Host resistance and susceptibility to tomato leaf curl New Delhi virus: molecular mechanisms, genetic resources, and strategies for durable control

1 Introduction

Tomato leaf curl New Delhi virus (ToLCNDV) is a member of the family Geminiviridae and genus Begomovirus and has been recently reclassified as Begomovirus solanumdelhiense and It has become a major threat to vegetable production worldwide (1, 2). It has quickly expanded its geographic range throughout Asia, the Mediterranean Basin, and portions of Africa and Europe since its initial identification in tomato (Solanum lycopersicum L.) and Nicotiana benthamiana in northern India in 1995 (3, 4). ToLCNDV affects more than 40 plant species in several nations, including Italy, Malaysia, Spain, Taiwan, and Tunisia, in addition to many other Asian countries (2). The arrival of ToLCNDV in Europe signaled a major change in its epidemiology. First detected in Spain in 2012, it has affected cucurbit crops, especially zucchini and melon in addition to tomato (5). Following this introduction, the virus quickly spread throughout the Mediterranean Basin, reaching nations including Italy, Tunisia, Morocco, Greece, Algeria, France, and Portugal (6). Similar epidemics in Spain and other Mediterranean nations demonstrated its ability to adapt to new agro-climatic conditions and cropping systems. At present, ToLCNDV is widely distributed throughout Asia, North Africa, and Southern Europe, presenting a severe and ongoing threat to vegetable production systems (7). It has been documented that ToLCNDV can lead to major outbreaks in vegetable crops (8, 9).

ToLCNDV has significant and rising economic effects. Depending on the time of infection, disease occurrence, and severity, reported yield losses vary from 80% to 100% (10). Tomato plants can become infected at any growth stage. The whitefly Bemisia tabaci (B. tabaci), a polyphagous species that can spread the virus throughout several farming systems and agroecological regions, is primarily responsible for its broad distribution (11). The continuous, circular pattern of transmission enhances the epidemiological importance and effectiveness of the vector (12, 13). Mechanical transmission of a specific begomovirus, ToLCNDV-OM, has been reported, particularly through sap inoculation in laboratory investigations. However, this approach is largely ineffective and does not constitute a main transmission path for most begomoviruses (14). Recent studies indicate that ToLCNDV can be transmitted via seed and pollen under experimental conditions. High transmission rates (60%) to seedlings have been reported in zucchini, suggesting the potential for vertical transmission (15), and similar findings in tomato and cucumber further support both seed- and pollen-mediated transmission (16). However, another report suggests that seed transmission is unlikely to impact natural field conditions (17). Moreover, reservoirs for viral inoculum and secondary transmission facilitation are provided by alternate hosts, including weeds and wild plants (5).

ToLCNDV can infect many different types of plants from the families Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae, Euphorbiaceae, and Papaveraceae. High rates of recombination and mutation, which promote fast genetic modifications and adaptation to new hosts, fuel this broad range of hosts (2, 5). First detected in tomatoes, the virus has now been found in a number of economically important solanaceous crops, including chili pepper, eggplant, and potato, where it typically causes severe symptoms such as leaf curling, vein clearing, chlorosis, and stunted growth, which often result in significant yield losses (2, 4, 18). Experimental research has shown that different plant species and cultivars show varied reactions to infection. Many studies have used Nicotiana benthamiana as a model host because it is highly sensitive and promotes effective viral replication. On the other hand, crops such as Capsicum annuum show varied vulnerability based on both the plant genotype and the viral strain, thereby mirroring host-specific compatibility and adaptive interactions (2, 4). Beyond cultivated crops, ToLCNDV attacks a varied spectrum of wild and weed species, including Datura stramonium, Solanum nigrum, and Sonchus oleraceus. Alternative hosts help keep the virus active between growing seasons and help start new outbreaks by acting as reservoirs for viral persistence.

