RNA silencing: the future potential strategy for engineering virus resistance in plants

Abstract

Plant viruses adversely affect worldwide agriculture and cause immense crop yield losses globally. Scientists have developed various strategies to combat the viral attacks on plants and one such ground-breaking discovery is the RNA interference (RNAi), also known as RNA silencing. RNA silencing has evolved as a major tool for developing viral resistance in plants through gene silencing that involves the intricate use of various small RNAs, such as small interfering RNAs (siRNAs), endogenous microRNAs (miRNAs), artificial miRNAs (amiRNAs), hairpin RNAs (hpRNAs), double-stranded (ds) RNA sprays (topical applications), and the less prevalent short hairpin (sh) RNAs. With tailor-made constructs, RNAi has opened the avenues for the immense potential to down-regulate the desired viral target genes, leading to reduced viral pathogenicity in several crop plants leading to enhanced sustainability in agriculture and food security for the teeming millions across the globe. In this article, we have reviewed the advances made in the generation of virus-resistant plants by using RNAi-based approaches, particularly siRNA- and amiRNA-mediated technologies. Despite certain issues with delivery, specificity, resistance, and safety that impede the RNAi-based treatments, targeted RNA silencing is expected to revolutionize the future agricultural research with tailor-made stress-tolerant crop plants.

Introduction

The growing population worldwide has increased the demand for food supply. This has further created pressure on the agriculture sector to increase crop productivity and minimiz yield losses. Globally, nations are investing in agricultural research to improve crop productivity without compromising its nutritional value. Unfortunately, the biotic stress causing agents impose a major threat to crop yield and quality. Among these biotic agents, viruses are more resilient and solely contribute to 47% of plant diseases (Jones RA 2021). Its rapid multiplication and fast-evolving traits have caused suppression of innate immunity in major agronomically important crops (Serfling et al. 2017; Hančinský et al. 2020). The globalization of agriculture and increased movement of plant material have significantly contributed to the rapid spread of plant viruses across regions, facilitating the transmission of these viruses between continents through infected crops and vectors. The monoculture cultivation of one crop in all seasons was the major reason for disease outbreak, which led to a substantial reduction in crop yield (Wu et al. 2023; Tatineni and Hein 2023). The mechanisms through which viruses cause yield losses are diverse, including interference with photosynthesis, disruption of nutrient uptake, and alteration of plant metabolism (Sastry 2013). One of the key factors contributing to crop yield reduction is the ability of viruses to manipulate the host plant's cellular processes, leading to stunted growth, reduced flower, and fruit formation (Sastry 2013). Beyond yield loss, viral infections have also profoundly impacted the quality of harvested crops (Wang et al. 2015; Mannini and Digiaro 2017).

The economic ramifications of viral infections in crops extend beyond the immediate losses experienced by farmers. The costs associated with disease management, including the use of pesticides and the implementation of quarantine measures, further strain agricultural economies. Moreover, the reduction in crop yield and quality amplifies food insecurity, as the affected regions may experience shortages and increased prices. Addressing viral infections in crops, therefore becomes crucial for ensuring global food security and sustaining agricultural economies (García-Estrada et al. 2022). Despite facing numerous challenges, plants have evolved sophisticated defence mechanisms to counteract viral infections such as the innate immune system, which includes pathogen-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (Nürnbergerand Kemmerling 2009). However, one of the most potent of all plant defences is the specialized resistance mechanism called RNA interference (RNAi), also known as RNA silencing, which involves small RNA molecules that specifically target and degrade viral RNA, thereby limiting viral replication and spread (Ryan et al. 2007; Rajam 2020). These small RNAs include small interfering RNAs (siRNAs) and microRNAs (miRNAs) that mediate post-transcriptional gene silencing and serve as antiviral agents within the host.

RNAi has emerged as a powerful, non-conventional strategy for plant virus management. siRNAs can be synthetically designed or endogenously expressed to silence viral genes, preventing their replication and accumulation (Rêgo-Machado et al. 2020; Deng et al. 2022; Majumdar et al. 2023). This has been successfully demonstrated in various crops including papaya, rice, and cassava (Pooggin 2017; Taliansky et al. 2021; Koeppe et al. 2023). Transgenic approaches using RNAi have enabled the stable expression of double-stranded RNA (dsRNA) constructs that mimic viral sequences, triggering a defensive RNAi cascade within the plant (Ibrahim and Aragão 2015; Singh et al. 2019). In addition to viral control, RNAi has shown efficacy in managing fungal and bacterial diseases (Kaur et al. 2021; Ali et al. 2024; Srimahesvari et al. 2024), as well as insect and nematode pests (Mamta and Rajam 2017; Liu et al. 2020; Yan et al. 2023). The environment-friendly nature, specificity, and adaptability of RNAi-based technologies make them a promising alternative to conventional chemical pesticides, supporting the goals of sustainable agriculture (Baulcombe 2015; Koeppe et al. 2023).

In parallel, resistance (R) genes form the cornerstone of conventional host-plant resistance strategies (Seo et al. 2016; Karki et al. 2021). These genes encode immune receptors, such as nucleotide-binding leucine-rich repeat (NLR) proteins, which detect specific viral effectors and trigger localized defense responses such as the hypersensitive response (HR) and systemic acquired resistance (SAR) (Moffett 2009; Seo et al. 2016). The genetic basis of resistance enables durable and heritable protection against specific viruses and has been widely employed in breeding programs for crops such as tomato, potato, and tobacco (Reinke et al. 2018). However, the constant evolution of viral populations and the emergence of resistance-breaking strains necessitate the continuous discovery and deployment of novel R genes from wild relatives and landraces (de Ronde et al. 2014; Karki et al. 2021). Marker-assisted selection (MAS) and genomic-assisted breeding have further accelerated the incorporation of R genes into elite cultivars, enhancing their resistance profiles without compromising agronomic performance (Dixon et al. 2016).

To enhance the effectiveness of viral disease control, an integrated approach that combines both conventional and non-conventional strategies is gaining prominence. Integrated pest and disease management (IPDM) programs combine the use of resistant varieties, vector control, RNAi, and novel genome-editing technologies such as CRISPR/Cas to create comprehensive and sustainable disease management pipelines (Zhang et al. 2018; Romeh 2019; Rêgo-Machado et al. 2020; Angon et al. 2023; Majumdar et al. 2023). These multifaceted strategies aim to minimize economic losses, safeguard crop productivity, and support global food security in the face of evolving viral threats.

RNAi and gene silencing

The history of RNAi dates to the late twentieth century when Andrew Fire and Craig Mello made ground-breaking discovery in the nematode Caenorhabditis elegans (Fire et al. 1998), leading to their Nobel Prize-winning work in 2006. Their research laid the foundation for understanding the role of small RNA molecules in gene silencing (Fire et al. 1998; Rajam 2020). RNAi serves as a defense mechanism against viruses and transposons, and plays a crucial role in the development and maintenance of genome stability. The discovery of RNAi opened avenues for harnessing this process for targeted gene silencing, both in basic research and various practical applications (Dykxhoorn and Lieberman 2005; Kurreck 2009; Mamta and Rajam 2017; Ali et al. 2024; Verma and Modgil 2024). Being sessile, plants have evolved sophisticated defense mechanisms to combat biotic stress, and RNAi has emerged as a crucial player in providing a highly regulated and efficient immunity in plant against viral invaders, besides being a powerful tool for gene regulation and functional genomics (Mann et al. 2008; Kaur et al. 2021; Chaudhary et al. 2024).

