The phenomenon of RNA-induced gene silencing (RNAi) gained prominence following its disclosed mechanism in pests, but it was in tobacco (Nicotiana tabacum) plants that this phenomenon was initially documented and published as early as 1928 (Wingard, 1928). This groundbreaking discovery has revolutionized our understanding of gene regulation and its implications for various biological processes, particularly gene silencing via RNAi. Harnessing this process has shown promise in diverse applications, including gene knock down, disease treatment, and crop improvement. Over the last decade, substantial efforts have been made to exploit RNAi for the development of novel crop protection methods. Moreover, RNAi technology has facilitated the creation of genetically modified crops with enhanced traits such as increased yield, improved nutritional content, and prolonged shelf life (Kumar et al., 2020).

Sustainable agriculture entails the development and implementation of environmentally friendly technologies and practices that are readily accessible and advantageous to farmers in terms of crop improvement and productivity (Fletcher et al., 2020). To minimize the adverse effects of synthetic pesticides on health and the environment, there has been a shift towards employing bio-based alternatives. This shift aims to promote agricultural sustainability, leading to the adoption of more environmentally friendly and innovative crop protection strategies by the scientific community. Plant genetic engineering has emerged as a promising avenue to address food shortage and mitigate the impact of plant stresses. Notably, the development of transgenic crops employing advanced biotechnological techniques, including RNAi, has been a significant contribution in this regard. Transgenic RNAi crops are still regarded as GM crops. Provided that Double-stranded RNA (dsRNA)-based products contain no GM organisms (e.g., bacteria for dsRNA production), they are not regarded as genetically modified organisms (GMOs). Interestingly, recent developments using exogenous dsRNA spray to control pathogens and pests have provided a non-transgenic alternative to GMOs (Rajam, 2020; Rodrigues and Petrick, 2020). Furthermore, RNAi induces the silencing of target genes, which is more advantageous than genome editing tools (Arpaia et al., 2020). These distinctive features of RNAi have made it a popular and effective strategy for crop enhancement and protection (Arpaia et al., 2020; Rajam, 2020).

In this review, we explained the mechanism of RNAi-mediated gene silencing and furnished a comprehensive report of the role of RNAi in protecting crops against diverse biotic stresses. Furthermore, we have directed attention towards the potential of RNAi as an innovative and potent alternative for global crop protection strategy, as well as its application in the development of futuristic smart crops resilient to various biotic stresses.

The ability of RNAi to specifically inhibit gene expression arises from the design 21-25 bp dsRNAs, ensuring only the intended target gene is silenced. The specificity grants RNAi a wide range of potential applications for genetic studies and agriculture, including the protection of beneficial insects against viruses and parasites (San-Miguel and Scott, 2016; Mehlhorn et al., 2021), novel and highly specific insect and parasites control, and traditional plant-incorporated protectants (PIPs: i.e. transgenic plants/RNAi-based plant traits) (Walawage et al., 2013). Another crucial aspect of RNAi is its conservation across different species, indicating its versatile use in manipulating gene expression not only within an organism but also for the organism’s survival and adaptation.

RNAi regulates gene expression through small noncoding RNAs (sRNAs) (Rajam, 2020) ( Figure 1 ). These sRNAs identify the target messenger RNA (mRNA) via homology-based binding and facilitate its degradation with effector proteins. Within the RNAi pathway, there are two primary classes of sRNAs: short-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) (Margis et al., 2006; Borges and Martienssen, 2015; Mamta and Rajam, 2018). While the basic pathway for sRNA biogenesis involves trimming long dsRNAs to form sRNAs, the type of sRNA present is determined by the source of the dsRNA (Parker and Barford, 2006). The application of RNA-based products for insect management requires dsRNA longer than 50 bp but not sRNAs (Ivashuta et al., 2015; Yoshida et al., 2023; Chi et al., 2023), although some studies have shown that sRNA can trigger gene silencing (Gong et al., 2013; Liu S. et al., 2021). In contrast, fungi and plants take up both dsRNAs and sRNAs (Wang and Jin, 2017; Subha et al., 2023; McLaughlin et al., 2023), suggesting different uptake mechanisms for these organisms (Wang and Jin, 2017).