The majority of Asian isolates of ToLCNDV show significant genetic variation and cluster together in a closely related group called ToLCNDV-OM (Oriental melon strain). Despite variations in host range, geographic source, and sampling period, these isolates have approximately 94% DNA-A sequence identity. Conversely, isolates from the western Mediterranean Basin form a distinct and very homogeneous group (>99% identity among themselves but <94% identity with other global isolates), thus categorizing them as a separate strain, ToLCNDV-ES (Spain strain) (2, 4). Isolates from India and Pakistan show further divergence, with some falling below the strain delimitation threshold. Based on current data, the ToLCNDV population has at least seven genetically distinct strains, which shows that it is constantly changing and diversifying. Importantly, emerging evidence suggests that ToLCNDV-OM and ToLCNDV-ES may differ in host adaptation, pathogenicity, and resistance-breaking potential. Mediterranean ToLCNDV-ES isolates are predominantly associated with cucurbit crops and generally exhibit lower genetic diversity, whereas Asian ToLCNDV-OM isolates display broader host adaptability and greater sequence variability. Comparative transcriptomic and pathogenicity studies indicate that Indian ToLCNDV isolates induce stronger host transcriptional reprogramming and more severe disease responses than ToLCNDV-ES isolates in cucumber, suggesting possible differences in virulence and compatibility with host resistance mechanisms (19, 20). However, definitive evidence for strain-specific resistance breakdown against major resistance loci remains limited and requires further investigation using standardized multi-isolate screening systems. While phylogenetic studies point to a shared evolutionary origin between cucurbit-infecting isolates in India and those in Spain, implying possible long-distance dissemination and shared evolutionary routes (21), the emergence of isolates with 91%–92% nucleotide identity further supports active genetic variation.

ToLCNDV has a bipartite genome at the molecular level comprising two circular single-stranded DNA (ssDNA) molecules, termed DNA-A and DNA-B, each approximately 2.7–2.8 kb long (4, 22; Figure 1). From an overlapping open reading frame, DNA-A encodes the replication-associated protein (Rep/AC1), transcriptional activator protein (TrAP/AC2), replication enhancer protein (REn/AC3), coat protein (CP/AV1), and the AC4 protein. DNA-B encodes the nuclear shuttle protein (NSP/BV1), which facilitates the movement of viral DNA between the nucleus and cytoplasm, and the movement protein (MP/BC1), which allows for cell-to-cell transmission via plasmodesmata. These proteins work together to ensure effective systemic infection, thereby enabling the virus to spread from first-infected cells to distant tissues through the vascular system. Due to its compact genome structure, which includes overlapping open reading frames and multifunctional proteins, ToLCNDV can use host cell resources effectively. The capacity of the virus to influence host cellular systems, including cell cycle regulation and antiviral defense responses, is also supported by this organization, thereby improving its replicative effectiveness and pathogenic potential (23). Although ToLCNDV is a bipartite begomovirus, the DNA-A component can act in a monopartite-like manner in the presence of a DNA β satellite, where the βC1 protein complements the movement functions normally provided by DNA-B, allowing systemic infection (24). Recombination, mutation, and genome reassortment-driven genetic diversity results in the development of novel variants with altered virulence, host specificity, and environmental adaptability. The discovery of unique strains adapted to cucurbit hosts in the Mediterranean area illustrates the virus’ capacity to cross host boundaries and take advantage of new ecological niches (23). This genetic adaptability compromises the long-term efficacy of containment measures and makes disease prediction difficult, while also promoting rapid geographical spread.

2 Disease cycle and cellular pathways facilitating infection

ToLCNDV’s infection cycle consists of a series of coordinated events that allow viral entry, replication, movement, and systemic colonization of the host plant. ToLCNDV enters plants via viruliferous whiteflies eating phloem tissues. The insect acquires viral particles via its stylet within 10–60 min during feeding. After acquisition, the virus undergoes a latent period inside the vector, which usually lasts between 8 and 19 h, depending on the virus and vector species, before it can be transmitted to a new host (12). The virus takes a circulative path inside the whitefly, passing from the midgut epithelium into the hemolymph, and finally, reaching the salivary glands. The virus enters a healthy plant through the saliva during later feeding, thereby starting the infection (Figure 2).

Once inside the plant cell, viral DNA is moved to the nucleus, where it uses the host DNA replication machinery to make more copies of itself. Rep/AC1 is crucial as it alters the host cell cycle and stimulates S-phase-like conditions in differentiated cells, thereby promoting effective viral genome duplication (22). The viral genome is replicated several times through a rolling-circle process, thus advancing viral multiplication. NSP and MP mediate the intracellular and intercellular movements of the virus. NSP helps move viral DNA between the nucleus and cytoplasm, whereas MP interacts with host cytoskeletal and membrane trafficking components to allow cell-to-cell movement through plasmodesmata (26). The phloem enables long-distance movement, thereby facilitating systemic infection. Particularly, RNA-directed DNA methylation (RdDM), host defense systems target viral genomes for transcriptional gene silencing (TGS). However, viral proteins such as AC2/TrAP interfere with important components of the methylation machinery, including ARGONAUTE (AGO) 4, which reduces DNA methylation and enhances viral transcription, thereby counteracting these defenses (27). Furthermore, viral effectors such as AC2 and AC4 suppress innate immune responses, including pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), thereby enabling viral persistence and systemic dissemination (28).

