60 bp) for nanocarrier delivery, as the parameters such as size or charge are easier to control when using RNAi molecules with lower molecular weight [135,152]. It remains to be demonstrated whether dsRNA encapsulated into larger nanocarriers or siRNA into smaller ones is more efficient in improving gene silencing. Currently, this decision is made on a case-by-case basis; however, other factors besides efficacy could also be determinants, such are biosafety, production costs, and reproducibility.

While stability and protection provided by nanocarriers are often the emphasized characteristics in designing an RNAi nanoformulation, there is another pivotal feature that should be equally explored, that are the mechanisms of nanocomplexes disassembly and RNA release. In most cases, intracellular anionic substances act as competitors for the cationic regions in the nanocarriers, displacing and consequently releasing the RNA molecules [180,181,182]. The advancements in the pharmaceutical industry have provided a number of controlled-release methods for different nanocarriers such as chitosan [183], silica [184], peptides [133], among others. These methods include temperature- or pH-induced release and nanocarrier’s surface modifications to exploit the recognition system mediated by receptors in cell membranes. Developing nanocarriers with tunable release mechanisms will be essential in the following years for the establishment of nanocarrier-mediated delivery of RNAi as a biopesticide. Although research in medical sciences has contributed to elucidating ways to achieve controlled release, further insights specific to applications against pathogens and insect pests are still needed. More on this will be discussed in the next section “topical RNAi to control insect pests”.

Finally, as for any pesticide, conducting risk assessment studies is crucial in the development of RNAi nanocarriers. To establish these studies and protocols, it is crucial to prioritize exploring how physicochemical characteristics, tissue accumulation, concentration, and the type of nanocarriers impact the specificity of RNAi against target pests and its toxicity towards non-target species. Recent publications have shown that chitosan/dsRNA nanocarriers specific to H. armigera did not produce any effects on non-target organisms (D. melanogaster and S. litura) [135,152]. However, we consider that risk assessments on species considered biomarkers are more appropriate to evaluate the safety of nanocarriers, due to their high sensitivity to chemical compounds, as in the case of beneficial insects (e.g., pollinators, aquatic organisms, and natural predators). For instance, star polycation nanocarriers for the control of whitefly only caused significant toxicity on predatory ladybird larvae at concentrations above that required for whitefly’s management [56,185]. Risk assessment protocols for nanocarriers containing molecules other than RNA have already been explored by comparing them with traditional agrochemicals [30,168,186]. Despite RNAi being considered a highly specific strategy for targeting pests, it is essential to conduct similar toxicity evaluations using RNAi-based nanocarriers to accurately evaluate their performance in comparison to current chemical pesticides. In conclusion, the use of dsRNA complexed with nanocarriers holds the potential to impact the fields of biotechnology and agriculture in the upcoming years, and biosafety considerations are important to accelerate this process.

3.4. Topical RNAi to Control Insect Pests

The foliar spray method has been the preferred approach for topical dsRNA delivery to control insect pests, characterized by its effectiveness not only against chewing insects but also relatively effective against sap-sucking insects due to the dsRNA’s ability to spread from the leaf surface into plant tissues [187]. Research by Hunter et al. [188] revealed that dsRNAs persist for an extended period in treated insects (5–8 days) and plants (at least 57 days post-treatment) under field conditions. However, it has been noted that most insect species from the Coleoptera and Hemiptera orders exhibit a robust RNAi response compared to Lepidoptera, which has a very limited response [127,189]. Therefore, despite advancements, there is still considerable room for further improvements in this research area, especially for RNAi-recalcitrant insects in open field conditions [113,116,190]. Current strategies to address this issue include focusing on the down-regulation of lethal-associated target genes [63,108,109,110], optimizing dsRNA delivery systems [191,192], and using nanocarriers to enhance the RNAi response in target insects [193,194]. Therefore, RNAi-based insect pest management is expected to become more cost-effective with the use of nanocarriers, as the required amount of dsRNA to control the pests is likely to be reduced. Nanocarriers-mediated dsRNA delivery in insect pests has been well described in mosquitoes [195], but also on aphids and whiteflies [196,197] and lepidopteran larvae [198,199,200].

The main objective of these previous studies was the protection of dsRNAs from the hydrolytic environment of the insect gut and hemolymph to enhance the silencing effect of target genes. Furthermore, most of these studies targeted lepidopteran and hemipteran pests and explored the efficiency of dsRNA/nanocarrier complex to be delivered by feeding (62%), while the alternative approach based on transdermal/direct application was less frequent (38%) [153,194,199,201,202]. Down-regulation of target genes by dsRNAs delivered using these methods was over 50% compared to control treatments, in approximately 68% of these studies, with star polycation nanocarriers being the most used (37%), followed by chitosan (21%). In particular, Jain et al. [203] showed that foliar application of dsRNA complexed with layered double hydroxide nanocarriers on cotton exhibited significantly better performance compared to naked dsRNA, due to better dsRNA uptake and movement into plant vascular bundles and insect tissues, causing higher mortality in B. tabaci. Moreover, these authors showed that the mixture of dsRNAs targeting three genes induced higher insect mortality compared to individual dsRNAs [203]. Likewise, the mixture of dsRNAs targeting two genes conjugated with star polycation nanocarriers increased significantly the RNAi-mediated mortality of C. suppressalis [199]. The cotton boll weevil was also treated with dsRNA targeting multiple target genes and encapsulated into chitosan and PEI nanocarriers, showing high protection levels against intestinal degradation [71]. Therefore, the use of nanocarriers and multiple dsRNAs targeting different insect genes is an interesting approach to enhance the RNAi response. However, how effective these strategies might be at increasing the RNAi efficiency in other insect species and the saturation limit of the RNAi pathway remains to be explored. Finally, Ledprona is the first and only topical RNAi-based active ingredient developed for foliar application to target L. decemlineata (a chewing insect) and is commercially approved for open field use in the USA [192,204].

