1. Introduction

The world is moving toward a more sustainable crop production system that requires specific and efficient tools to battle plant pathogens. RNAi can be used for such purposes. The molecule is used in nature, degrades quickly, can disrupt the pathogen at a genetically specific level, and can complement the current agronomic crop protection practices used for organic, conventional, ecological, or technological production [1]. The reader may be familiar with the concept of DNA and genes located in the nucleus of eukaryote cells, containing the instructions to create organic molecules, mainly proteins. RNA messenger works as an intermediator, carrying the nucleus’s message to the cytoplasm to be read by the ribosomes to assemble the protein. The RNAi eukaryotic machinery is a complex system for virus defense and gene expression control, sometimes called post transcriptional gene silencing (PTGS). The system can be triggered by external specific dsRNA, resulting in its RNA messenger being blocked before it gets to the ribosome, leaving the organism, such as a pathogen, disarmed [2]. The extravesical delivery of dsRNA to disarm the expression system was proven to be natural and bidirectional from plant to fungal pathogens and vice versa cross-kingdom communication [3,4,5,6,7,8].

Consequently, RNAi represents an opportunity to emulate or improve the natural plant pathogen control system by providing well-designed external dsRNA [9]. Here, we aimed to present advantages in crop protection mediated by RNAi. There are two RNAi plant-based technologies: host-induced gene silencing (HIGS) since the 1990s and emerging spray-induced gene silencing (SIGS). Both can provide sustainable solutions to control pathogens, such as insects, viruses, and fungi. We will focus on SIGS because it is becoming an emerging affordable option with a cost reduction of approximately USD 0.5–1 per gram [10]; the small amount of dsRNA necessary seems to be near 2–10 g per hectare; it is safe; and it has fast environmental degradation [11,12,13]. When dsRNA is applied externally to plants, one proposed mechanism is that plant cells can take it and use it directly to tackle the pathogen through secreted vesicles containing the RNA at the site of the infection and plasmodesmata [14,15,16]. The amount of sprayed RNA may vary depending on the target species’ sensitivity to RNAi, the capacity to trigger the defense system, and the efficiency of the delivery method. Other challenges for this technology are the need for science-based risk assessment procedures for topical RNAi applications within existing plant protection product legislation, regulatory approaches [12,13], and strategies to use more than one target sequence to avoid resistance of uptake [17].

2. How it Works

Figure 1. Spray-induced gene silencing (SIGS) mechanism.

3. Potential Targets

The potential targets of RNAi can be viruses, fungi, bacteria, nematodes, and endogenous genes. Next, we describe a general table ( Table 1 ) containing several targets to demonstrate that the technology is flexible enough to start exploring other plagues, and broader reviews exist in case of interest by the reader [23]. Out of these possibilities, we would like to remark on the potential of using SIGS to produce a new generation of crop protection-specific products.

Table 1. Potential targets for spray-induced gene silencing (SIGS) in plants.

4. Encapsulation Technology to Improve Efficiency

According to the data available in the references, we identified four principal encapsulation systems: the formation of liposomes, virus-like particles, polyplex nanoparticles, and bioclay. As mentioned in Table 2, these systems coincide in the biodegradability of the materials, the ability to improve the stability of RNAi, and its synergistic effect to induce control. Figure 2 presents a diagram of the most reported systems.

Figure 2. Primary encapsulation system for RNAi delivery.

4.1. Liposomes

Liposomes are spherical artificial vesicles synthesized from cholesterol and nontoxic natural phospholipids. They present hydrophilic and hydrophobic characteristics (amphiphilic molecules) that facilitate their interaction in multiple chemical environments [57]. 

The hydrophobic part consists of two fatty acid chains (10–24 carbon atoms and 0–6 double bonds in each chain), while the hydrophilic section is mainly phosphoric acid bound to a water-soluble molecule [58]. They are made up of at least one phospholipid layer. The type of lipid defines properties such as surface charge, solubility, and size of the vesicles; therefore, it is critical in the functionality of the particle. For example, unsaturated phosphatidylcholine lipids form much more permeable but less stable bilayers, while saturated phospholipids with long acyl chains form more rigid and waterproof structures. According to their size, they are classified as very small (0.025 m) or large (2.5 m) vesicles [57]. Multiple types of lipids are used as raw materials, the most frequent in the literature being phosphatidylglycerol (PG), phosphatidylcholine (PC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dicetylphosphate, cholesterol (CH) stearylamine, or their mixture [59].