3 Resistance

The most cost-effective and environmentally friendly approach to control begomovirus-induced leaf curl disease is host plant resistance. Therefore, considerable emphasis has been placed on identifying resistant germplasm, landraces, wild relatives, and breeding lines in vegetable crops (29; Table 1). Resistance to ToLCNDV is mediated through both dominant and recessive mechanisms involving major resistance genes, quantitative trait loci (QTLs), RNA silencing pathways, and host susceptibility factors. Wild Solanum species remain the main source of resistance in tomato breeding programs. Through conventional and marker-assisted breeding methods, resistance loci derived from S. chilense, S. peruvianum, S. habrochaites, and S. pimpinellifolium have been extensively used (37). Conversely, cucurbit resistance is typically polygenic, quantitative, and genotype-dependent; many resistance sources are derived from tolerant landraces and wild relatives (30). Resistant or tolerant accessions have been identified in melon, cucumber, pumpkin, squash, and Luffa cylindrica, where resistance is commonly associated with reduced viral accumulation and delayed symptom development (4, 38).

Table 1. Reported sources of resistance and genetic determinants against ToLCNDV.

3.1 Dominant and monogenic resistance

Monogenic resistance is controlled by major genes, providing strong but occasionally strain-specific protection. The Ty gene family (Ty-1, Ty-2, Ty-3, Ty-4, and Ty-6), derived primarily from wild Solanum species, is the primary source of the dominant resistance in tomatoes (39–41). Of these, Ty-1 and Ty-3 encode DFDGD-class RNA-dependent RNA polymerases (RdRp/RDRs) that increase RdDM and TGS, thereby inhibiting viral replication by increasing cytosine methylation of viral genomes (42). By contrast, Ty-2 functions through nucleotide-binding leucine-rich repeat (NB-LRR)-mediated immune reactions (43).

Several important resistance loci have also been identified in cucurbits. Accession CGN23089 has a significant QTL on chromosome 2 associated with recessive resistance in cucumber (30). More recently, the Cy-1 locus on chromosome 1 was found to encode CsRDR3, a DFDGD-class RDR functionally homologous to Ty-1/Ty-3 in tomato (31). Similar to tomato resistance genes, CsRDR3 boosts antiviral RNA silencing and RdDM-mediated defense systems using conserved catalytic domains engaged in siRNA amplification and viral genome methylation. Resistance is severely compromised by the functional silencing of CsRDR3, thereby confirming its antiviral role (31, 42).

3.2 Quantitative resistance

Quantitative resistance involves multiple loci with partial effects and is generally considered more durable because it imposes a complex barrier to viral adaptation. In melon, resistance to ToLCNDV is associated with major QTLs on chromosomes 11, 2, and 12 (30). Similarly, cucumber resistance involves several loci on chromosomes 1 and 2, indicating a multifactorial defense system comprising both major and minor genetic components (30, 31). Polygenic resistance typically results in reduced viral accumulation, delayed symptom expression, and attenuated disease severity rather than complete immunity. These traits are often associated with genes involved in RNA silencing, hormone signaling, transcriptional regulation, and other defense-related pathways (44).

A comparative analysis reveals important differences between tomato and cucurbit resistance mechanisms. Tomato resistance is largely governed by well-characterized major loci such as Ty-1/Ty-3, which provide relatively strong and broad-spectrum resistance through enhanced antiviral siRNA amplification and RdDM-mediated silencing. In contrast, cucurbit resistance is predominantly quantitative, more genotype-dependent, and potentially more vulnerable to environmental variation and strain-specific interactions (30, 42). Although Cy-1/CsRDR3 shares mechanistic similarities with Ty-1/Ty-3, the molecular pathways regulating resistance durability and viral movement restriction in cucurbits remain comparatively less understood (31). These differences suggest that breeding strategies may require gene pyramiding in tomato and stabilization of polygenic resistance in cucurbits.

3.3 Molecular mechanisms of host resistance

Host resistance to ToLCNDV involves multilayered defense systems, including RNA silencing, innate immunity, epigenetic regulation, and restriction of viral replication and movement (Figure 3). RNA silencing constitutes the primary antiviral defense mechanism. During infection, viral double-stranded RNA (dsRNA) intermediates are processed by Dicer-like (DCL) proteins into small interfering RNAs (siRNAs), which guide post-transcriptional gene silencing (PTGS) and TGS pathways to reduce viral accumulation.