Gene silencing through RNAi can follow two different pathways, namely endogenous and exogenous pathways. These two pathways differ primarily in their mechanisms of origin of dsRNAs that are involved in triggering the silencing of genes. Endogenous RNAi pathways utilize dsRNA derived from the organism's own genome, such as pre-microRNAs, miRNAs or transcripts from transposable elements. These are basically involved in regulation of plant developmental processes and responses to environmental cues (Kaur et al. 2020; Rajam 2020). On the other hand, the exogenous RNAi pathways are triggered by dsRNAs that are introduced from outside the cell, like synthetic dsRNA or viral RNA (Alam et al. 2023). These offer a defense mechanism against viruses and allow for the manipulation of gene expression through external application of dsRNAs. Both pathways ultimately lead to gene silencing through similar core components such as Dicer and Argonaute (AGO) proteins, which are explained in detail in the following sections. However, both the pathways rely on Dicer to cleave dsRNA into small RNAs and AGO proteins to guide these small RNAs to target mRNA for degradation or translational repression (Alam et al. 2023).

Gene silencing mechanisms of RNAi

Small interfering RNA (siRNA) pathway

The RNAi process involves the generation of small RNA molecules, primarily siRNAs and miRNAs. Dicer-like (DCL) proteins, a class of ribonucleases, process viral double-stranded RNA (dsRNA) into siRNAs, typically 21–24 nucleotides in length (Pumplin and Voinnet 2013). The generated siRNAs guide the RNA-induced silencing complex (RISC) to complementary viral mRNA sequences, leading to endonucleolytic cleavage and degradation (Rosa et al. 2018; Kaur et al. 2020, 2021; Matsumura and Kormelink 2023; Fig. 1). This process impedes viral replication and spreads within the plant (Ding and Voinnet 2007). In short, DCLs generate primary siRNAs, and the RNA-dependent RNA polymerases (RdRPs) amplify the signal by producing secondary siRNAs from cleaved viral RNAs (Wang et al. 2010). DCLs, such as DCL4 and DCL2 are involved in antiviral defense in plants like Arabidopsis (Andika et al. 2015). Hence, RNAi not only acts locally at the site of infection but also induces SAR. Mobile siRNAs, termed phloem-mobile silencing signals (PMSS), move systemically through the plant, enhancing antiviral defenses in distal tissues (Molnar et al. 2010).

Fig. 1 .

Plant antiviral RNAi: siRNA generation and systemic silencing. Viral RNA or DNA enters host plant upon infection and utilizes host machinery to replicate its genome. Plant RNAi machinery acts on viral RNA transcript to produce dsRNA, which is processed preferentially by DCL4 (~ 21 nt) or by DCL2 (~ 22 nt), in case DCL4 is absent or saturated. This results in RISC-mediated cleavage of viral mRNA by the primary siRNAs. Further, RdRP-mediated replication of cleaved RNAs produce dsRNA substrates for DCL4/DCL2 to generate secondary siRNAs, reinforcing the silencing signal. A subset of siRNAs move through the phloem as phloem-mobile silencing signals to trigger systemic acquired resistance in distal tissues of the host plant. [Key to abbreviations used in Fig. 1: RdRP RNA-dependent RNA polymerase; DCL Dicer-like protein; Aux Auxiliary protein; AGO Argonaute protein; RISC RNA-induced silencing complex; RDR6 RNA-dependent RNA polymerase 6]

miRNA pathway

The miRNAs are pivotal regulators of gene expression in plants, intricately involved in various biological processes such as plant development and responses to various biotic as well as abiotic stress mechanisms (Kaur et al. 2020; Luo et al. 2024). The miRNA genes are transcribed mostly by RNA polymerase II (rarely by RNA polymerase III) forming the primary miRNAs (pri-miRNAs), which further initiate a cascade of events (Jones-Rhoades et al. 2006). Pri-miRNAs exhibit a characteristic hairpin-like structure and are recognised by DCL1 protein complex that cleaves the pri-miRNA, producing precursor miRNA (pre-miRNA), ~ 70 nucleotide stem-loop structure (Kaur et al 2020). Then, DCL1, in association with other proteins, cleaves the pre-miRNA, generating a short double-stranded duplex miRNA. In the cytoplasm, exportin-like protein and HASTY facilitate the transport of miRNA duplex out of the nucleus. This duplex consists of the mature miRNA strand or the guide strand and passenger strand. The guide strand is loaded into an AGO protein within the RISC and leads to its activation. AGO1, a member of the AGO protein family, stands out as a central player in miRNA-mediated gene regulation in plants (Mallory and Vaucheret 2006). The activated RISC complex, armed with the guide strand, scans the cellular environment for target mRNAs. The recognition of target mRNAs by the miRNA-loaded RISC complex is based on sequence complementarity. This interaction results in the degradation of the target mRNA or the inhibition of its translation (Kaur and Rajam 2020, 2021; Ding et al. 2024; Fig. 2).

Fig. 2.

miRNA biogenesis and antiviral defense in plants. In the nucleus, miRNA genes are transcribed by RNA polymerase II (or rarely RNA Pol III) into primary miRNAs (pri-miRNAs) with hairpin structures. The DCL1 protein complex (comprising of SE, HYL1 and DCL1) sequentially cleaves pri-miRNAs into precursor miRNAs (pre-miRNAs) and then into ~ 21 nt miRNA duplexes, which are then methylated by HEN1 methyltransferase. Transport proteins, like Expo1 and HASTY, mediate the export of these methylated miRNA duplexes from nucleus to cytoplasm. The guide strand is incorporated into an AGO1-containing RISC, which targets complementary viral or host mRNAs for cleavage or translational repression, while the passenger strand is subjected to SDN1 degradation. [Key to abbreviations used in Fig. 2: MIR gene miRNA gene; pri-miRNAs primary miRNAs; pre-miRNAs precursor miRNAs; Expo1 Exportin-like protein 1; RISC RNA-induced silencing complex; AGO1 Argonaute protein 1; DCL1 Dicer like protein 1; SE Serrate (a C2H2 zinc-finger protein); HYL1 Hyponastic Leaves 1 (a type of dsRNA binding protein); HEN1 Hua Enhancer 1; SDN1 Site-directed nuclease 1]

Gene silencing strategies

Virus-induced gene silencing (VIGS)