On the other hand, sucking insects feed on phloem or xylem sap do not have much access to cellular contents, partially depriving themselves of the RNAi present in plant cells [126,188,190]. Therefore, dsRNA delivery by topical spray may be, in some cases, insufficient to significantly down-regulate the target genes of these insect pests [205]. Furthermore, delivery of naked dsRNAs through topical spray in sufficient quantities into the vascular system of the host plant remains a challenge [205]. In turn, delivery of dsRNA in liquid solution through soaking for plant cuttings [190,191], detached leaves [206], and roots [207], or trunk injection [208] is an effective laboratory-scale method to provide systemic movement of dsRNAs through the phloem and to control sap-sucking insects. Although the economic viability of these phloem-restricted methods still needs to be evaluated for open field application, it can be enhanced and benefited by reducing the cost of dsRNA production and by optimizing nanocarrier-mediated delivery.

Optimization of nanocarriers for dsRNA/siRNA delivery in insects must consider the heterogeneous physiology among species. Particularly, internal pH (gut or hemolymph) influences the stability and efficacy of nanocarriers. Many nanomaterials used for pest management have come from studies with medical purposes; hence, their buffering capacity has been selected for the slightly acidic or physiological (pH 7.0) environment surrounding mammalian cells [131]. However, some pest species, notably from orders Lepidoptera and Orthoptera, exhibit highly alkaline midguts [209]. Structural modifications of nanocarriers are often required to overcome these conditions; for instance, the addition of functional groups such as guanidine provides an increased pKa value for polymeric nanocarriers [193]. Other nanocarriers, like branched amphiphilic peptide capsules, can be directly engineered in their primary structure to grant them stability at specific pH ranges, turning additional modifications unnecessary [162]. Further, pH not only affects nanocarriers’ stability but also RNA release. In fact, pH-responsive nanocarriers have been widely used for drug delivery [210]. By taking advantage of pH differences between insect cells and the extracellular environment, it would be possible to favor tighter encapsulation efficacy before cell uptake and promote easier release at the physiological pH of target cells. In summary, the implementation of topical RNAi for insect pest management can or not require the use of nanocarriers to be reliable for field applications. This will depend on the target species, environmental conditions, and delivery method.

3.5. Topical RNAi to Control Major Plant Pathogens

Smaller advances have been made in topical RNAi to control plant-parasitic nematodes in open fields, given the complexity of its delivery via soil [211]. However, this approach has shown promise at least through proof-of-concept tests carried out under in vitro conditions such as nematode soaking in solutions containing dsRNA [212,213]. In contrast, topical RNAi to control plant viruses has shown more significant advances, mainly using nanocarrier-mediated RNAi delivery [108,214]. Furthermore, its efficiency has been largely dependent on the selection of the target viral RNA, as well as the overtime stability of dsRNAs [89,215]. In particular, Konakalla et al. [216] showed that topical application of dsRNA molecules engineered from TMV p126 and coat protein genes enhanced tobacco resistance against TMV. Likewise, Rego-Machado et al. [217] improved tomato resistance by spraying dsRNA molecules that target coat protein genes of Tomato mosaic virus and Potato virus Y. Comparatively, the topical RNAi triggered by dsRNA engineered from begomovirus genome was not effective against phloem-limited begomoviruses [217]. Meanwhile, topical RNAi to control plant pathogenic fungi and oomycetes has advanced with promising results for multiple pathogen species, being a promising alternative to associate with chemical control [218,219]. However, the topical RNAi effectiveness in controlling these pathogens is highly dependent on the dsRNA stability, the efficiency of dsRNA uptake by the pathogen, and the function of the gene targeted by RNAi [110,220,221]. The lifetime of dsRNA on sprayed plants particularly conferred by nanocarriers and E. coli-derived anucleated minicells is another determining factor for the effectiveness of dsRNA uptake and control of these pathogens [222,223]. In particular, Koch et al. [11,224] showed that spraying barley plants with dsRNA engineered to target three Fusarium graminearum CYP450 genes required for ergosterol biosynthesis successfully inhibited fungal growth. Therefore, topical RNAi to control plant pathogens has made notable advances and is a promising alternative to overcome the issue of transgenic acceptance by consumers and reduce reliance on chemical pesticides, but it still needs major advances for use in open field crops. Examples of topical RNAi applications to control plant pathogens and an overview of dsRNA delivery methods, target genes, crops, and plant phenotypes are reported in Tables S3 and S4.

4. Intellectual Property of RNAi-Based Technologies

4.1. Patent Search Methodology

The patent search covered a 20-year period (2004–2024) and utilized both public databases (Espacenet, USPTO, and INPI) and the commercial platform Questel Orbit to ensure broad coverage. The strategy combined keyword searches (e.g., “RNA interference”, “dsRNA”, “agricultural RNAi”, and crop-specific terms like “soybean” or “sugarcane”), International Patent Classification (IPC) codes (e.g., A01H 1/00 for plant genetic modification), and Jurisdictional filters (South America-focused filings). Duplicates and irrelevant patents were manually excluded. Trends were analyzed quantitatively (annual filings) and qualitatively (technology evolution). Due to the 18-month confidentiality window for patent applications, data beyond 2022 were treated as preliminary. This approach aligns with established methodologies for patent landscaping, as described in Trippe [225].