RdDM plays a particularly important role against geminiviruses by targeting viral minichromosomes for cytosine methylation and transcriptional repression. Enhanced RdDM activity is strongly associated with Ty-1/Ty-3-mediated resistance (42). Simultaneously, pathogen-triggered immunity (PTI) activates reactive oxygen species (ROS) production, callose deposition, and defense-related gene expression, whereas ETI limits viral spread through hypersensitive responses and localized cell death (45). Host defenses also restrict viral replication and systemic movement by modulating plasmodesmatal permeability, interfering with viral protein activities, and limiting long-distance phloem transport (46).

3.4 Recessive resistance and susceptibility factors

Recessive resistance results from the alteration or loss of host susceptibility (S) genes required for viral infection and replication (47). Unlike dominant resistance based on R–Avr interactions, recessive resistance disrupts compatible host–virus interactions and is often more durable. In cucurbits, important recessive loci have been identified on chromosomes 11, 12, and 2 in melon and chromosomes 7 and 8 in pumpkin (Cucurbita moschata) (33, 48). Molecular markers linked to pumpkin resistance loci show prediction accuracies approaching 94%, supporting their utility in marker-assisted breeding. In cucumber, inheritance studies involving the resistant genotype DC-70 demonstrated monogenic recessive resistance with segregation ratios consistent with single-gene control (49). At the molecular level, the recessive resistance allele ty-5 corresponds to a mutation in the pelota gene, which affects translation and viral replication efficiency (41, 50, 51). Geminiviruses also exploit the host replication machinery through interactions between viral proteins and cellular factors such as POLA2 and retinoblastoma-related protein (RBR), which promote viral DNA replication and cell-cycle reprogramming (22, 52, 53).

Autophagy-related pathways further influence susceptibility. ATG8 proteins may either promote antiviral defense through degradation of viral proteins or be hijacked to facilitate infection (54). In ToLCNDV, interactions between SlATG8f and viral TrAP (AC2) suggest a role for autophagy in antiviral defense, although detailed mechanistic validation remains limited (55). Similarly, eukaryotic translation initiation factors (eIF4E and eIF4G) represent conserved susceptibility factors across plant–virus systems (47). However, functional validation of many candidate susceptibility factors in ToLCNDV-specific systems remains limited.

4 Viral counter-defense strategies in ToLCNDV

Recent developments in plant virology have revealed that the outcome of ToLCNDV infection is determined by a complex molecular interaction between host antiviral defense mechanisms and viral counter-defensive tactics. Through rapid mutation, recombination, and interactions with satellite molecules that boost virulence and weaken host immunity, ToLCNDV has evolved complex counter-defense systems. These strategies function on several levels, viz., RNA silencing suppression, transcriptional reprogramming, hormone control, and epigenetic interference (Table 2; Figure 4).

Table 2. Host defense mechanisms and viral counter-defense strategies in ToLCNDV–host interactions.

4.1 Silencing suppressors

Several ToLCNDV-encoded proteins, including AV2, AC2 (TrAP), AC4, and βC1 (associated with betasatellites), function as viral suppressors of RNA silencing (VSRs) (2, 62). These proteins interfere with key components of the host RNA silencing machinery, including DCL enzymes, AGO proteins, and RNA-induced silencing complexes (RISC), thereby preventing the degradation of viral nucleic acids (64, 65). Among these, AC2 is a central regulator that suppresses both PTGS and TGS. It obstructs components of the DNA methylation pathway, thereby opposing epigenetic silencing of the viral genome, and interferes with AGO1-mediated RISC activity. AC4 works by binding small RNAs and preventing their integration into the RISC complex, whereas AV2 interferes with RDR1-mediated antiviral siRNA amplification. These proteins work together to reduce host resistance and enhance viral replication and systemic infection (66, 67).

4.2 Manipulation of host transcription and hormonal networks

Infection with ToLCNDV causes significant changes in the transcription and hormone networks of the host. In cucumbers, in particular, transcriptomic studies have shown major alterations in the expression of genes linked to defense and genes implicated in metabolism and protein synthesis (20). The AC2 protein acts as a transcriptional regulator, thereby changing host gene expression to promote viral replication and inhibit immune responses (62). Furthermore, ToLCNDV interferes with hormone signaling networks, especially those involving jasmonic acid and ethylene, thereby compromising plant immunity. Changes in these pathways compromise the plant’s capacity to mount strong immune responses (68). The virus interferes with RdDM, which reduces methylation of its DNA and keeps transcription active (62). Variations found in the 63-nt area of the C-terminal domain of the TrAP/REn of ToLCNDV alter interactions with important host proteins such as PCNA and AGO1, thereby regulating infection efficiency and facilitating changes in host range. These results imply that susceptibility is not controlled by a single gene but rather relies on dynamic virus–host interactions that regulate viral accumulation, symptom expression, and host adaptation (69).