VIGS is a transient and non-transgenic RNAi method that utilizes modified plant viruses as vectors to deliver gene-specific sequences into the host plant (Zulfiqar et al. 2023). For instance, modified plant viral vectors such as, tobacco mosaic virus (TMV), tobacco rattle virus (TRV), or potato virus x (PVX), were used to silence the expression of targeted genes in host plant species, such as Nicotiana benthamiana, Arabidopsis, tomato, etc. (Unver and Budak 2009; Zulfiqar et al. 2023). Upon infection, the recombinant virus replicates and spreads systemically, inducing RdRP activity, which leads to dsRNA production (Voinnet 2005). These dsRNA are further processed by plant RNAi machinery to yield siRNAs, and utilizes host's RNA-silencing machinery to silence endogenous genes (Ruiz-Ferrer and Voinnet 2009). VIGS is widely used in functional genomics that enables rapid and efficient gene silencing without the need for stable plant transformation. It is especially valuable for high-throughput screening of gene function, including in species that are recalcitrant to genetic transformation (Burch-Smith et al. 2006; Becker and Lange, 2010; Zulfiqar et al. 2023). Although VIGS is primarily used to silence endogenous plant genes rather than those of pathogens or pests, it remains a powerful tool for dissecting plant gene function and understanding host defense responses. Recent advances include the development of second-generation VIGS vectors, such as TRV, which exhibit broader host ranges and milder symptoms, thereby reducing off-target effects and enhancing reliability (Senthil-Kumar and Mysore 2011).

Host-induced gene silencing (HIGS)

HIGS is a transgenic RNAi-based strategy in which host plants are genetically engineered to produce dsRNA molecules that target essential genes in interacting pathogens or pests, such as fungi, nematodes, insects, or viruses. These dsRNAs are processed by the plant’s RNAi machinery into siRNAs that specifically degrade the corresponding mRNA in the invading organism, thereby disrupting critical biological processes and conferring resistance. Unlike transient silencing approaches, HIGS enables stable, systemic, and heritable gene silencing due to the continuous endogenous production of RNAi triggers in the transgenic plant. This method capitalizes on the plant’s natural immune system and offers a sustainable, environmentally friendly alternative to chemical controls by precisely targeting pathogen virulence factors or metabolic genes without impacting host physiology (Koch and Kogel 2014; Nowara et al. 2010; Ghag et al. 2014; Wang et al. 2016a, b). Recent advancements include the design of polycistronic amiRNA constructs that can simultaneously target multiple genes within a single RNAi cassette, enhancing the durability and breadth of resistance (Rajam 2015; Yin et al. 2011; Cisneros and Carbonell, 2020; Miao et al. 2021).

Spray induced gene silencing (SIGS)

SIGS, a recent and novel RNA silencing strategy for disease control, involves the external application of dsRNA (Dalakouras et al. 2020; Miao et al. 2021; Rêgo-Machado et al. 2020) with or without nanoparticles (Ghosh et al. 2023; Xu et al. 2023; Quilez-Molina et al. 2024; Verma and Modgil 2024). Plant pathogens uptake the externally applied dsRNA, leading to the silencing of targeted genes crucial for disease improvement (Hunter et al. 2010). This non-transgenic and eco-friendly strategy offers minimal off-target effects and has been successfully applied to pathogens affecting both monocots as well as dicots (Mitter et al. 2017). SIGS provides an environmentally sustainable approach to crop protection at both pre-harvesting and post-harvesting stages (Kachroo and Kachroo 2009). Recent advances include successful resistance against various viruses, such as cymbidium mosaic virus (Lau et al. 2014), sugarcane mosaic virus (SCMV) (Gan et al. 2010), alfalfa mosaic virus, pepper mild mottle virus, tobacco etches virus (Ghag et al. 2014), papaya ringspot virus (PRSV) (Shen et al. 2014; Valdamundi et al. 2020), TMV (Konakalla et al. 2016; Niehl et al. 2018), zucchini yellow mosaic virus (ZYMV) (Kaldis et al. 2018), and cucumber green mottle mosaic virus (CGMMV) (Delgado-Martín et al. 2022). Optimization of high-pressure spraying technologies has enhanced targeted gene silencing, overcoming previous challenges associated with low-pressure spraying (Dalakouras et al. 2020). Although, the topical application of dsRNA for the control of plant viruses is promising strategy, there are certain limitations such as the quality of dsRNA, uptake of dsRNA by the plant, cost of dsRNA production, short lifespan of dsRNA, and lack of adequate product formulation to develop this technology for commercial use (Rêgo-Machado et al. 2020). However, these issues can be addressed with the appropriate methodologies like the use of nanoparticle-encapsulated dsRNA for its stability (Quilez-Molina et al. 2024). Another potential alternative to the dsRNA sprays can be the use of sprays comprising of the amiRNAs, which are engineered small RNAs designed to specifically target genes of interest. However, to the best of our knowledge, the use of synthetic amiRNAs (monocitronic and polycitronic), with or without nanoparticle carriers, as foliar sprays for disease and pest control has not yet been demonstrated in published studies. While this approach could hypothetically offer advantages over dsRNA sprays, including increased specificity and reduced off-target effects, its efficacy remains to be validated in peer-reviewed research.

Role of RNAi in immunity against viruses

RNAi is a highly conserved, sequence-specific post-transcriptional gene silencing mechanism that plays a central role in plant innate immunity against viral pathogens (Ryan et al. 2007; Pooggin 2017). During viral infection, dsRNA, or highly structured regions of single-stranded viral RNAs are recognized by plant DCL enzymes, which cleave them into 21–24 nucleotide siRNAs. These siRNAs are incorporated into AGO proteins within the RISC, which guides the complex to complementary viral RNAs for targeted cleavage and degradation (Moffett 2009; Baulcombe 2015; Pooggin 2017).

This mechanism acts as a robust antiviral defense strategy by limiting virus replication and systemic spread. The potential of RNAi has been exploited in biotechnology to generate virus-resistant crops. Transgenic plants expressing hairpin RNAs (hpRNAs), synthetic siRNAs, or amiRNAs targeting viral genomes have demonstrated effective and specific resistance against a wide range of plant viruses (Ibrahim and Aragão 2015; Singh et al. 2019; Deng et al. 2022). Such RNAi-based resistance strategies are not only environmentally sustainable but also offer precise alternatives to chemical pesticides, especially for managing viruses that lack effective vector control strategies (Rêgo-Machado et al. 2020; Majumdar et al. 2023).

Viral suppressors of RNA silencing (VSRs)

Despite the efficacy of RNAi-mediated immunity, many plant viruses have evolved counter-defensive strategies in the form of viral suppressors of RNA silencing (VSRs), a group of —specialized proteins that inhibit various stages of the host RNAi pathway to facilitate viral replication and infection (Csorba et al. 2015; Sarkar et al. 2025; Table 1). These VSRs are diverse in structure and function, reflecting the evolutionary arms race between plant hosts and viral pathogens. The various known plant VSRs have been summarized in Table 1.

Table 1.