4.2. Patent Scenario

Intellectual property is essential to protect RNAi-related technological knowledge and guarantee the right of use [226,227]. Private and public companies invest in a robust portfolio of patents in their technological area to attract and safeguard investments made in research and development, to increase the value of the company, or to overcome obstacles imposed by other competitors [86]. These companies can also expand the economic value of this portfolio by licensing their patented products to other partners, thereby increasing the impact of the technology and earning royalties [228]. Scientific research in RNAi depends on crucial inventive aspects, such as the sequence identity of dsRNA to the target gene and non-target gene, structural engineering of the dsRNA sequence, nanocarriers to protect and deliver the dsRNA, and the optimized method for large-scale RNA production [229]. Over the past 20 years, RNAi has been exploited in agriculture to control insect pests and plant pathogens, which cause significant agricultural losses worldwide each year [230]. Therefore, this study describes the patents that currently protect RNAi innovation and the current scenario of intellectual property-based RNAi businesses focused on South America.

There are currently approximately 100,000 patent families worldwide identified open access and private databases using the keyword “RNAi” through Boolean searches, based on the International Patent Classification (Figure 3A). Of this number, approximately 18,291 patent families refer to RNAi-based technologies for agriculture (Figure 3B), of which 13,644 are operationally active. Within this subset, 114 patents are for in planta RNAi, with 48% owned by the top ten players, and 65 patents are for topical RNAi, with 47% owned by the top ten players. Additionally, 1321 patents refer to dsRNA nanoformulations for agriculture, with 26% owned by the top ten players. In particular, Bayer CropScience is the leading applicant worldwide, with 4268 granted patents on RNAi-based solutions and 278 patents still pending approval. MS Technologies and Stine Seed Farm companies rank second and third, respectively, in terms of the number of RNAi patents granted or pending, followed by Bayer CropScience, Pioneer Hi-Bred International, and BASF Plant Science (Figure 3C). USA (9290), China (3646), and European Union (3117) are the top three countries with the highest number of patents related to RNAi for agriculture, followed by Canada (2088), India (1888), Japan (1690), Mexico (1581), Australia (1510), Brazil (1366) and Korea (1288). In addition to Brazil, South America is also represented by Chile (491), Argentina (425), and Colombia (370) (Figure 3D). The number of RNAi patents filed In South America for agriculture reached its highest number in 2012 (251), followed by a linear decline until 2022 (Figure 3E). In Brazil, there are approximately 10,754 active patents (granted or pending) identified by searching for the keyword ‘RNAi’. Among these, nearly 12% (1366) are destined for agriculture. Bayer CropScience holds the highest number of active patents (234), representing 8% of all active RNAi patents for agriculture in Brazil. Pioneer Hi-Bred International (187), Bayer CropScience (169), Corteva (152), and BASF (141) rank next in terms of the largest number of active RNAi patents. Embrapa is the only South American among the global companies with the highest number of RNAi patents for agriculture, with 11 patents. The DevGen company, which specializes in formulations for topical RNAi, holds eight RNAi patents for agriculture in Brazil. GreenLight Biosciences, a world leader in large-scale dsRNA production based on the cell-free synthesis method, holds seven patents in Brazil. RNAissance BioScience, the second-largest company for dsRNA production, holds four RNAi patents in Brazil.

Based on the global RNAi intellectual property scenario, South America portrays a growing interest in research and development, with an increase of approximately 8% in patents each year over the last 20 years. However, only approximately 23% of all RNAi-related patents worldwide are also protected in South America. While this percentage certainly reflects the difference in investment in research and development made in countries like the USA and China, it may also be influenced by variations in protective legislation relating to genetic material, particularly in South American countries compared to the USA. Taking Brazil as an example, the issue of patentability of genetic material, in particular DNA sequences, has been a source of controversy and legal ambiguity. Although the Brazilian Industrial Property Law [231] allows the patenting of transgenic materials and processes, the implementation of this law has been a challenge. In particular, Brazilian patent legislation requires that an invention not be obvious to a person with skills in the area, preventing patentability when any individual with average knowledge in the relevant scientific or technical field can combine different known information to achieve the same result. Thus, the question arises as to whether the isolation of a natural DNA sequence would be merely a routine task for competent biologists or geneticists, and would not qualify as an inventive activity. Furthermore, due to the history of biopiracy, there is increased scrutiny for granting patents on genetic resources. Finally, the Brazilian Patent Office (National Institute of Industrial Property) has defined unified guidelines for patent examiners on the patentability of genetic materials, reducing disparities in examination criteria and uncertainty for applicants.

In the USA, the main criteria for patentability are novelty, non-obviousness, and utility [232,233]. Therefore, a genetic sequence can be patented if it has a practical application. According to USA law applied through USPTO, it is possible to find the following explanation: “an isolated DNA molecule that has the same sequence as a naturally occurring gene is eligible for a patent because (i) an excised gene is eligible for a patent as a composition of matter or as an article of manufacture because that DNA molecule does not occur in this isolated form in nature, or (ii) synthetic DNA preparations are eligible for patents because their purified state is different from the naturally occurring compound” [234]. Many identified and isolated genetic sequences have been patented in the USA, particularly in biotechnology and diagnostics. The USA also has a large volume of jurisprudence related to the patenting of genetic materials. In particular, the Supreme Court ruling in an episode of Myriad Genetics [235] determined that naturally occurring DNA is not patentable, while complementary DNA can be patented. Therefore, this scenario of RNAi patents in South America highlights the great potential for local technological development, and the importance of agribusiness in these countries represents a promising opportunity for stakeholders who wish to participate in this market.