4.3 Recombination, evolution, and satellite effects

A major evolutionary force driving the development of new ToLCNDV variants with increased adaptability, virulence, and the capacity to overcome host immunity is recombination. Closely related begomoviruses or different ToLCNDV strains can exchange genes, creating new genomic configurations. These changes can lead to altered pathogenicity determinants, a wider range of hosts, and increased fitness under different environmental conditions (70).

In addition to genomic recombination, ToLCNDV’s pathogenicity and ability to overcome resistance are highly influenced by its interactions with related DNA satellites, especially betasatellites and alphasatellites. These subviral compounds are important in modulating disease severity, viral accumulation, and host–virus interactions. Data show that whereas the tomato leaf curl virus-encoded C4 protein imparts the same movement capability to ToLCNDV DNA A (24), βC1 encoded by DNA β satellites may replace DNA-B activities to allow systemic infection. Recent research has revealed that the presence of betasatellites notably increases viral DNA accumulation and accelerates disease development, with viral titers usually correlating more strongly with betasatellite abundance than with the concentration of the helper virus itself (71, 72).

Additionally, growing evidence indicates that ToLCNDV can interact with several betasatellites simultaneously, leading to complex interactions that affect replication efficiency, disease expression, and infection dynamics. These multi-satellite interactions can cause diverse viral populations to form within a single host, thereby increasing adaptability and facilitating the development of more virulent or resistance-breaking mutations (70). Specifically, betasatellites help the virus overcome host defense systems and infect previously resistant types, thereby promoting resistance breakdown. By contrast, alphasatellites operate more subtly and modulatively within the virus-satellite complex. Although they can replicate autonomously, they do not encode their own Rep. By inhibiting host gene silencing mechanisms, alphasatellites can indirectly promote viral survival and reproduction (73). However, by lowering betasatellite accumulation and competing for host replication resources, they frequently have an opposing effect on disease–severity (74). This rivalry may result in reduced viral loads and diminished symptoms in some host–virus combinations. Research, especially on ToLCNDV, indicates that alphasatellites are generally maintained at relatively low levels and that their survival is substantially reduced in the presence of betasatellites, thereby suggesting a competitive hierarchy within the virus-satellite complex (75). This hierarchy favors betasatellites, which drive disease severity and control infection dynamics. Therefore, although alphasatellites can alter infection outcomes, their influence is typically subordinate to the strong effects of betasatellites.

5 Management strategies for ToLCNDV

Controlling ToLCNDV is particularly challenging due to the absence of direct antiviral treatments in plants; therefore, reliance on preventative approaches is vital. The main goal of current control methods is to keep whitefly population numbers low by using a combination of approaches, including deploying physical barriers such as insect-proof nets, using biological controls, implementing cultural practices, and applying insecticides judiciously. The removal of infected plants is also essential for lowering inoculum pressure. However, because of variables such as the rapid development of insecticide resistance in B. tabaci, environmental issues associated with chemical control, and the efficiency of virus transmission even at low vector densities (76), these approaches frequently have only limited success under field conditions. Although vector suppression and sanitation practices continue to be important, these measures alone seldom provide long-lasting defense against strong disease threats. Therefore, the development of new and cutting-edge management technologies is essential for long-term ToLCNDV suppression (Table 3).

Table 3. Major research advances and emerging priorities in ToLCNDV biology, resistance, and management (2020–2026).

5.1 Host resistance breeding

For managing ToLCNDV, the use of resistant cultivars remains the most eco-friendly and cost-effective method. Resistance to begomoviruses has been studied extensively in tomato, primarily using wild Solanum species, especially S. chilense, S. habrochaites, and S. pimpinellifolium. Ty-1 and Ty-3, two major loci encoding RdRp involved in TGS, have exhibited broad-spectrum activity against various begomoviruses and limited efficacy against ToLCNDV (42). The recessive allele ty-5, associated with the mutation of the pelota gene, also confers resistance by limiting viral replication efficiency (41). Although symptom escape and strain-dependent variability have been reported (40), field studies in India showed that tomato lines carrying Ty loci had substantially lower disease incidence and lower viral titers than vulnerable types under natural whitefly pressure.

Specifically in cucurbit crops, significant advances have been made in identifying resistant germplasm and mapping QTLs linked with resistance. These developments have accelerated the development of resistant varieties and facilitated the implementation of marker-assisted selection (86). Resistance in cucurbits is generally quantitative and genotype-dependent rather than controlled by single dominant genes. Several melon accessions exhibited moderate to strong resistance, typified by delayed symptom expression and lowered viral accumulation, whereas QTLs linked with resistance were detected on chromosomes 11, 2, and 12 (30). The recently identified Cy-1 locus encoding CsRDR3, a DFDGD-class RdRp, conferred strong resistance in cucumber; silencing of CsRDR3 restored susceptibility, thereby confirming its functional involvement in antiviral defense (31).