Viral suppressors of plant RNA silencing

S. No	Plant Virus	Suppressors	Plant	References
1	Tomato yellow leaf curl virus	V2 protein	Tomato	Wang et al. (2025)
2	Tomato leaf curl New Delhi virus	AV2, AC2 and AC4	Tomato	Sarkar et al. (2025)
3	Tomato bushy stunt virus	P19 protein	Tomato	DeMell et al. (2024)
4	Tombusvirus	P19 protein	Arabidopsis	Jay et al. (2023)
5	Tobamovirus	P122/P130	Tomato	Vaisman et al. (2022)
6	Ipomovirus sweet potato mild/feathery mottle virus	P1	Sweet potato	Rodamilans et al. (2021)
7	Turnip crinkle virus	P38	Nicotiana benthamiana	Iki et al. (2017)
8	Carnation Italian ringspot virus	P19	Nicotiana benthamiana	Law et al. (2013)
9	Cauliflower mosaic virus	P6	Cauliflower	Laird et al. (2013)
10	Potato virus x	TGB-1 protein	Potato	Park et al. (2013)
11	Potato virus x	p25 protein	Potato	Chiu et al. (2010)
12	Tobacco etch potyvirus	P1/HC-Pro	Tobacco	Torres-Barcelo et al. (2008)
13	Poleroviruses	P0	Arabidopsis	Baumberger et al. (2007)
14	Potyvirus	HC-Pro protein	Tomato	Kasschau et al. (2007)
15	Cucumber mosaic virus	2b protein	Cucumber	González et al. (2010), Zhang et al. (2006)
16	Pothos latent aureus virus	P14	Nicotiana benthamiana	Mérai et al. (2005)
17	Citrus tristeza virus	P20, P23, coat protein (CP)	Citrus	Lu et al. (2004)
18	Turnip crinkle virus	P38, HC-Pro	Nicotiana benthamiana	Thomas et al. (2003)

Some VSRs, such as the P19 protein from tomato bushy stunt virus, bind siRNAs with high affinity and specificity, preventing their incorporation into AGO proteins and thereby, neutralizing RISC activity (DeMell et al. 2024). The 2b protein of cucumber mosaic virus (CMV) interferes with multiple components of the RNAi machinery by directly binding to AGO1 and inhibiting its slicer activity, as well as impairing siRNA and miRNA pathways (González et al. 2010; Zhang et al. 2006). Likewise, the HC-Pro protein of potyvirus acts as a multifunctional VSR by sequestering siRNAs, suppressing DCL activity, and destabilizing AGO1 (Kasschau et al. 2007).VSRs not only suppress antiviral RNAi but can also interfere with endogenous gene regulation, potentially leading to developmental defects and increased susceptibility to other pathogens (Csorba et al. 2015; Sarkar et al. 2025; Table 1). This interference underscores the broader consequences of VSR activity beyond the immediate scope of viral replication.

In response, plants have evolved a secondary layer of defense involving host RNA-binding proteins and protein–protein interaction modulators that mitigate the effects of VSRs and restore RNAi activity (Huh and Paek 2013; Azevedo et al. 2010). Moreover, genome editing and synthetic biology approaches are being explored to engineer RNAi components or mimic resistant alleles that evade VSR-mediated suppression (Sanan-Mishra et al. 2017; Yıldırım et al. 2023). Understanding the diversity and mechanism of VSRs is essential for developing robust RNAi-based antiviral strategies. The incorporation of multi-targeting constructs, amiRNAs, and polycistronic RNAi systems are ongoing effort to bypass viral suppression and achieve long-lasting resistance (Rajam 2015; Yin et al. 2011; Mamta and Rajam 2017; Miao et al. 2021; Sarkar et al. 2025).

siRNA-mediated virus resistance in plants

Plant viruses severely impact the productivity of economically important crops such as rice, wheat, maize, tomato, potato, cotton, and others. RNAi has emerged as a powerful strategy to confer resistance against viral infections in plants by specifically suppressing viral gene expression through sequence-specific RNA silencing mechanisms (Rosa et al. 2018; Halder et al. 2022).

Potato (Solanum tuberosum), a major staple crop, is highly susceptible to multiple plant viruses. Arif et al. (2012) demonstrated that a single transgene construct containing sequences from the ORF2 gene of PVX, HC-Pro gene of potato virus y (PVY), and the coat protein (CP) gene of potato leafroll virus (PLRV) could confer simultaneous resistance through RNA silencing. Similarly, Hameed et al. (2017) achieved nearly 100% resistance to PVX, PVY, and potato virus S (PVS) by expressing a 600 bp inverted repeat of fused CP coding sequences under a 35S promoter. Petrov et al. (2015) showed that RNAi-based vaccination targeting the HC-Pro gene of PVY reduced viral replication without altering desirable traits. Chung et al. (2013) conferred resistance to multiple viruses in potato by using a hairpin construct containing tandem 200 bp fragments from PVY, PLRV, and potato virus A. Marker-free plants resistant to PVY were generated by targeting the host eIF4E1 gene (Miroshnichenko et al. 2020).

Rice (Oryza sativa), a staple for over half of the global population, is frequently affected by viruses such as rice black-streaked dwarf virus (RBSDV), rice stripe virus (RSV), rice grassy stunt virus (RGSV), and rice tungro bacilliform virus (RTBV). Ahmed et al. (2017) developed RBSDV resistant marker-free transgenic rice lines expressing hpRNA constructs targeting RBSDV S7-2 and S8 genes, encoding proteins P7-2 and P8, respectively, which function in plant-virus interactions via the ubiquitin pathway. Wang et al. (2016a, b) reported a strong resistance against RBSDV by introducing hpRNA constructs targeting four RBSDV genes (S1, S2, S6, and S10), encoding RdRP, core protein, RNA silencing suppressor, and outer capsid protein, respectively. Shimizu et al. (2011, 2013) demonstrated RSV and RGSV resistance via inverted repeat constructs targeting pC3 (nucleocapsid protein) and pC4 (movement protein), and pC5 and pC6, respectively. For RTBV, Tyagi et al. (2008) used RNAi constructs against ORF IV, reducing viral titers nearly 50-fold.

In soybean (Glycine max), resistance to soybean mosaic virus (SMV) was achieved by RNAi-mediated silencing of the viral CP gene (Kim et al. 2013). Yang et al. (2018) engineered resistance to multiple potyvirus strains using a 302 bp inverted repeat of the SMV P3 cistron. Transient expression of hpRNA targeting the AC2 gene of mungbean yellow mosaic india virus (MYMIV) conferred significant resistance (Ramesh et al. 2019), while Kumari et al. (2018) used intron-spliced hp constructs of the MYMIV CP gene in transgenic lines. In cowpea (Vigna unguiculata), Kumar et al. (2017) developed MYMIV-resistant lines using three intron-spliced hpRNA constructs targeting AC2, AC4, and AC2 + AC4, resulting in complete resistance.

RNAi has also been used to combat viral infections in other crops. Transgenic sugarcane expressing hpRNA constructs of the CP gene of SCMV exhibited resistance under a ubiquitin promoter (Widyaningrum et al. 2021). Leibman et al. (2011) developed transgenic cucumber and melon lines expressing hpRNA constructs of the HC-Pro gene of ZYMV, which conferred resistance not only to ZYMV but also to watermelon mosaic virus and papaya ringspot virus-w (PRSV-W). These lines showed elevated levels of RdRP1, AGO1, and siRNAs, suggesting active RNAi-mediated silencing.