siRNAs were originally observed during transgene- and virus-induced silencing in plants (Mello and Conte, 2004). They are generally formed from the dsRNA obtained from various sources, including viruses, transposons, transgenes, aberrant mRNAs, and inverted repeats (IRs), among others (Preall and Sontheimer, 2005; Matranga and Zamore, 2007; Golden et al., 2008; Wilson and Doudna, 2013). In addition to siRNAs, a multitude of miRNAs have been identified in various eukaryotes, and their sequences are available online. miRNAs are short, endogenous, single-strand RNAs, typically 21-24 nucleotides long, derived from hairpin transcripts. They play a regulatory role in gene expression in animals and plants (Bartel, 2009; Feng and Riddle, 2020). Furthermore, multiple miRNAs can regulate the same gene (Samad et al., 2021). The miRNA pathway operates at the post-transcriptional level and is involved in a range of physiological and pathophysiological processes (Ambros, 2004; Yu et al., 2017). Even though siRNAs and miRNAs were first found in separate research studies, these two small RNAs are closely connected in their biological functions, formation of RNA-protein complexes, and capacity to regulate gene transcription negatively (Meister and Tuschl, 2004). Both siRNA and miRNA molecules are initially generated from dsRNAs processed by the ribonuclease III enzyme Dicer into 20-30 nucleotide duplexes (Margis et al., 2006).

In plants, whereas for the miRNAs, the related endogenous genes are transcribed into long primary microRNAs (pri-miRNAs) under the action of RNA Polymerase II (Pol II) (Wassenegger and Krczal, 2006). These pri-miRNAs, which are single-stranded and polyadenylated RNA molecules, form hairpin-like structures. Dicer-Like1 (DCL1) cleaves the pri-miRNAs, generating precursor microRNAs (pre-miRNAs) which undergo further processing by DCL1 (Zotti and Smagghe, 2015). This leads to the production of mature miRNA duplexes consisting of the active miRNA strand and its complementary strand miRNA*. The pre-miRNAs are subsequently transported to the cytoplasm through HASTY (HST), the ortholog of human exportin-5, where they undergo further processing by the cytoplasmic RNase III Dicer to produce approximately 22-nucleotide miRNAs duplexes ( Figure 1 , Left). The export of pre-miRNAs is facilitated by HST (Cambiagno et al., 2021).

For siRNAs, these RNAi-triggering dsRNAs are formed in the nucleus through several mechanisms ( Figure 1 , Right). The 21/22-nt sRNAs are primarily associated with mRNA cleavage and are involved in posttranscriptional gene silencing (PTGS) (Hamilton and Baulcombe, 1999). The 24-nt sRNAs are primarily associated with RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (Henderson et al., 2006; Hamilton et al., 2015). Both metazoan and plant Dicer-Like proteins display domains such as DEAD-box, helicase-C, domain of unknown function 283 (DUF283), PIWI/Argonaute/Zwille (PAZ), RNase-III, and dsRNA-binding domain (dsRBD) domains (Parker et al., 2005). These sRNAs, which consist of 21-24 nucleotide duplexes, are subsequently incorporated into the RNA-induced silencing complex (RISC). With the RISC, they undergo unwinding (Borges and Martienssen, 2015). Following this step, an Argonaute (AGO) protein cleaves the passenger (sense) strand, while retaining the guide (antisense) strand within RISC (Kedde et al., 2007; Ketting, 2011; Gong, 2021). The guide strand of the sRNA then directs RISC to target mRNA through Watson-Crick base pairing, resulting in the cleavage of the target mRNA by the AGO protein and subsequent degradation. This degradation of the target mRNA leads to specific post-transcriptional gene silencing (Huvenne and Smagghe, 2010; Borges and Martienssen, 2015; Tyagi et al., 2019; Kaur et al., 2020). The AGO protein Nrde-3, which is necessary for nuclear RNAi in Caenorhabditis elegans, was identified through a genetic screen and found to reside in the cytoplasm until siRNA binding induced its translocation to the nucleus (Guang et al., 2008). The dynamic localization of RNAi factors suggests a highly regulated and adaptable system for gene regulation in cells, allowing for efficient targeting and silencing of specific genes or transcripts.