5. Biosafety and Risk Assessment of In Planta and Topical RNAi-Based Technologies

In planta RNAi-based products are considered transgenics worldwide and regulated by well-known biosafety laws applied to genetically modified crops [77]. In turn, topical RNAi-based products have great potential for pest and disease control with minimal environmental impact because they have the potential to be much more specific than chemical pesticides and are less residually persistent in the environment [115,236,237,238]. Therefore, in theory, these topical products are comparatively safer than chemical pesticides [239] and fit the world’s demands for eco-friendly products [14]. However, topical RNAi-based products require appropriate and specific biosafety assessment and authorization procedures for use [240,241]. The need for specific biosafety regulation led the European Food Safety Authority and the Organization for Economic Co-operation and Development (OECD) to bring together expert scientists to discuss and define guidelines for risk assessment related to topical RNAi [242,243]. These meetings stimulated scientific debate and some articles have already described the proposals for regulatory frameworks in different countries [18,77].

5.1. Risk Assessment for Human Health

The longstanding history of safe consumption of dsRNAs naturally present in all food products, even those with complementarity to human and vertebrate transcripts, strongly supports the safety of these molecules for application as agricultural biopesticides [237]. Moreover, the safe consumption of transgenic plants expressing dsRNAs in high doses has been approved over two decades (

Table 1. Current crops with improved agronomic traits developed by host-induced gene silencing (HIGS) and successfully approved for commercial use in different countries. Information from the International Service for the Acquisition of Agri-biotech Applications (ISAAA, 2024).