Cotton plants expressing intron hpRNA constructs targeting the intergenic region of cotton leaf curl Rajasthan virus displayed high resistance (Khatoon et al. 2016). In maize, transgenic expression of hpRNA constructs targeting the CP gene of maize dwarf mosaic virus conferred resistance, with longer constructs proving more effective (Zhang et al. 2011a).

In cassava (Manihot esculenta), resistance to cassava brown streak virus (CBSV) and its Ugandan variant was conferred through expression of hpRNA constructs targeting the near full-length CP gene (Yadav et al. 2011). Patil et al. (2011) further demonstrated the effectiveness of multiple CP-targeted RNAi constructs against CBSV in transgenic tobacco. Resistance to African cassava mosaic virus (ACMV) was achieved by expressing hp-dsRNAs homologous to the ACMV AC1 gene (Vanderschuren et al. 2009). In common bean (Phaseolus vulgaris), Bonfim et al. (2007) obtained resistance to bean golden mosaic virus (BGMV) via downregulation of AC1. Reyes et al. (2009) reported resistance in tobacco through self-complementary hpRNA constructs from CP and 54 K genes of citrus psorosis virus.

A comprehensive summary of RNAi-mediated virus resistance in different plants is presented in Table 2.

Table 2.

Development of virus-resistant plants by using siRNA-based approaches

S. No	Virus targeted	Host plant	Target gene	References
1	Maize dwarf mosaic virus	Maize	CP	Balassa et al. (2024)
2	Cotton leaf curl Multan virus and Cotton leaf curl Multan betasatellite	Cotton	AC3 and an overlapping region ofAC2–AC1 of CLCuMuV and βC1 of CLCuMB;	Jain et al. (2024)
3	Strawberry mottle virus, Strawberry crinkle virus and strawberry virus 1	Strawberry	vsiRNA	Koloniuk et al. (2023)
4	Tobacco curly shoot virus	Tobacco	vsiRNA18	Wu et al. (2023)
5	African cassava mosaic virus	Cassava	DNA-A and DNA-B	Mohamed et al. (2022)
6	African cassava mosaic virus	Tobacco	Hotspot regions of viral genome	Mohamed et al. (2022)
7	Tobacco mosaic virus pathotype p0	Pepper	vsiRNA	Kim et al. (2021)
8	Cauliflower mosaic virus	Wheat	Catalase 3 (CAT3)	Leonetti and Pantaleo (2021)
9	Wheat yellow mosaic virus	Wheat	thioredoxin-like gene (TaAAED1)	Liu et al. (2021)
10	Sugarcane mosaic virus	Sugarcane	CP	Widyaningrum et al. (2021)
11	Rice stripe virus	Nicotiana benthamiana	eIF4A	Zhang et al. (2021)
12	Potato virus y	Potato	eIF4E	Miroshnichenko et al. (2020)
13	Tomato mosaic virus	Tomato	CP	Rêgo -Machado et al. (2020)
14	Mungbean yellow mosaic India virus	Soybean	AC2	Ramesh et al. (2019)
15	Tomato leaf curl New Delhi virus and Tomato leaf curl Gujarat virus	Tobacco	AC2 and AC4	Singh et al. (2019)
16	Tomato yellow leaf curl virus	Tomato	Tomato lncRNA (designated as SlLNR1)	Yang et al. (2019)
17	Soybean mosaic virus	Soybean	P3	Yang et al. (2018)
18	Sugarcane mosaic virus	Rice	CP and Helper-component proteinase (Hc-Pro)	Akbar et al. (2017)
19	Papaya ring spot virus	Papaya	CP	Jia et al. (2017)
20	Mungbean yellow mosaic India virus	Cowpea	AC2, AC4	Kumar et al. (2017)
21	Cotton leaf curl Rajasthan virus	Cotton	IR	Khatoon et al. (2016)
22	Triticum mosaic virus	Wheat	CP	Shoup Rupp et al. (2016)
23	Rice black-streaked dwarf virus	Rice	S1, S2, S6, S10	Wang et al. (2016a, b)
24	Tomato yellow leaf curl virus-Oman	Tomato	CP, V2 and replication-associated gene of virus	Ammara et al. (2015)
25	Potyvirus, Sugarcane mosaic virus, Sorghum mosaic virus	Sugarcane	CP	Guo et al. (2015)
26	Rice grassy stunt virus	Rice	CP and Mp	Shimizu et al. –(2013)
27	Wheat streak mosaic virus	Wheat	Viral replicase (Nib) full-length gene	Fahim et al. (2012)
28	Banana bunchy top virus	Banana	Viral replication initiation protein (Rep protein)	Shekhawat et al. (2012)
29	Cassava brown streak virus	Tobacco	CP	Patil et al. (2011)

Artificial miRNA (amiRNA)-mediated virus resistance in plants

The amiRNAs can mediate specific and effective gene silencing in plants and are widely being used to achieve viral resistance in plants (Rajam 2015; Taliansky et al. 2021). Due to their precise functions involving down-regulation of site-specific genes and their variants, amiRNA strategy has also gained importance in studies involving gene function. One of the earliest studies on amiRNA technology was done by Schwab et al. (2006) where a genome-wide expression profiling was done and the results revealed the specificity of amiRNAs as high as that of natural plant miRNAs, and established the fact that amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be down-regulated. Niu et al. (2006) modified an Arabidopsis miR159 precursor to express amiRNAs targeting viral mRNA sequences encoding two genes of silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). The resulting transgenic Arabidopsis plants expressing these two amiRNAs showed improved tolerance to TYMV and TuMV, respectively.

The amiRNA vectors are generally developed based on the natural miRNA precursor structures. In these vectors, miRNA/miRNA* sequence from precursor backbone is replaced with amiRNA/amiRNA* sequence, known as the seed duplex, which is complementary to the target gene. The amiRNAs are designed as per guidelines discussed in online available software- Web MicroRNA Designer (WMD3—http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). For effectiveness, any mismatches in the seed duplex (9–11 bp) of amiRNA should be avoided. Other essential characteristic features in amiRNA designing include "A" at the 10th bp position and an unstable 5'-end. The amiRNA precursor should be thermodynamically stable and the selection of precursor miRNA should be selected from the same species (Yogindran and Rajam 2021) (Fig. 3). One such example was demonstrated by Liu et al. (2010), where a simple amiRNA vector based on the structure of Arabidopsis miR169d precursor was used to drive efficient silencing of target genes in transgenic Arabidopsis plants. Similar study was shown in Fragaria vesca (strawberry) plants by expression of a simple amiRNA vector based on Fv-miR166 precursor of strawberry designed by annealing of eight synthetic oligonucleotides, that included GUS, GFP, and miR390 (Li et al. 2019). Transient transformation of this vector, under the control of a CaMV35S promoter, in strawberry fruit resulted in the down-regulation of GUS and GFP, in addition to effectively targeting the endogenous TAS3 gene by amiR390. Nowadays, several databases are being developed, which can be used to design oligos for amiRNA synthesis using backbones of natural plant miRNAs, thereby facilitating the strategic designing of amiRNA genes (Yasir et al. 2022).