Beyond these classical miRNAs and siRNAs, other classes of RNA are continually being discovered, participating in a wide range of pathways and regulatory mechanisms. Recent research has uncovered further intricacies within the RNAi machinery. For example, the involvement of long non-coding RNAs (lncRNAs) in plant immunity, the mechanisms of lncRNA action in various stages of immunity, and different interactions between plants, microbes and insects (Wu et al., 2020; Statello et al., 2020). Additionally, a study found that aphids translocated a lncRNA into plants, which functioned as lncRNA virulence factors by enhancing aphid fecundity (Chen et al., 2020).

The RNAi mechanism operates at the mRNA level through a sequence-dependent mode of action, rendering it unique in potency and selectivity compared to regular agrochemicals. One advantage of RNAi, whether through transformative or non-transformative approaches, is its potential to enable farmers to target pathogens, nematodes, and pests more specifically. The technology can be designed by using RNA sequences that match specific gene sequences in the target pathogens, nematodes, and pests, thereby minimizing harm to other species. Careful selection of unique regions of insect genes results in highly targeted effects, while avoiding unintended consequences. RNAi in crop protection can be achieved through plant transformation via PIPs, such as transgenic plants, or through non-transformative strategies employing a spray-induced gene silencing (SIGS) process. Exogenously applied dsRNA can be taken up by two means: pathogenic cells directly uptake dsRNAs to evoke RNAi in pathogens. On the other hand, exogenously applied dsRNAs can be taken up by plant cells and then transferred to interacting pathogens to induce RNAi responses. Irrespective of the delivery strategy, the use of RNA-based products to provide plant protection against pests and pathogens represents a potential alternative to conventional pesticides. Moreover, these dsRNAs can function as resistance repressors for resistant insect and weed strains. In this category of non-PIPs, dsRNA-containing end-use products (dsRNA-EPs) are anticipated to enter the market in four categories: (i) direct control agents; (ii) resistance factor repressors; (iii) development disruptors; and (iv) growth enhancers (Zotti and Smagghe, 2015; San-Miguel and Scott, 2016). This review will focus exclusively on developments related to crop protection ( Figure 2 ).

The RNAi strategy, known as host-induced gene silencing (HIGS), has been successfully employed in transgenic plants to protect crops from specific insects (Head et al., 2017; Rodriguez Coy et al., 2022; Darlington et al., 2022), plant pathogens (Ray et al., 2022; McLaughlin et al., 2023), viruses (Tenllado and Díaz-Ruíz, 2001; Khalid et al., 2017), and nematodes (Sikandar et al., 2021; Morozov et al., 2019), as reviewed recently. The use of transgene-expressed dsRNA for inducing virus resistance and gene silencing in plants has proven to be effective against various viral infections and pests (Waterhouse et al., 1998). Scientists have successfully enhanced the natural defenses of plants by targeting specific genes involved in pathogen defense or insect resistance pathways, thus eliminating the need for chemical pesticides. However, approval from various regulatory agencies is required for crops that express dsRNA due to laws and regulations concerning genetically modified organisms. These factors complicate broader applications of HIGS worldwide, despite undeniable practicality and efficiency of HIGS strategies. Furthermore, the spray application of exogenous dsRNA or sRNA has initiated an era of RNAi-based fungicide strategies for controlling crop disease (Wang and Jin, 2017; Islam and Sherif, 2020). Table 1 summarizes some examples of successful application of HIGS and SIGS through RNAi-based approaches in insects or fungi.