One advantage of this type of particle is its semipermeable character that simulates biological membranes, allowing smart delivery of the compounds it carries due to its cellular penetration capacity [60]. Additionally, its low solubility in hydrophilic environments is an advantage when looking for a slow-release mechanism, which helps immobilize compounds and facilitate their environmental biodegradation. However, this technology also has some technical disadvantages, such as a short half-life due to oxidation and hydrolysis of phospholipids, vulnerability to temperature, and others related to its cost of production [57]. The most cited synthesis methods are injection, electroformation, reversephase evaporation, hydration or thin-film hydration (Bangham method), microfluidics, freeze drying of double emulsions, heated membrane extrusion, detergent depletion, and supercritical fluid preparation. The challenge of the methods is to achieve stable vesicles with controlled and functional sizes [59,61].

The applications of liposomes are very versatile; for example, in the case of human and animal cells, they include a vast inventory of drug delivery systems to treat multiple diseases by reducing collateral damage in nontarget tissues and excessive doses [60]. In food science, they are used for the protection and controlled delivery of enzymes, vitamins, functional compounds, antibiotics, and metabolites to improve the properties of food [62]. In plants, in addition to the reports in Table 2 for RNAi delivery, they are used as carriers of CRISPR/Cas gene-editing systems, proteins, DNA, or mRNA [63]. Additionally, lipofection (liposome-mediated DNA delivery) in protoplasts [64] and nutrient

transport/internalization are cited [65].

4.2. Virus Like-Particles (VLPs)

Virus-like particles (VLPs) are defined as supramolecular self-assemblies of proteins of approximately 10–200 nm in diameter, presenting the same or similar structure as that of native virions. VLPs are not infectious since they lack infective genetic material [66]. In addition to being nanocarriers, VLPs can be functionalized on the surface by ligand proteins, exhibiting additional properties such as immunological and labeling reactivity to changes in the chemical environment [67,68].

VLPs can be produced by heterologous expression systems such as baculovirus, bacteria, yeast, plants, and animal cells (insects, mammals, etc.). The expression vector to be used must be selected depending on the type of the desired protein and the target organism since the system’s success may depend on post-translational modifications. According to the type of virus from which they are derived, VLPs are classified as single-stranded RNA positive-sense viruses, single-stranded RNA negative-sense viruses, double-1 stranded RNA viruses, and single- and double-1 stranded DNA viruses. Additionally, VLPs can be produced in crude protein extracts (cell-free system), mainly when heterologous expression implies the production of toxic compounds for the vector [69]. Furthermore, the expression systems have different yields and are generally more efficient in viral and bacterial vectors.

VLP surface conjugation can be covalent and noncovalent; the first option is to functionalize VLPs with large molecules, even complete proteins. This type of conjugation is frequent through bacteriophage expression systems (MS2 and Q-beta) [66]. Regarding noncovalent conjugation, a biotinylation process is required, which needs a linking agent such as streptavidin to ensure surface fixation [70].

Some of the advantages of VLPs are self-assembly, repetitive structural stability, and the resulting polydispersity systems. This improves uptake by target cells compared to that of naked molecules [71,72]. Using cell recognition proteins represents lower barriers to biointeraction [54]. However, its production is not exempt from limitations related to vectors and strategic aspects in terms of system complexity, speed of expression, performance, scalability, and regulation [67].

The main application of this technology is for prophylactic vaccines; however, their valuable properties allow them to be used as nanocarriers in drug and gene therapy, as scaffolds for bioimaging, and for the synthesis of bionanomaterials [72–74]. Regarding its uses in plants and plant cells, in addition to being vehicles for the transport of RNAi, it is used to carry the CRISPR/Cas complex as a DNA-free gene editing system [75].