Fig. 3.

Engineering virus resistance in plants using artificial microRNA (amiRNA) technology: amiRNA mono- and poly- cistronic construct design and mode of action. AmiRNA genes are engineered by replacing the native miRNA/miRNA* sequences in a plant miRNA precursor backbone with sequences complementary to viral target genes (amiRNA/amiRNA*). Monocistronic amiRNA constructs express a single amiRNA designed to silence a specific viral gene, while polycistronic constructs incorporate multiple amiRNA precursors or tandem amiRNA sequences within a single transcription unit to simultaneously target several viral genes or multiple viruses. Following transcription (primarily by RNA polymerase II), the pri-amiRNA is processed in the nucleus by the DCL1 complex into pre-amiRNA and subsequently into a ~ 21 nt mature amiRNA duplex. The methylated amiRNA duplex is then transported from nucleus to cytoplasm, where the guide strand is loaded into AGO1 within RISC. The activated amiRNA-RISC complex binds to the complementary viral RNAs, leading to target mRNA cleavage and degradation, thereby inhibiting viral replication. [Key to abbreviations used in Fig. 3: aMIR gene amiRNA gene; pri-amiRNAs primary amiRNAs; pre-miRNAs precursor amiRNAs; RISC RNA-induced silencing complex; AGO1 Argonaute protein 1; DCL1 Dicer like protein 1; WMD3 Web MicroRNA Designer 3]

Tomato plants are highly prone to viral attacks, which cause yield losses. Transgenic tomato plants expressing amiRNA targeting the overlapping region of the AV1 (coat protein) and AV2 (pre-coat protein) transcripts showed high tolerance to tomato leaf curl New Delhi virus (ToLCNDV) (Vu et al 2013). Expression of amiRNAs targeting ATP binding domain of AC1 gene, involved in virus replication and pathogenicity, conferred resistance to Tomato leaf curl disease caused by tomato leaf curl virus (ToLCV) as well as ToLCNDV in transgenic tomato plants (Sharma and Prasad 2020). A recent study applied amiRNA technology in two ways, i.e., amiRNA in introns and amiRNA in exons (Khalid et al. 2023). The group expressed 14 amiRNAs targeting conserved regions in seven genes and their satellite DNA belonging to tomato yellow leaf curl virus (TYLCV) like species and the resulting transgenic tomato plants were evaluated for their tolerance levels to mixed TYLCV infection. The results suggested that amiRNAs in introns showed more effective tolerance levels than transgenic lines expressing amiRNAs in exons. Liu et al. (2021) developed transgenic wheat lines using four amiRNA expression vectors carrying vsiRNAs from wheat yellow mosaic virus (WYMV), and the laboratory and field tests showed that two transgenic wheat lines expressing amiRNA1 were highly resistant to WYMV infection. In an interesting study where the combination of the amiRNA-mediated silencing technology and the clay nanosheet-mediated delivery was employed, Liu et al. (2020) prepared plant expression vectors expressing pre-amiRNAs targeting the conserved region of the AV1 gene and a partial region of the AV2 gene of TYLCV, and recombinant plasmid DNAs were then loaded onto clay nanosheets, and these were sprayed onto virus infected plant leaves. The sprayed plants showed a significant reduction in viral infection and titers due to the accumulation of amiRNAs against AV1 and AV2 genes of TYLCV in tomato plants (Liu et al. 2020). Latif et al. (2024) developed stable transgenic Nicotiana benthamiana lines expressing amiRNA targeting P1 gene of SMV, and the results showed the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner.

Transgenic Arabidopsis plants expressing the amiRNAs targeting the 3′ untranslated region (UTR) of the CMV genome conferred high resistance to the CMV (Duan et al. 2008). Transgenic expression of an amiRNA targeting sequences encoding the silencing suppressor 2b of CMV conferred effective resistance to CMV infection in transgenic tobacco plants (Qu et al. 2007). The amiRNA-mediated virus resistance was enhanced in plants by an asymmetric bulge AB in the miRNA precursor that in turn was shown to affect the interaction between P19 protein of tomato bush stunt virus (TBSV) and the miRNA/miRNA* duplex, thus improving the TBSV resistance abilities of amiRNA (Zhang et al. 2020). Transgenic papaya lines expressing two different untranslatable chimeric constructs containing the truncated CP coding region of the PRSV and potyvirus sp., papaya leaf-distortion mosaic virus (PLDMV), respectively, showed double resistance to both the viruses (Kung et al. 2009). Fahim et al. (2012) developed an amiRNA strategy against wheat streak mosaic virus, incorporating five amiRNAs within one polycistronic amiRNA precursor of rice miR395, which was transformed into wheat under a constitutive promoter, thereby conferring viral resistance in transgenic wheat plants.

Recent studies have engineered multiple amiRNAs for simultaneous silencing of multiple genes to enhance plant antiviral resistance. Three amiRNAs containing Arabidopsis miR159 as a backbone and expressing genes targeting P25, HC-Pro and Brp1 of PVX, PVY and potato spindle tuber viroid (PSTVd), were transformed into potato plants, thereby resulted in developing resistance to multiple viruses, namely PVX, PVY and PSTVd (Jiang et al. 2023). Three dimeric amiRNA precursor expression vectors that simultaneously target the CP genes of RSV and RBSDV based on the structure of the rice miRNA namely, osa-mir528 precursor (Sun et al. 2016a, b). Assays showed degradation of viral RNAs by the action of amiRNAs, together with the secondary siRNAs, and showed that transgenes and amiRNA-mediated virus resistance could be stably inherited in the transgenic plants. In a separate study, three amiRNAs targeting CGMMV RNA (amiR1-CP, amiR4-MP and amiR6-Rep) showed reduced CGMMV replication and retarded the development of disease in virus-infected Nicotiana benthamiana plants (Liang et al. 2019a, b).

More et al. (2021) identified a new strain of a Jatropha leaf curl Gujarat virus (JLCuGV), which encoded six ORFs with each one having RNA silencing suppressor activity. So, they designed three amiRNA constructs for C1/C4, C2/C3 and V1/V2 employing overlapping regions, each targeting two ORFs of JLCuGV genomic DNA that resulted in viral resistance in transgenic tobacco plants. RNAi induced down-regulation of HC-Pro and p25 genes was obtained in transgenic Nicotiana tabacum by expression of two types of amiRNA designed using A. thaliana miR159a, miR167b and miR171a precursors as backbones. The strategy proved successful in conferring highly specific resistance against PVY as well as PVX infection under conditions of increased viral pressure (Ai et al. 2012).