Research efforts are underway to explore non-transformative approaches to control insects, diseases, nematodes, and weeds. It is anticipated that RNAi-based products will soon be available on the market as sprays for foliar application, trunk injection, root dipping, or seed treatment to directly employ as controlling agents. Since the discovery of RNAi and its regulatory potential, scientists have been investigating diverse applications of this powerful tool in insect control. The immense potential of RNAi lies in its capacity to selectively target and suppress genes responsible for insect survival and reproduction. An important advantage of utilizing RNAi in insect control is its distinct mode of action in comparison to traditional chemical pesticides (Kupferschmidt, 2013; Cagliari et al., 2019). RNAi-based products can be designed to selectively target particular pests while sparing non-target species. This targeted approach not only minimizes environmental impact but also mitigates the risk of pest resistance development.

dsRNA-based products, such as RNAi therapeutics or RNAi-based pesticides, utilize this mechanism to selectively suppress disease-causing genes or pest-specific genes. By designing dsRNA molecules that are complementary to the target gene’s mRNA, researchers can effectively shut down the expression of the target gene, potentially leading to the treatment of genetic disorders or enhanced crop protection. One of the earliest studies in the use of sprayable RNA molecules to control insect pests involved the application of siRNA, which led to effective RNAi silencing in the diamondback moth (Plutella xylostella). When larvae were fed with Brassica spp. leaves sprayed with chemically synthesized siRNAs targeting the acetylcholine esterase gene AchE2, mortality rates of about 60% were observed (Gong et al., 2013).

Moreover, the use of RNA to target pathogen resistance to conventional fungicides is currently in development (Song et al., 2018). Botrytis cinerea transfers small RNAs into plant cells, which then bind to the plant’s AGO1 to suppress host genes essential for plant immunity (Weiberg et al., 2013). This application is sprayed onto the surface of fruits (such as tomato, strawberry, and grape), and vegetables (such as lettuce and onion), resulting in a significant inhibition of grey mold disease development (Wang et al., 2016).

Recent advances in nanoparticle technology have significantly improved the potential applications for plant protection. To address issues related to dsRNA stability, double-layered hydroxide (LDH) nanoparticles were developed and combined with dsRNA molecules to produce “BioClay” (Mitter et al., 2017; Dubrovina and Kiselev, 2019; Yong et al., 2021; Jha et al., 2023). Nanoparticles are particles with diameters ranging from 1 to 100 nanometres (nm), with high stability and transport in plants (Cheng et al., 2024). LDH also degrades safely under mildly acidic conditions, thereby minimizing risk of the excessive persistence of dsRNA in the environment (Abd El-Monaem et al., 2023). Foliar spraying of LDH-dsRNA could disrupt Bemisia tabaci at multiple whitefly developmental stages by enhancing the delivery of dsRNA to cotton leaves (Jain et al., 2022). Additionally, optimizing nanostructures for agricultural use enhances the stability of RNA-based treatments, boosting control effectiveness and minimizing environmental impact (Wang et al., 2023).

Up to now, RNA-based molecules (dsRNA or siRNA) commonly utilized in insect and disease management studies have been expensive synthetic molecules or have been produced through time-consuming, laborious procedures. To address the limitations of these methods, the potential of delivering dsRNA expressed in bacteria has been investigated, providing an alternative method for large-scale target gene screening. For instance, the Escherichia coli HT115 (DE3) strain has been used to produce large quantities of dsRNA due to its lack of the enzyme RNase III which degrades dsRNA (Ahn et al., 2019; Figueiredo-Prates et al., 2023). Nanomaterials derived from plant viruses offer a promising alternative to synthetic nanoparticles. Unlike synthetic nanoparticles, plant viruses offer a higher environmental biocompatibility and degradability, yet they are exceptionally robust in the harsh environment. And most importantly, they are non-infections to mammals. Indeed, some of the most widely used plant viruses have been cowpea mosaic virus (CPMV) (Beatty and Lewis, 2019; Ortega-Rivera et al., 2021) and tobacco mild green mosaic virus (TMGMV) (Caparco et al., 2023). Practical applications of RNAi have rapidly advanced with transgene-expressed dsRNA employed not only for inducing virus resistance and gene silencing in plants but also for enhancing crop traits such as yield improvement and nutritional enhancement. Although the precise mechanism of external RNA recognition, uptake, and transport is yet to be determined, recent studies demonstrated that exogenous RNA application is a promising strategy for the regulation of plant properties, indicating the need for further research.