Crop	Trade Name	Event Code (Name)	Developer	Target Gene	Modified Plant Trait	Regulatory Approvals	First Approval
Carica papaya	Rainbow, SunUp	CUH-CP551-8 (55-1)	Cornell University and the University of Hawaii	CP	Improved plant resistance to Papaya ringspot virus	Canada, Japan, and USA	1996
	not available	CUH-CP631-7 (63-1)	Cornell University and the University of Hawaii	CP		USA	1996
	Huanong No. 1	Huanong No. 1	South China Agricultural University	Rep		China	2006
	not available	UFL-X17CP-6 (X17-2)	University of Florida	CP		USA	2008
Solanum lycopersicum	FLAVR SAVR™	CGN-89564-2 (FLAVR SAVR™)	Monsanto	PGs	Delayed fruit softening	Mexico, Canada, and USA	1992
	not available	SYN-ØØØØB-6 (B)	Zeneca Plant Science and Petoseed	PGs	Delayed fruit softening	Mexico and USA	1994
	not available	SYN-ØØØDA-9 (Da)	Zeneca Plant Science and Petoseed	PGs	Delayed fruit softening	Mexico and USA	1995
	not available	SYN-ØØØØF-1 (F)	Zeneca Plant Science and Petoseed	PGs	Delayed fruit softening	Mexico, Canada, and USA	1995
	not available	1345-4	DNA Plant Technology Corporation	ACC	Reduced synthesis of endogenous ethylene	Mexico, Canada, and USA	1995
	not available	Huafan No. 1	Huazhong Agricultural University	ACO	Delayed ripening by suppressing the production of ethylene by silencing the ACO gene	China	1997
	not available	PK-TM8805R (8805R)	Beijing University	CP	Improved plant resistance to Cucumber mosaic virus	China	1999
Cucurbita pepo	not available	SEM-ØZW2Ø-7 (ZW20)	Seminis Vegetable Seeds (Canada) and Monsanto (Asgrow)	CP	Improved plant resistance to Zucchini yellow mosaic virus, and Watermelon mosaic virus 2	USA	1994
	not available	SEM-ØCZW3-2 (CZW3)	Seminis Vegetable Seeds (Canada) and Monsanto (Asgrow)	CP	Improved plant resistance to Cucumber mosaic virus, Zucchini yellow mosaic virus, and Watermelon mosaic virus 2	Canada and USA	1996
Dianthus caryophyllus	not available	FLO-ØØØ66-8 (66)	Florigene Pty Ltd.	ACC	Reduced synthesis of ethylene	Australia and Norway	1995
Capsicum annuum	not available	PK-SP01	Beijing University	CP	Improved plant resistance to Cucumber mosaic virus	China	1998
Solanum tuberosum	Hi-Lite NewLeaf™ Y potato	HLMT15-15 HLMT15-3 HLMT15-46	Monsanto	CP	Improved plant resistance to Potato virus Y	USA	1998
	Shepody NewLeaf™ Y potato	NMK-89935-9 (SEMT15-02) SEMT15-07 NMK-8993Ø-4 (SEMT15-15)	Monsanto	CP	Improved plant resistance to Potato virus Y	Australia, Canada, Japan, Mexico, New Zealand, Philippines, South Korea, and USA	1998
	New Leaf™ Y Russet Burbank potato	NMK-89653-6 (RBMT15-101) NMK-89684-1 (RBMT21-129) RBMT21-152 NMK-89896-6 (RBMT22-082) RBMT22-186 RBMT22-238 RBMT22-262	Monsanto	ORF1 and ORF2	Improved plant resistance to Potato leaf roll virus	Australia, Canada, Japan, Mexico, New Zealand, Philippines, South Korea, and USA	1998
	Amflora™	BPS-25271-9 (EH92-527-1)	BASF	gbss	Reduced levels of amylose and increased levels of amylopectin in starch granules	European Union	2010
	Starch Potato	BPS-A1Ø2Ø-5 (AM04-1020)	BASF	gbss		USA	2014
	Simplot Innate	SPS-ØØØZ6-5 (Gen2-Z6)	J.R. Simplot Co.	asn1, ppo5, PhL, and Vlnv	Improved black spot bruise tolerance, reduced levels of cold-induced sweetening, and reduced levels of acrylamide	Canada and USA	2020
	not available	SPS-ØØØW8-4 (W8)	J.R. Simplot Co.	asn1, ppo5, R1, PhL, and Vlnv		Australia, Canada, New Zealand, and USA	2015
	Innate® Acclimate	SPS-ØØX17-5 (X17)	J.R. Simplot Co.	asn1, ppo5, R1, PhL, and Vlnv		Australia, Canada, New Zealand, Philippines, and USA	2016
	Innate® Hibernate	SPS-ØØØY9-7 (Y9)	J.R. Simplot Co.	asn1, ppo5, R1, PhL, and Vlnv		Australia, Canada, New Zealand, Philippines, and USA	2016
	Innate® Cultivate	SPS-ØØE12-8 (E12)	J.R. Simplot Co.	asn1, ppo5, PhL, and R1		Australia, Canada, Japan, Malaysia, Mexico, New Zealand, Philippines, Singapore, and USA	2014
	not available	SPS-ØØE24-2 (E24)	J.R. Simplot Co.	asn1, ppo5, PhL, and R1		USA	2014
	not available	SPS-ØØE56-7 (E56)	J.R. Simplot Co.	asn1, ppo5, PhL, and R1		Australia and New Zealand	2017
	Innate® Generate Innate® Accelerate	SPS-ØØF10-7 (F10) SPS-ØØØJ3-4 (J3)	J.R. Simplot Co.	asn1, ppo5, PhL, and R1		Australia, Canada, Mexico, New Zealand, and USA	2014
	not available	SPS-ØØF37-7 (F37) SPS-ØØG11-9 (G11) SPS-ØØH37-9 (H37) SPS-ØØH50-4 (H50) SPS-ØØJ78-7 (J78)	J.R. Simplot Co.	asn1, ppo5, PhL, and R1		USA	2014
	not available	SPS-ØØJ55-2 (J55)	J.R. Simplot Co.	asn1, ppo5, PhL, and R1		Canada and USA	2014
	Innate® Invigorate	SPS–ØØV11–6 (V11)	J.R. Simplot Co.	asn1, ppo5, PhL, and R1		Australia, New Zealand, and USA	2016
	not available	TIC-AR233-5	Technoplant Argentina	CP	Improved plant resistance to Potato virus Y	Argentina	2018
Nicotiana tabacum	not available	Vector 21-41	Vector Tobacco Inc.	NtQPT1	Reduced production of nicotinic acid	USA	2002
Medicago sativa	HarvXtra™	MON-ØØ179-5 (KK179)	Monsanto and Forage Genetics International	CCOMT	Reduces content of guaiacyl (G) lignin	Australia, Canada, Japan, Mexico, New Zealand, Philippines, Singapore, South Korea, and USA	2013
Zea mays	not available	MON-87411-9 (MON87411)	Monsanto	DvSnf7	Improved plant resistance against Diabrotica virgifera virgifera	Argentina, Australia, Brazil, Canada, Colombia, Japan, Mexico, New Zealand, Philippines, South Korea, Taiwan, and USA	2014
Malus domestica	Arctic™ “Golden Delicious” Apple	OKA-NBØØ1-8 (GD743)	Okanagan Specialty Fruits Incorporated	PPOs	Apples with a non-browning phenotype	Canada and USA	2015
	Arctic™	OKA-NBØØ2-9 (GS784)	Okanagan Specialty Fruits Incorporated	PPOs	Apples with a non-browning phenotype	Canada and USA	2015
	Arctic™ Fuji Apple	OKA-NBØØ3-1 (NF872)	Okanagan Specialty Fruits Incorporated	PPOs	Apples with a non-browning phenotype	Canada and USA	2018
Ananas comosus	Rosé	FDP-ØØ114-5 (EF2-114)	Del Monte Fresh Produce	b-Lyc and e-Lyc	Increased lycopene accumulation	Canada and USA	2016
Gossypium hirsutum	not available	TAM-66274-5 (TAM66274)	Texas A&M AgriLife Research University	dCS	Reduced gossypol biosynthesis	USA	2018
Carthamus tinctorius	not available	GOR-73226-6 (Event 26) GOR-7324Ø-2 (Event 40)	Go Resources Pty Ltd.	fatB and fad2.2	Modified oil/fatty acid	Australia	2018
Glycine max	not available	DD-Ø26ØØ5-3 (260-05)	DuPont (Pioneer Hi-Bred International Inc.)	gm-fad2-1	Blocked conversion of oleic acid to linoleic acid	Australia, Canada, Japan, New Zealand, and USA	1997
	Treus™, Plenish™	DP-3Ø5423-1 (DP305423)	DuPont (Pioneer Hi-Bred International Inc.)	gm-fad2-1	Blocked conversion of oleic acid to linoleic acid	Australia, Brazil, Canada, China, Colombia, European Union, Indonesia, Iran, Japan, Malaysia, Mexico, New Zealand, Philippines, Singapore, South Africa, South Korea, Taiwan, Turkey, and USA	2008
	Vistive Gold™	MON-877Ø5-6 (MON87705)	Monsanto	fad2.1A and fatb1-A	Blocked conversion of oleic acid to linoleic acid and decreased transport of saturated fatty acids out of the plastid	Australia, Canada, China, Colombia, European Union, Indonesia, Japan, Mexico, New Zealand, Nigeria, Philippines, Singapore, South Korea, Taiwan, Turkey, Vietnam, and USA	2011
Phaseolus vulgaris	BRS FC401 RMD	EMB-PVØ51-1 (EMBRAPA 5.1)	Embrapa	AC1	Improved plant resistance to Bean golden mosaic virus	Brazil	2011
Prunus domestica	not available	ARS-PLMC5-6 (C-5)	USDA—Agricultural Research Service	CP	Improved plant resistance to Plum pox virus	USA	2007