5.2 CRISPR/Cas-mediated resistance

Genome editing represents a promising approach for developing resistance to ToLCNDV. Particularly aimed at genes necessary for replication or pathogenicity, CRISPR/Cas systems can be developed to target conserved viral sequences (84). CRISPR/Cas9 constructs targeting replication-associated genes reduced viral accumulation in transgenic plants infected with related geminiviruses, thereby demonstrating the viability of this technique for DNA viruses (87). Although comprehensive, validated ToLCNDV-specific field applications remain limited, similar techniques are readily transferable because of the conserved genome architecture. Simultaneous multiplex CRISPR targeting of several viral regions could lower the risk of escape mutants. In addition to direct viral targeting, editing host susceptibility genes implicated in replication or intracellular trafficking may offer recessive, broad-spectrum resistance (87, 88). Because ToLCNDV is a bipartite ssDNA virus that reproduces in the nucleus via double-stranded DNA intermediates, it is particularly receptive to DNA-targeting CRISPR systems, including Cas9 and Cas12a (89). The first demonstrations of CRISPR-mediated antiviral defense against geminiviruses showed that transgenic Nicotiana benthamiana and Arabidopsis thaliana expressing Cas9 with guide RNAs targeting viral intergenic regions, coat protein genes, or Rep genes displayed marked reductions in viral accumulation and symptom severity (90). A recent CRISPR/Cas9 study targeting Chilli leaf curl virus (ChiLCV) in Nicotiana benthamiana showed that single and multiplex guide RNAs lowered viral DNA accumulation; dual-guide constructs consistently outperformed single-guide systems. The most effective dual combination targeting CP and Rep genes reduced viral titer by 49.3% at 4 dpi and 47.9% at 20 dpi, while also decreasing symptom severity and producing precise edits in the viral genome (91). The utilization of Cas12a (Cpf1) and related nucleases presents another encouraging path. Cas12a generates staggered cuts, recognizes different PAM sequences, and can process multiplex guide arrays more efficiently than Cas9. These characteristics may increase targeting adaptability across different ToLCNDV strains. Early research on other plant DNA viruses indicates that Cas12a possesses strong antiviral capabilities and may complement Cas9-based systems (92).

Despite the considerable promise of CRISPR/Cas-mediated antiviral resistance, unintended off-target genome editing remains an important concern. Off-target mutations may alter endogenous plant genes, potentially affecting growth, development, stress responses, or agronomic performance. The risk of unintended edits can increase when guide RNAs target conserved genomic regions with partial sequence similarity elsewhere in the host genome. Therefore, careful guide RNA design, the use of high-fidelity Cas variants, whole-genome validation approaches, and rigorous molecular screening are necessary to improve editing precision and biosafety. In addition, long-term evaluation of genome-edited plants under field conditions is required before large-scale deployment of CRISPR-based resistance strategies against ToLCNDV (93).

5.3 RNA silencing-based strategies

For both control and functional analysis of ToLCNDV, RNA silencing-based technologies have proven to be highly effective instruments. By exploiting the plant’s natural RNAi machinery, these methods either inhibit viral genes or momentarily silence host genes implicated in resistance or vulnerability. Direct antiviral defense is provided by Host-Induced Gene Silencing (HIGS) using transgenic or dsRNA-mediated techniques, while Virus-Induced Gene Silencing (VIGS) primarily serves as a rapid reverse-genetics tool for confirming host genes and accelerating resistance breeding.

HIGS involves plants expressing dsRNA homologous to essential viral genes, which is processed into siRNAs that degrade viral transcripts or promote transcriptional silencing during infection. This approach is particularly relevant for begomoviruses such as ToLCNDV, which encode multifunctional proteins necessary for replication, movement, and suppression of host defense. Unlike ToLCNDV, Tomato leaf curl Gujarat virus (ToLCGV) caused symptom remission by activating host antiviral silencing mediated by NtRDR1 (Nicotiana tabacum-RNA-Dependent RNA Polymerase 1), which led to increased methylation of the viral promoter and subsequent plant recovery. This was shown in a comparative study of tobacco. Conversely, the AV2 (pre-coat) protein of ToLCNDV inhibited NtRDR1-dependent defense, allowing the development of severe disease and exposing a host–virus molecular arms race regulating symptom remission (66). A ToLCNDV investigation revealing that the viral suppressor proteins AV2, AC2, and AC4 also function as key pathogenicity determinants provided further proof for RNAi-based control. Hairpin RNAi-mediated knockdown significantly decreased viral titers and symptom severity in Nicotiana benthamiana. Notably, exogenous application of dsRNA targeting these genes, especially a mixed dsRNA (dsCk) and dsAC4, substantially reduced viral load and disease severity, demonstrating the potential of a non-transgenic RNA-based management approach for ToLCNDV (62). A related subcellular localization study showed that AV2 localizes to the cell membrane and nucleus, AC2 predominantly to the nucleus, and AC4 mainly to the cell membrane, suggesting coordinated interactions with host regulatory pathways that suppress antiviral RNA silencing (94).