Resistance to wheat dwarf virus (WDV) was observed even at low temperatures when a polycistronic amiRNA precursor construct was expressed in transgenic barley plants (Kis et al. 2016). The construct was designed to express three amiRNAs simultaneously, which were designed using a barley miRNA precursor backbone targeting different conservative sequence elements of the WDV strains. Transgenic tobacco plants resistant to cymbidium mosaic virus (CymMV) were obtained by expressing amiRNAs targeting RdRP gene of CymMV and odontoglossum ringspot virus, and containing Oryza sativa miR528 as backbone (Petchthai et al. 2018). The amiRNA strategy was extended to imparting resistance to Grapevine fanleaf virus by transient expression of amiRNA precursors targeting the viral CP gene using the pre-miR319a backbone (Jelly et al. 2012). Over-expression of rice miR171b by an amiRNA provided resistance to RSV and alleviated the disease symptoms in transgenic rice thereby leading to enhanced yield (Tong et al. 2017). The detailed information on the development of virus-resistant plants by using amiRNA-based approaches has been summarized in Table 3.

Table 3.

Development of virus-resistant plants by using amiRNA-based approaches

S. No	Virus targeted	Host plant	Target gene	References
1	Tomato yellow leaf curl virus	Tomato	AC1 and Rep	Al-Roshdi et al. (2023)
2	Potato virus x, Potato virus y and Potato spindle tuber viroid	Potato	P25, HC-Pro and Brp1	Jiang et al. (2023)
3	Tomato yellow leaf curl virus	Tomato	AMIE, AMIN, TY1	Khalid et al. (2023)
4	Plum pox virus	Nicotiana benthamiana	Nib and CP	Mesel et al. (2022)
5	Rice stripe virus	Rice	MP	Zhou et al. (2022)
6	Wheat yellow mosaic virus	Wheat	wheat thioredoxin-like (TaAAED1) gene	Liu et al. (2021)
7	Cucumber green mottle mosaic virus	Cucumber	Rep, MP, CP	Miao et al. (2021)
8	Jatropha leaf curl Gujarat virus	Tobacco	RNA silencing suppressors (RSS)	More et al. (2021)
9	Physostegia chlorotic mottle virus and Tomato brown rugose fruit virus	Tomato	L, M, G	Gaafar et al. (2020)
10	Tomato yellow leaf curl virus	Tomato	AV1 and AV2	Liu et al. (2020)
11	Tomato spotted wilt virus	Arabidopsis and Tobacco	RdRP	López-Dolz et al. (2020)
12	Tomato leaf curl New Delhi virus	Tomato	AC1	Sharma and Prasad (2020)
13	Cucumber green mottle mosaic virus	Tobacco	CP, MP, Rep	Liang et al. (2019a)
14	Cymbidium mosaic virus and Odontoglossum ringspot virus	Tobacco	RdRp	Fetchthai et al. (2018)
15	Rice stripe virus	Rice	OsSCL6-IIa, OsSCL6-IIb, and OsSCL6-IIc	Tong et al. (2017)
16	Wheat dwarf virus	Barley	Different conserved Sequences	Kis et al. (2016)
17	Rice stripe virus and Rice black streaked dwarf virus	Rice	Cp	Sun et al. (2016a)
18	Cassava brown streak virus and Ugandan cassava brown streak virus	Cassava	P1 and NIb genes of CBSV and the P1 and CP genes of UCBSV	Wagaba et al. (2016)
19	Watermelon silver mottle virus	Tobacco	Rep	Kung et al. (2015)
20	Cotton leaf curl Burewala virus	Tobacco	V2	Ali et al. (2013)
21	Turnip mosaic virus	Arabidopsis	HC-Pro	Lafforgue et al. (2013)
22	Tomato leaf curl virus	Tomato	AV1 and AV2	Vu et al. (2013)
23	Wheat streak mosaic virus	Wheat	5’UTR, pipo region of P3 cistron, P1, P3 cistron, HC-Pro	Fahim et al. (2012)
24	Grapevine fanleaf virus	Grapevine	CP	Jelly et al. (2012)
25	Cucumber mosaic virus	Tomato	2a/2b and 3’UTR	Zhang et al. (2011b)

Discussion and future prospects

Plant viral diseases continue to pose a serious threat to global agriculture, causing major yield losses and threatening food security. Advances in biotechnology have introduced RNAi and genome editing as powerful tools for managing viral infections in crops. RNAi strategies such as siRNA and amiRNA enable targeted, sequence-specific silencing of viral genes by introducing virus-derived dsRNA or designing specific amiRNAs that downregulate viral transcripts (Baulcombe 2015; Duan et al. 2008). Meanwhile, CRISPR/Cas genome editing allows precise modification of plant genomes to confer resistance, including editing viral genomes or host susceptibility factors (Zhang et al. 2018; Majumdar et al. 2023). In fact, siRNAs and miRNAs are both short duplex RNA molecules that enable gene silencing at the post-transcriptional level by targeting the desired mRNA (Rajam 2020). Despite sharing similarities, these approaches differ in certain aspects, such as their biogenesis and target specificity, which in turn influences their mechanism and therapeutic application (Lam et al. 2015).

First and foremost, siRNAs are usually synthesized exogenously, which enables them to show nearly perfect complementarity to their target, mostly a single-stranded mRNA (Rajam 2020). On the contrary, amiRNAs must be expressed from an engineered precursor transcript that mimics an endogenous miRNA. This requires careful design to ensure correct processing by the Drosha and Dicer enzymes, thereby making the biogenesis of amiRNAs a more complex process (Bravo-Vázquez et al. 2025). Secondly, with perfect or near-perfect complementarity to their target mRNA, siRNAs can achieve highly specific and complete knockdown of a single target gene (Back and Manfredi 2021). This makes them strong candidates for treat single-gene disorders. On the other hand, due to its imperfect complementarity with the target site, a single amiRNA can affect multiple genes. This makes them more potent as a knockdown tool for modulation of multiple genes within a single or multiple biological pathways. Thus, amiRNAs are valuable research tools for studying complex biological processes and hold promise for treating multigenic diseases (Teotia et al. 2023). However, this also introduces challenges in identifying the most relevant targets among the many potential ones.

Thirdly, siRNAs display an efficient mechanism of action as they are directly incorporated into the RISC complex to cleave the target mRNA (Friedrich and Aigner 2022). This bypasses the cellular processing steps required for endogenous amiRNA precursors, thereby making siRNA-mediated silencing faster and more direct in approach. Fourthly, being expressed from vectors, amiRNAs can provide more stable and long-term gene silencing compared to transiently delivered synthetic siRNAs (Bravo-Vázquez et al. 2025). For long-term effects using the siRNA approach, siRNAs must also be expressed via a vector system, such as a short hairpin RNAs (shRNAs), which are processed intracellularly into functional siRNAs (Sheng et al. 2020). Lastly, despite their high specificity, siRNAs can still cause unintended off-target effects (Knoblich et al. 2025). This occurs if the guide strand partially complements other mRNAs, mimicking the miRNA pathway, or if high concentrations are used, which can saturate the RNAi machinery. On the other hand, amiRNAs show reduced off-target effects due to its vector-based delivery. When delivered via an expression vector, amiRNA mimics the natural pathway more closely than a synthetic shRNA. Some studies have shown that amiRNAs expressed from vectors may cause less saturation of the RNAi machinery and fewer off-target effects than shRNAs expressed at high levels (Ahmad et al. 2025). To summarize, the applicability of the siRNAs and amiRNAs depends on the research or therapeutic goal. siRNAs are preferred when the aim is precise and specific knockdown of a single, well-defined gene and when a temporary effect is sufficient. This makes them ideal for studying the function of individual genes or treating disorders caused by mutations in single gene. In contrast, amiRNAs are more appropriate when the goal is to modulate multiple genes or entire pathways simultaneously, such as in complex diseases like cancer. This approach offers a broader and more sustained regulatory effect, especially when delivered via a vector.