) [244]. The safe consumption is attributed to the presence of multiple biological barriers at the gastrointestinal, bloodstream, and cellular levels in mammals [237]. Indeed, small siRNAs and miRNAs are ubiquitous in plant- and animal-derived foods. Therefore, if there were no natural barriers for these molecules, the consumption of natural foods would pose a permanent risk to human health. The OECD 2019 Conference addressed the potential exposure of sensitive human populations to dsRNA-based biopesticides and no significant issues were raised regarding the safety of this technology [242]. The risk assessment for in planta RNAi-based products is typically conducted on a case-by-case basis by these authorities considering the laws applied to genetically modified crops. In turn, topical RNAi-based biopesticides can be incorporated into formulations to improve the cellular uptake of dsRNA or to protect dsRNA against nucleolytic degradation [114,193,245]. Therefore, the use of these formulations might potentially increase human exposure and elevate risks. However, considering the presence of multiple biological barriers in mammals, it is unlikely that formulated RNAi-based products developed for agricultural purposes will effectively deliver dsRNA into human cells upon ingestion [246]. To date, regulatory authorities have not adopted standardized methods for assessing the safety of topical RNAi-based products for agriculture. Nevertheless, a substantial consensus suggests that the current regulatory framework for agrochemicals could serve for risk assessment of topical RNAi-based products. Finally, transgenic rootstocks containing RNAi-based strategies to pest control or improve agronomic traits restricted to roots are a viable alternative since the aerial part is not transgenic and does not have equivalent amounts of these dsRNAs compared to the root [85]. In this sense, a particular risk assessment analysis must be carried out to authorize the transgenic planting and the commercialization of its non-transgenic fruits.

5.2. Environmental Risk Assessment

Some authors argue that the principles and guidelines used for environmental risk assessment of RNAi-based transgenic plants can be effectively adapted to topical RNAi-based products [240,247]. The primary environmental risks associated with topical RNAi-based products are linked to their potential impact on non-target organisms [243,248]. Numerous scientific articles showed that bioinformatic analyses are indispensable in the risk assessment for non-target organisms [249]. In fact, the action mode of RNAi is well understood, relying on the base pairing of siRNA with the target organism’s mRNAs [240,250]. Therefore, it is theoretically easy to design dsRNAs to specific sequences on target pests, thus mitigating off-target effects [238,251]. However, there are still knowledge gaps that justify the inability to predict the impact on non-target organisms using bioinformatics alone [241,242]. Despite the significant update of sequence databases containing information on non-target organisms, many remain unavailable. In addition, the action mode of the RNAi might vary across different organisms. For example, there may be differences in the number of mismatches to still be able to activate the RNAi machinery [249,250] and in the efficiency of systemic siRNA transport [252], which may not be accurately identified through bioinformatic analysis. For example, siRNAs resulting from DCL2 processing have demonstrated variable lengths (ranging from 20 to 22 nucleotides) across different insect species [253]. Another study revealed that differences in the number of mismatches could lead to varying effects on the mortality of non-target organisms [254]. Besides these sequence-specific mechanisms, some studies suggest that effects on the non-target organisms could occur due to sequence-unrelated mechanisms, making it challenging to predict all possible effects using only bioinformatics [252]. One theoretically possible sequence-unrelated mechanism would be immune stimulation or saturation of the enzymes involved in the RNAi machinery, which could negatively affect the fitness of non-target organisms [255]. However, considering the multiple natural barriers against exogenous dsRNAs, the likelihood of this occurring in non-target organisms is probably low [240]. Therefore, despite bioinformatics being an essential tool for risk assessment, it is not sufficient to predict all possible effects on non-target organisms from RNAi-based applications and needs to be employed in conjunction with empirical testing.