Indeed, efficient methods to facilitate the uptake of exogenous dsRNA for RNAi-mediated crop protection are essential for practical applications. In recent years, there has been a growing interest in the market for dsRNA, promoting both established companies and startups to focus on enhancing production efficiency and developing stable delivery systems. With the potential of dsRNA for crop protection, many companies and academic researchers are exploring cost-efficient methods for large-scale dsRNA production. It is speculated that externally applied synthetic dsRNA and siRNA may enter plant tissues and cells through natural mechanisms similar to those used by extracellular nucleic acids from plant microbial pathogens, insects, or viruses (Tatematsu et al., 2018; Dubrovina and Kiselev, 2019; Dad et al., 2021; Liu G. et al., 2021). However, the existing literature offers limited insight into the natural mechanisms responsible for the recognition, uptake, and translocation of exogenous nucleic acids in plant tissues.

Environmental risks associated with chemical and microbial pesticides are typically evaluated using a tiered approach (Pathak et al., 2022). This approach often involves testing the maximum hazard dose of known environmental exposure concentration using non-target indicator species from different ecological guilds such as pollinators, predators, and parasitoids (Lundgren and Duan, 2013). Predicting toxic effects and designing maximum hazard assays for the numerous potentially exposed organisms is a challenging task. It has been reported that foliar applied transgene-encoding dsRNA was detectable in RNA probes obtained from treated Arabidopsis leaves at 1 day and 7 days post-treatment, but its presence sharply decreased after 14 days (Lundgren and Duan, 2013). The “RNAgri” agricultural industry has developed microbial fermentation technology for large-scale production of dsRNA, utilizing a protein to bind the desired RNAs and protect them against degradation. The resulting dsRNA products are considered safer to use and more stable than naked dsRNA (Numata et al., 2014). Additionally, bacterial minicells have shown promise as a technology for both the production and encapsulation of dsRNA (Islam et al., 2021). If successful, this technology could provide improved shielding and slow, sustained release of dsRNA for agricultural purposes under open-field conditions. It is important to note that siRNAs might exhibit off-target binding in the genome of non-target species. However, considerations for microbial production of dsRNA include potential by-products from fermentation and the additional concern of GMOs (Figueiredo-Prates et al., 2023). In addition to the standard quality control measures aimed at ensuring dsRNA purity, bacterial production systems require meticulous attention to exclude potential contaminants and living GMOs. Longer dsRNA (>200 nt) yields many siRNAs after cleavage, enhancing RNAi response and reducing resistance (Darlington et al., 2022). Transgenic plants, with continuous dsRNA, increase selection pressure and resistance. RNAi resistance may come from reduced cellular uptake (Khajuria et al., 2018), mRNA mutations, RNAi suppressor production (Tayler et al., 2019), target gene overexpression, silencing machinery gene downregulation (Timani et al., 2023), increased nuclease activity, or behavioral changes (Spit et al., 2017; Kadoić-Balaško et al., 2020). Recently, cell-free platforms for dsRNA synthesis have been established, allowing for cost-effective, GMO-free production of significant quantities of dsRNA suitable for use in agricultural applications. The efficiency of RNAi naturally varies among the target species, life stage, and delivery strategy. Choosing appropriate combination of these factors can significantly expedite research and conservation of resources. Regardless of the delivery strategy or target species, the successful implementation of a non-transformative RNAi strategy hinges on the identification of unique regions within essential target genes. This ensure that even minor changes in expression levels will elicit substantial consequences.