In practice, RNAi construct design often involves creating intron-spliced hpRNA structures containing viral gene fragments, for example, CP genes or replication-related genes, placed under strong promoters such as CaMV35S. These hairpin constructs trigger post-transcriptional gene silencing (PTGS) effectively. Marker-free hpRNA constructs targeting host factors like eIF4E1 in potato have also been developed to reduce regulatory concerns and improve biosafety (Miroshnichenko et al. 2020).

Field level validations have been conducted for many crops. In rice, RNAi constructs targeting multiple viral genes of RBSDV, including RdRP and RNA silencing suppressors, have shown significant disease reduction and yield improvements in multilocation trials under natural infection pressure (Wang et al. 2016a, b). Similarly, SMV resistance has been engineered via hpRNA targeting the multifunctional P3 protein, with durable resistance observed through greenhouse and confined field evaluations (Yang et al. 2018). Despite such successes, commercialization of RNAi-based virus-resistant crops remains limited. Regulatory approval pathways demand comprehensive molecular characterization, biosafety assessments, and environmental impact studies. A notable commercial example is the Rainbow papaya, engineered for PRSV resistance, which established a precedent for RNAi-based crop deployment.

Challenges remain in the high genetic diversity and rapid evolution of plant viruses, which necessitate continuous target identification and strategy updates. Viruses being highly evolving entities, manage to escape the routine strategies designed to combat them. In such scenarios, new viruses and viroids need to be identified using the high throughput sequencing (HTS) techniques, so that effective RNAi measures can be tailor-made against their emerging menace (Maina et al. 2024; Pacheco-Dorantes et al. 2025; Sohi et al. 2025). Additionally, off-target effects and delivery efficiency of RNA molecules and genome editing reagents are technical hurdles that researchers are actively addressing through improved bioinformatics, high-fidelity Cas variants, and innovative delivery systems such as nanoparticles and viral vectors (Liang et al. 2017; Cameron et al. 2017). Besides the conventional exogenous RNAi delivery methods such as foliar spraying and mechanical application, the newer delivery systems have ensured the success of the desired RNA silencing in plants (Kumar et al. 2025; Mathur et al. 2025). These include the delivery systems such as nanoparticles, viral vectors, loop-ended dsRNA, biolistic delivery, clay nanosheet-mediated delivery and RNA-based nanocarriers, such as liposomes and carbon nanotubes (Smith et al. 2025; Mathur et al. 2025). The various nanocarriers for effective RNAi delivery include the lipid nanoparticles, polymeric nanoparticles, inorganic nanoparticles, virus-like nanoparticles, carbon nanotubes and nanogels among others (Mathur et al. 2025). Thus, exogenous RNAi offers non-transgenic, an environment friendly and sustainable alternative to conventional chemical control methods in plant disease management.

A promising frontier lies in the synergistic integration of RNAi technologies with genome editing. For example, the combination of SIGS with CRISPR/Cas systems offers a flexible and non-transgenic approach: SIGS transiently silences viral genes via topical dsRNA or siRNA applications, while the synthetic biology tool of CRISPR introduces permanent genomic edits disrupting viral integration or host susceptibility factors (Touzdjian Pinheiro Kohlrausch Távora et al. 2022). Multiplex genome editing further enables broad-spectrum resistance by targeting multiple genes involved in virus-host interactions, reducing the likelihood of viral escape mutants (Wang et al. 2019). Additionally, RNA-based nanocarriers, such as liposomes and carbon nanotubes, can enhance the delivery and durability of RNA silencing-based disease resistance in plants (Morales-Becerril et al. 2022). By encapsulating RNA molecules, the nanocarriers confer their safety from degradation by nucleases and improve their cellular uptake by mechanisms such as endocytosis (Wang et al. 2023). Furthermore, these tiny particles also ensure that the RNA molecules are delivered to the desired cells or tissues, minimizing off-target effects, leading to more effective and sustained gene silencing (Wang et al. 2023; Morales-Becerril et al. 2022).

Looking ahead, the integration of siRNA, amiRNA, VIGS, SIGS, and advanced genome editing technologies such as base editing and prime editing, holds immense potential not only for viral disease resistance but also for improving crop traits like yield, stress tolerance, and nutritional quality (Anzalone et al. 2019; Gaudelli et al. 2020; Voytas 2013). Success will depend on the international collaboration, robust regulatory frameworks, multilocation field trials to validate performance and stability, and transparent communication with stakeholders to foster acceptance.

Apart from the above involvements of RNAi in combating biotic stress, RNA-mediated gene silencing can significantly contribute to climate-resilient agriculture by enabling the development of crops with enhanced resistance to various abiotic stresses as well as improved yield (Kaur et al. 2020; Kumar et al. 2021). This approach offers a targeted and environment friendly alternative to traditional methods, potentially leading to more sustainable and productive farming practices in the face of climate change.

Conclusions

RNAi has established itself as a targeted and effective method for controlling plant viral diseases by enabling sequence-specific degradation of viral RNAs. Its application across variety of crops has demonstrated the feasibility of durable resistance through carefully designed RNAi constructs targeting viral coat proteins, replication enzymes, or host susceptibility genes (Garcia-Ruiz 2018; Majumdar et al. 2023).

However, several challenges remain. The rapid evolution of viral suppressors, genetic variability among virus strains, potential off-target effects, and regulatory hurdles continue to limit widespread commercialization. To overcome these obstacles, a multifaceted approach is essential along with clear and standardized regulatory guidelines for the development of non-transgenic RNA silencing based viral resistant plants and crops. Future research should explore the enhanced RNAi delivery mechanisms and synergistic combinations with newer disease control strategies such as CRISPR/Cas.

The future of plant viral disease management will, therefore, depend on the judicious convergence of RNA-based silencing methods such as siRNA, amiRNA, VIGS, and SIGS with precise genome editing technologies and HTS. Supported by multilocation trials, regulatory approvals, and cooperation from global scientific community, these integrated approaches hold promise to deliver virus-resistant crops that enhance global food security and contribute to sustainable agriculture.

Author contributions

MVR has conceived the concept. RK and SC written the manuscript. MVR, RK and SC revised the manuscript. All the authors approved the final manuscript.

Funding

This work was generously supported by the Department of Biotechnology, New Delhi (Grant No. BT/PR7063/PBD/16/1012/2012).

Declarations

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.