4.3. Polyplex Nanoparticles

The term polyplex nanoparticles refers to the encapsulation of genetic material in polymeric particles. The polymers used are polycations that electrostatically interact with negatively charged nucleic acid molecules. Thus, neutralization of charges (phosphodiester groups) and the consequent compaction in a colloidal complex occur. It is also stabilized by hydrogen bonding interactions between the components [76]. The final properties of the polyplex depend on the physicochemical characteristics of the pristine polymers and the resulting nanoparticle features: size, surface charge, polydispersion, and hydrophilicity [77].

There is a large number of polymers used for the manufacturing of polyplexes, and a limited list of the most commonly used polymers includes poly(ethyleneimine) (PEI) [77], poly-L-lysine (PLL), poly(amidoesters) (PAE) [78], polyamidoamine (PAMAM), poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) [79], polyethylene glycol (PEG), and chitosan [80], as well as copolymeric combinations such as phosphorylcholine-modified polyethyleneimine (PEI) PEGylated PEI-based, PEGylated poly(dimethylaminomethyl methacrylate) containing folate, PEI-grafted ,  -poly(N-3-hydroxypropyl)-DL-aspartamide, galactose-modified trimethyl chitosan-cysteine-based, and others [81]. Theoretically, any

positively charged polymer could form a polyplex, of particular interest being biodegradable polymers such as PEG, poly(glutamic acid) (PGA), poly(caprolactone) (PCL), poly(D,Llactide- coglycolide) (PLGA), poly(lactic acid) (PLA), N-(2-hydroxypropyl)-methacrylate copolymers (HPMA), polystyrene-maleic anhydride copolymer, and poly(amino acids) (PAAs) [81]. Polycations can also be synthesized by ring-opening polymerization controlling the resulting charge [82].

The synthesis methods of polyplexes are very diverse, defined by the raw material and the application. There are promising methodologies, such as the three-dimensional hydrodynamic focusing (3D-HF) technique supported by microfluidic devices. It seeks to optimize the properties and the resulting system’s effectiveness; since conventional methods can become a limitation since some of them involve an organic-aqueous interface, shear stress, or pH and temperature levels that can damage the genetic material [78].

Another limitation is the reduced biocompatibility and biodegradability of some polymers, as well as their cytotoxicity, requiring the production of derivatives to cope with these disadvantages [83]. Nevertheless, they are a widely used transport mechanism for genetic material because they manage to prevent the degradation of genetic material caused by extracellular enzymes and precisely direct the load to the target cells, increasing the effectiveness of gene expression systems [78]. Nanoparticle uptake occurs by endocytosis involving interactions with cell membrane glycoproteins and the formation of the endosome, followed by its release into the cell cytoplasm and further translocation to the target organelles [76].

Its main application is as a nonviral vector for DNA transfer in gene therapy, including regenerative medicine [78], AIDS, cancer, and hereditary disorder treatments [79]. It has also been used for transfection and editing of genomes in plants [84,85], as well as for the gene silencing systems mentioned above aimed at disease and pest control [22,47,52,86,87].

4.4. Bioclays

Bioclay systems are based on 10–100 nm nanoclay particles able to transport selected compounds such as nucleic acids. Nanoclays are stratified aluminosilicates of a single (0.7 nm thick) or double layer (1 nm thick) with variable plasticity and swelling capacity. Its chemical formula is (Ca, Na, H) (Al, Mg, Fe, Zn)2(Si, Al)4O10(OH)2-xH2O. There are both natural and synthetic materials, and their structures consist of alternating tetrahedral SiO2 and octahedral AlO6 sheets with varying ratios [88,89].

Its properties depend on the nature of the atoms on the surface and the exchangeable cations between layers. Clays are negatively charged due to the substitution of Al3+ or Mg2+ ions by Si. Consequently, the surface of the nanolayer is hydrophobic due to Si-O covalent bonds; however, the addition of exchangeable hydrophilic cations can change the charge to positive [88]. The most commonly used raw materials are bentonite, hectorite, montmorillonite, kaolinite, laponite, halloysite, laponite, sepiolite, saponite, and vermiculite. They are low-cost, nontoxic, and charge versatile vehicles [89,90].