VIGS is primarily a functional genomics technique; however, it has significant indirect utility for ToLCNDV management because it rapidly signals resistance genes, susceptibility variables, and defense mechanisms. VIGS vectors, usually based on tobacco rattle virus (TRV) or related systems, carry fragments of host genes that trigger transient silencing within days after inoculation. This makes VIGS especially useful in tomato, Nicotiana benthamiana, and cucurbit species, where stable transformation can be slow or genotype dependent (95). VIGS has been widely used in begomovirus research to investigate the functions of components of the RNA silencing machinery (96). Many studies have focused on the roles of genes encoding RDRs, DCLs, AGOs, receptor-like kinases, and transcription factors in antiviral defense (97). VIGS has been especially helpful in validating resistance loci identified through transcriptomics and QTL mapping in ToLCNDV pathosystems. The Cy-1 resistance locus in cucumber is a prominent example; transient suppression of CsRDR3, a DFDGD-class RdRp, increased the susceptibility of resistant plants to ToLCNDV, resulting in greater disease severity and higher viral accumulation than in non-silenced controls (31).

Non-transgenic RNA-based protection strategies, including spray-induced gene silencing (SIGS), are emerging as environmentally friendly alternatives. Local or systemic RNA silencing reactions can be induced through the exogenous application of dsRNA molecules targeting viral genes that are taken up by plant tissues. In various plant-virus systems, dsRNA sprays lowered viral titers by 40%–80% and slowed the onset of symptoms. In theory, dsRNA molecules targeting Rep or AC2 should be effective against ToLCNDV because these genes are both crucial and highly conserved. Improving RNA stability under field conditions through formulation technologies remains a research focus (98).

Despite these advances, the durability of resistance remains an issue. Strong selection pressure exerted by resistant cultivars might result in the development of resistance-breaking viral types (99). Therefore, relying on host resistance alone might not provide long-term defense. Integrating resistant varieties with complementary disease control techniques, such as vector control, crop rotation, and the removal of alternative host reservoirs, represents a more sustainable approach (39, 100). Integrated management systems are essential for limiting the spread of ToLCNDV and ensuring consistent crop yield in affected locations.

6 Future perspectives

ToLCNDV has emerged as one of the most economically detrimental plant viruses. Long-term control is especially challenging because of its rapid geographic expansion, wide host range, effective transmission by B. tabaci, and substantial evolutionary potential through recombination and mutation (2, 4, 11). Although considerable progress has been made in understanding the molecular biology, epidemiology, and host resistance of ToLCNDV, many important scientific and practical challenges remain unresolved.

6.1 Durable resistance breeding and genetic improvement

The development of broad-spectrum and long-lasting host resistance remains a major priority for ToLCNDV management. However, currently available resistance sources in tomato and cucurbit crops are often incomplete, dependent on the genotype, and vulnerable. Ty-genes studied from wild Solanum sp provide resistance to several begomoviruses, but their efficacy against ToLCNDV varies depending on the viral isolate and host genetic background (40, 42). Likewise, resistance in cucumber and melon is largely quantitative and controlled by several loci with modest effects rather than by total immunity (30, 31).

Resistance breeding is further complicated by strain-specific heterogeneity. In terms of host range, virulence, and symptom expression, Mediterranean ToLCNDV-ES isolates vary from Asian isolates. Comparative studies have shown that different strains cause distinct disease reactions in cucumber, underscoring the need to assess breeding materials against several representative isolates (4, 20). Therefore, breeding approaches should emphasize gene pyramiding, combining major resistance genes with minor QTLs, aided by marker-assisted selection, genomic prediction, and speed breeding. The use of landraces and wild relatives will remain essential for expanding the resistance gene pool.