5.3. Possible Off-Target Effects of RNAi-Based Products Versus Decision Making

Surveying all potential theoretical adverse effects against non-target organisms does not contribute effectively to risk assessment to support decision making. For regulatory risk assessment of RNAi-based products, generating a comprehensive list of possible off-target effects would be challenging and inefficient in supporting decision making. Instead, problem formulation should establish criteria that define an acceptable risk framework and develop a plan to test the hypothesis that the product meets those criteria. The key to effective risk assessment of RNAi-based products is determining whether their properties indicate acceptable or unacceptable risk [247]. There is a robust body of evidence that these products, in principle, pose lower risks to non-target species compared to chemical pesticides [241,256]. However, this hypothesis needs to be tested before these products are commercially released to ensure that their use does not cause unacceptable harm to the environment [257]. Therefore, considering that agricultural activities are in practice employed in food production resulting in non-negligible environmental impacts, the main hypothesis that must be tested to support decision making is whether the use of RNAi-based products offers a significantly lower environmental impact than traditional activities. If the impact of RNAi-based biopesticides is significantly lower than that of chemical pesticides, the risk of these new technologies should be considered acceptable. Romeis and Widmer [257] suggested an empirical approach based on toxicity studies carried out in the laboratory that can be employed to test whether the effect of a specific RNAi-based product on non-target organisms constitutes an acceptable or unacceptable risk. After bioinformatics analysis, a group of closely related to phylogenetically distant species should be selected, exposed to the RNAi, and their responses analyzed [257]. This approach allows a clear comparison between the RNAi-based product and traditional chemical pesticides, making it possible to test the hypothesis that the impact of the new product is significantly lower than the current one. A combination of bioinformatics and small field trials can be performed to test the hypothesized acceptability of in planta and topical RNAi-based products for decision making. Field trials designed to assess the impact on non-target organisms are carried out in the risk assessment of transgenic plants in most countries where cultivation is permitted. It is believed that these field tests can be adapted to determine whether an RNAi-based product poses an acceptable risk. The rationale of the proposed approach is to carry out a field trial comparing two treatments: (i) RNAi-based product and (ii) conventional chemical pesticide used to control a target. Subsequently, an assessment of the microbiota and insect populations is carried out using methodologies commonly employed for transgenic plants. If these results demonstrate that the impact of the RNAi-based product is significantly lower than that of using a chemical pesticide, the environmental risk of the new technology would be considered acceptable. If the difference is large or the effect in non-target organisms is negligible, these results can in principle be extrapolated to other crop areas where the experiments were not carried out following the principle of transportability of environmental data on transgenic plants [258,259]. This principle is based on the assumption that if no biologically relevant differences are observed between a transgenic plant and its conventional counterparts in one crop region, data from these studies can be used to inform the risk assessment in another region or country, regardless of the agroclimatic zone [259]. Therefore, this principle can also be perfectly employed in the risk assessment of RNAi-based biopesticides. These studies also consider that field trials present important practical advantages over toxicity studies carried out in the laboratory to evaluate adverse effects against non-target organisms. Field trials enable the assessment of several species almost simultaneously, providing a broad view of the impact on phylogenetically distant non-target microorganisms and insects. They also allow addressing the effects of RNAi in non-target cross-species and cross-kingdoms [17,77,241]. Finally, field trials avoid the need to develop protocols for laboratory cultivation of the species to be analyzed.

5.4. Current Regulatory Status of In Planta and Topical RNAi in Latin America

In planta RNAi-based products are regulated as transgenic plants worldwide and undergo biosafety and risk assessments as determined by each country’s legislation [19]. The planned release or commercial use of transgenics needs to be addressed and approved by each country’s National Biosafety Commission. The submission and approval of a transgenic product in different countries have standard guidelines at global levels but may include some particular national legislative measures [18,19]. This global format is necessary for countries to comply with standards for importing and exporting agricultural commodities. Recently, these countries have begun to adopt additional laws to categorize and incorporate innovative technologies, such as topical RNAi and genome editing. Therefore, these new laws aim to judge on a case-by-case basis and define whether or not these new technologies are transgenic [17].

In Latin America, some countries have already adopted this new law to regulate these precision breeding technologies [19]. For both genome editing and RNAi-based products, this new law adopts as a prerogative the presence or absence of foreign DNA with the replication capacity in the final product to classify it as a transgenic or non-transgenic product. However, this same prerogative is not adopted by some other Latin American and European countries [18,260]. Once considered non-transgenic, these topical RNAi-based products must undergo prior approval from other national agricultural and environmental oversight boards to ultimately be considered commercial. Finally, the formulation of topical RNAi-based products must be addressed taking into account the encapsulation mediated by proteins, microorganisms and nanocarriers, target genes, off-target genes, non-target organisms, surfactants, and stabilizers. In particular, the regulations of these RNAi-based biopesticides are still being elaborated in Latin America countries. This regulatory subdivision, which must be drawn up on a scientific basis, will be important to allow their classification to differentiate them from non-selective synthetic chemicals.

5.5. Resistance Against Exogenous dsRNA and RNAi

The development of resistance against RNAi has already been reported in mammals [261], plants [262], plant pathogens [263,264], and insect pests [41,265]. The loss of RNAi efficiency is not completely understood in all organisms. However, the encoding of proteins with suppressive activity of the RNAi pathway, genetic variability and diversity caused by selection pressure on target genes of RNAi, hijacking of the siRNAs originating from the dsRNA interfering, disruption of amplification or systemic movement of RNA-silencing signal, and the epigenetic silencing of the transgene encoding dsRNA interfering are some examples of mechanisms that lead to resistance to RNAi [264,266,267]. Likewise, resistance against dsRNA in D. virgifera virgifera was considered polygenic, located on a single locus (CEAS 300), and associated with the reduced uptake of dsRNA in midgut cells [41]. Similarly, variation in RNAi sensitivity in L. decemlineata correlates with the expression of RNAi machinery genes and staufenC, a dsRNA-binding protein involved in dsRNA processing to small interfering RNA [268,269]. These data reveal that pyramiding or concatenating different dsRNA sequences targeting different mRNAs may not be sufficient or long-lasting to control pests. Therefore, future research is needed to understand the molecular mechanisms of resistance against dsRNA and RNAi, to measure the implications of interaction between plants versus non-target organisms, and to develop strategies to overcome these limitations. Furthermore, resistance management strategies must be implemented for the long-term sustainability of RNAi-based products in agriculture [127].