Surface functionalization is frequently performed to adapt them to specific applications. Processes such as silylation prevent the aggregation of clays through covalent modification of the surface. For example, modified montmorillonite shows improvement in the polymer mechanical performance as well as in water absorption, cationic exchange, and ionic retention capacities. Other clays, such as sepiolite, are functionalized by grafting organosilane due to its many silanol groups on the surface, making them excellent heavy metal removers. Halloysite is modified mainly by covalent grafting of organosilane via condensation with hydroxyl groups [91]. Synthetic clays, such as Laponite® (phyllosilicate composed of layered synthetic silicate), are obtained from inorganic mineral salts and are superficially modified by ion exchange or covalent linkage [91]. Their applications mainly cover materials science, chemistry, physics, and biology, where they become strategic materials for developing smart nanoarchitectures. Among the most documented uses are drug delivery, environmental remediation, wastewater treatment, and food packaging [91–93]. Plant biotechnology has also been used for the manufacture of nanocomposites to improve resistance properties in wood [94,95], vehicles for the controlled dosage of nutrients, and regulating soil properties [96–98]. It is relevant to mention the transformation of desert soils into cultivable fields through liquid nanoclay [99].

5. Regulatory Approaches

The time and cost associated with obtaining the data for a registry of a biomolecule such as dsRNA can be low compared with that of a conventional pesticide of 4 versus 12 years and USD 3–7 million versus USD 280 [10]. An important consideration is that dsRNA generally has low environmental persistence in soil, sediment, and water [100,101].

The biomolecule shows a record of safe consumption of short and long RNAs in the diet from food and lacks oral immunostimulation [11]. Another positive input when regulating this technology is the technical discussion that has occurred over the last decade, where the USEPA and OECD (Organization for Economic Co-operation and Development) proposed using and adapting the existing plant protection product norms and procedures as described next. The United States Environmental Protection Agency (EPA) has analyzed addressing this technology based on problem formulation for human health and ecological risk assessment. At the same time, OECD proposed using risk assessment to evaluate the toxicity profile and exposure of the molecule by adapting the current regulatory framework for small-molecule agrochemicals as a general framework for dsRNA-based agricultural products and proposed taking into consideration the experience gained through the review

of dsRNA-based GE crops. The EFSA literature review on GM plants is a document to be taken into consideration as well. A summary is presented next in Table 3.

Table 3. Regulatory approaches.

6. Conclusions and Future Perspectives

RNAi technology is a powerful and versatile alternative for pest and disease control in crops. Its use in the agricultural field extends to viruses, bacteria, fungi, insects, nematodes, and plants. It grows steadily with other complementary technologies, such as the recombinant production of RNAi in vectors, transgenesis, and micro/nanoencapsulation of candidate si/dsRNA. The main issue preventing its adoption in the past was the cost of production and stability. The cost of production is getting lower with the development of new technologies, while stability encapsulation strategies provide a solution to avoid degradation.

Together with the positive approaches to regulation, the emergence of more interdisciplinary alternatives that combine gene silencing by RNAi is also expected. For instance, the induction of resistance in crops by elicitation and metabolic control methods, using the strengths of both. Following this approach, we are developing a technology that uses elicitor nanoparticles made of natural polymers to induce defense in plants, which will also carry a double control mechanism based on RNAi to unblock inhibitors of systemic defense against systemic defense pathogenic species of the genus Fusarium . There is also interest in the scientific community to produce multiple knockdowns that protect systemically against a consortium of pathogens under the same application. Another challenge for this technology is to keep reducing production costs, for which biotechnology is emerging as one of the main allies to improve profitably on a large scale.

Together with the positive approaches to regulation, the emergence of more interdisciplinary alternatives that combine gene silencing by RNAi is also expected. For instance, the induction of resistance in crops by elicitation and metabolic control methods, using the strengths of both. Following this approach, we are developing a technology that uses elicitor nanoparticles made of natural polymers to induce defense in plants, which will also carry a double control mechanism based on RNAi to unblock inhibitors of systemic defense against systemic defense pathogenic species of the genus Fusarium. There is also interest in the scientific community to produce multiple knockdowns that protect systemically against a consortium of pathogens under the same application. Another challenge for this technology is to keep reducing production costs, for which biotechnology is emerging as one of the main allies to improve profitably on a large scale.