RNAi is an emerging technology that offers new opportunities for innovative insect control strategies and the development of desired traits in genetically modified plants. The next generation of RNAi-based products will harness dsRNA to induce gene suppression in insect species, either through in planta production or application via plant spraying. Regulatory agencies for biotechnological products, such as the U.S. Environmental Protection Agency (US-EPA) and the European Food Safety Authority (EU-EFSA), have extensive experience in assessing ecological risk associated with newly introduced PIPs, the unique mode of action of dsRNA may necessitate a distinct assessment framework, as indicated by several years of crop assessments (Devos et al., 2014; Papadopoulou et al., 2020). To fully harness the potential of RNAi-based products, effective communication among agricultural consumers, RNAi-based product users (farmers), RNA pesticides producers, and public institutions will be essential to ensure accurate information exchange and mutual understanding. This should be firmly rooted in scientific evidence pertaining to RNAi-based products. Additionally, the implementation of appropriate management, oversight, and legislation, along with safety evaluation and approval processes, will be imperative for the successful adoption and regulation of RNAi-based products.

In conclusion, the utilization of exogenous dsRNA-induced RNAi technology holds great potential for revolutionizing various aspects of agriculture and crop management. By harnessing this innovative approach, we can pave the way for more eco-friendly and sustainable practices in gene regulation. By targeting genes related to plant diseases, exogenous dsRNA-induced RNAi offers a promising solution for combating plant pathogens, potentially reducing reliance on chemical pesticides and minimizing their harmful effects on human health and the environment (OECD, 2020; 2023). This technology also benefits crop improvement by selectively targeting specific genes involved in desirable traits such as drought tolerance or insect resistance, thus accelerating breeding programs, and developing new varieties with enhanced resilience and productivity (Jothi et al., 2023). However, before exogenous dsRNA-induced RNAi can be widely adopted in agricultural practices at large-scale levels such as greenhouses and fields, several essential considerations need to be addressed. One critical aspect is production technologies-optimizing methods for synthesizing quantities of high-quality dsRNA molecules efficiently will be essential to meet the demands of commercial applications. Additionally, cost-effectiveness plays a vital role in determining whether this technology becomes economically viable on a larger scale. Research efforts should focus on finding ways to reduce production costs while maintaining efficacy. Enhancing the stability of dsRNA is critically significant because it directly impacts the effectiveness of applications. In challenging environmental conditions, dsRNA is susceptible to degradation, resulting in diminished efficacy. Advancements in dsRNA delivery mechanisms are essential. Currently, the available methods exhibit low efficiency and poor targeting capabilities. In agricultural applications, encapsulating dsRNA within liposomes can facilitate better adherence to plant foliage and enable timely release, thus allowing for precise action against pests and enhancing pest management efficiency. The development of novel carriers or the optimization of existing delivery system parameters can further enhance the effectiveness of dsRNA.

The United Nations Environment Programme (UNEP) and the Food and Agriculture Organization (FAO) have recognized the crucial necessity of rehabilitating degraded ecosystems. They claim that the required restoration efforts are indispensable for attaining sustainability goals, including food and water supply security, biodiversity preservation, poverty alleviation, and climate change mitigation. Comprehensive risk assessments are necessary to ensure environmental safety when deploying exogenous dsRNA-induced RNAi technology extensively. When adequate conservation biocontrol strategies are implemented in combination with RNAi technology, the effectiveness of sustainable crop protection can be greatly enhanced. This is especially significant considering that RNAi products have relatively lower environmental risks compared to traditional pesticides.The U.S. EPA has officially approved the innovative RNA-based biopesticide Ledprona by Greenlight Biosciences to combat the increasingly severe destructive pest, the Colorado potato beetle (CPB, Leptinotarsa decemlineata) (Kadoić-Balaško et al., 2020; Rodrigues et al., 2021). The EPA emphasizes that RNA-based biopesticides provide farmers with additional tools to address the challenges of climate change and contribute to resistance management. In summary, while there are still challenges ahead regarding production technologies and cost-effectiveness issues surrounding exogenous dsRNA-induced RNAi technology’s implementation in large-scale agricultural settings like greenhouses and open fields, continued research efforts will play a pivotal role in realizing its potential to achieve more sustainable farming practices worldwide.