6. Major Contributions of RNAi to More Sustainable Agriculture

Through genetic engineering, several in planta RNAi-based traits were introduced in 16 crops and approved for commercial use in several countries (Table 1). These traits focused on improving plant resistance to viruses, delaying fruit softening and ripening, reducing oxidative browning of fruits, improving oil quality, and improving maize resistance to D. virgifera virgifera. The common bean resistant to BGMV (Brazil, in 2011) and potato resistant to Potato virus Y (Argentina, in 2018) were pioneers in Latin America. Bayer CropScience, Zeneca, Seminis, BASF, J.R. Simplot, DuPont/Corteva, and Okanagan Specialty Fruits are the main agencies developing these RNAi-based technologies. Despite this release, commercial use in the countries where they were approved still faces restrictions for being transgenic. However, the benefits brought by these in planta RNAi-based traits for food security and greater environmental sustainability are highlighted by several studies [14,270]. The adoption of these transgenic crops makes it possible to improve the product quality and durability until they reach the consumer, reduce the use of synthetic agrochemicals, and decrease economic losses caused by pests. In this context, topical RNAi-based products are an alternative to overcome the barriers faced by in planta RNAi and to promote greater environmental sustainability (Figure 4). Conversely, topical RNAi-based biopesticides can theoretically be applied to any pest susceptible to RNAi. Another advantage of topical RNAi-based biopesticides over the in planta RNAi is the shortest development time. Given these advantages, several agribusiness companies have added efforts in research and development in RNAi. Bayer CropScience developed the first technology portfolio based on topical RNAi for controlling insect pests, weeds, and pathogens (BioDirect™ technology). GreenLight Biosciences has launched the first commercially approved active ingredient and product, respectively Ledprona and Calantha^TM, to control the Colorado potato beetle in potatoes [192,204]. Other topical RNAi-based products are being implemented by these companies to control plant pathogens and insect pests. Likewise, new topical RNAi-based products that enhance the effects of synthetic agrochemicals on the target pest are also expected for commercial release. More recently, other companies such as Syngenta, Corteva, Sempre AgTech (WIN), AgroRNA, Nufarm, RNAissance BioScience, AgroSpheres, Agrivalle, PlantArcBio in partnership with ICL Planet, NanoSUR, Trillium Ag, and RNAgri also opened portfolios of topical RNAi-based solutions and materials to meet RNAi industry. These companies have focused on large-scale and low-cost dsRNA production using cell-free systems, while others have focused on developing nanocarriers for RNAi delivery. Therefore, these efforts and investments have made byproducts available at more affordable costs, including the production of dsRNA, nanocarriers, and stabilizers.

7. Concluding Remarks and Perspectives

Global agriculture has undergone remarkable transformations over recent years, witnessing several impactful changes across the world [271]. Notably, technological advances in various agribusiness sectors, together with the challenges arising from geopolitical conflicts, pandemics, socio-economic instability, and the profound impacts of climate change, placed sustainable food production at the forefront of discussions [14,272]. Given this scenario, the search for technologies that, in addition to providing high yield, are eco-friendly, has been intense across all agricultural sectors and countries [273]. Consequently, the agribusiness sector is currently transitioning to Agriculture 6.0, which is based on Environmental, Social, and Corporate Governance principles, that aim to prioritize technologies that minimize negative impacts on the ecosystem, such as RNAi-based products [13,14]. Despite reluctance against transgenic-based technologies, the potential of in planta RNAi to revolutionize agriculture by improving crop yield, mitigating losses caused by pathogens and insect pests, and reducing dependence on non-selective synthetic agrochemicals is becoming increasingly evident [274]. Meanwhile, topical RNAi-based products have established themselves with consistent solutions for agriculture challenges, and their continued advancements will contribute to renewable agriculture [270]. These technological advances rely on basic research to identify suitable target genes in target pests, as well as reducing the production costs of dsRNAs and nanocarriers, improving the delivery of these molecules to the target and prolonging the durability of the applied molecules [17]. Furthermore, substantial financial investments are needed to resolve existing drawbacks and facilitate the transfer of knowledge from laboratory to field [116]. These investments are crucial for transforming research findings into useful products and solutions for agriculture (Table S6) [14]. Thanks to technological advances achieved, some topical RNAi-based products are already in the regulatory approval phase for commercial use [192,204]. However, essential adjustments to the regulatory laws remain a bottleneck to be resolved in several countries [77]. By overcoming these obstacles, it will become increasingly evident that numerous RNAi-based solutions will become available, contributing significantly to agricultural and environmental sustainability [127]. Furthermore, the participation of several companies and startups focused on in planta and topical RNAi-based technologies will facilitate the offering of high-quality solutions at competitive prices (Table S7). Importantly, these new products will control insect pests, plant pathogens, and weeds, as well as enhance agronomic traits, ranging from seed treatment using non-transgenic approaches to transgenic crops [14,17]. Finally, there are also numerous opportunities with RNAi as biostimulants and priming technology for use in crops [275], which will also contribute significantly to sustainable agricultural and food production.