6.2 Omics-based approaches for deciphering the molecular mechanisms of ToLCNDV infection

Modern omics technologies offer unprecedented opportunities to understand the molecular basis of ToLCNDV pathogenicity and host defense. Of these, transcriptomics has already provided valuable insights into differential host responses to different strains of viruses. We compared RNA-seq data from ToLCNDV-India and ToLCNDV-ES infections in cucumber and found that the Indian isolate regulated approximately 10 times more differentially expressed genes than the ES strain, particularly in the pathways of defense signaling and flavonoid biosynthesis, which may contribute to more severe symptoms and greater host compatibility at the molecular level (20). Future progress will likely depend on multi-omics integration. Genomics can identify resistance genes, QTLs, and viral adaptive mutations; transcriptomics can reveal hormone signaling, RNA silencing, and stress-responsive pathways; proteomics can uncover interactions between viral proteins such as AC2, AC4, AV2, NSP, and MP and their host targets; metabolomics can track shifts in flavonoids, sugars, amino acids, and phytohormones during infection; epigenomics can characterize RdDM, chromatin remodeling, and defense priming; and small RNA omics can profile antiviral siRNAs and regulatory miRNAs involved in host immunity. Together, these approaches can generate systems-level models of susceptibility and resistance.

Single-cell transcriptomics and spatial omics are particularly promising, as they can reveal tissue-specific responses in phloem-associated cells where begomoviruses replicate and move. These technologies could uncover hidden defense niches, barriers to cell-to-cell movement, and early infection signatures that may be obscured by bulk tissue analyses. Their application in plant virology remains limited but represents a major research frontier (101).

B. tabaci remains the primary driver of ToLCNDV epidemics. However, B. tabaci is a cryptic species complex, and its different members vary in transmission efficiency, insecticide resistance, fecundity, and climatic adaptability. This variation may help explain differences in disease outbreaks across regions and production systems (12, 102). Future integrated research combining vector genomics, microbiome analysis, and behavioral ecology should improve the prediction of virus distribution. Climate change adds further complexity to ToLCNDV epidemiology due to increased temperature and rainfall fluctuations, milder winters, and more protected horticulture, all of which favor whitefly population growth and prolong the period during which transmission can occur. These climatic changes are likely to promote earlier seasonal outbreaks and expansion into regions that have historically been suitable for whitefly activity in Mediterranean and subtropical environments. Climate-based predictive models incorporating weather data, vector populations, and crop growth parameters could greatly assist in the development of improved early-warning systems (103).

It is unlikely that any single preventive measure will provide long-term control for ToLCNDV. Achieving long-term, sustainable management of the disease will require the use of multiple integrated disease management approaches, including the use of resistant varieties, certified clean seed, rapid diagnostic tests, vector control, management of alternate weed hosts, crop sanitation, the use of pest-free production inputs, and a coordinated area-wide surveillance program to monitor ToLCNDV activity in affected regions. By removing infected plants and managing weed hosts between cropping seasons, the causal pathogen that leads to disease (inoculum pressure) can be diminished. Furthermore, the use of row covers and reflective mulches has been shown to provide some degree of protection against high levels of whiteflies during periods of elevated insect pressure (104). The development of portable diagnostic tools will allow growers to more rapidly identify infected plants in the field and enable more precise decision-making for disease control. These diagnostic tools, in combination with genomic monitoring, will support more responsive disease management at both farm and regional levels (Table 4).

Table 4. Emerging omics and advanced technologies for understanding and managing ToLCNDV.

6.3 Future research gaps and emerging challenges

Although substantial progress has been made in comprehending the epidemiology of ToLCNDV, host interactions, and resistance mechanisms, several significant knowledge gaps remain unaddressed. First, the molecular determinants driving strain-specific pathogenicity and host adaptation between ToLCNDV-OM and ToLCNDV-ES remain inadequately defined. Particularly in terms of viral factors responsible for varied virulence, host range expansion, and resistance-breaking potential, comparative functional genomics investigations are still scarce. Second, although numerous resistance loci have been identified in tomato and cucurbit crops, the toughness and stability of these resistance mechanisms under varied environmental settings and mixed-virus infections remain poorly understood (31). In cucurbits especially, resistance is typically quantitative and highly affected by genetic background, which complicates marker-assisted breeding and resistance pyramiding (30). Third, the mechanistic foundation of host antiviral immunity against ToLCNDV requires further research. Although RNA silencing, RdDM, autophagy, and hormone-mediated defense mechanisms have been implicated, the dynamic crosstalk among these pathways during infection remains largely unknown. Further functional validation is especially needed to clarify the exact molecular interactions between VSRs and host immune controls.

Vector–virus–host interactions represent another major research gap. The effects of climate change, environmental stress, cryptic species variations, and whitefly population dynamics on ToLCNDV transmission effectiveness and epidemic development requires further investigation. Likewise, more thorough research is needed on the role of mixed infections and virus–virus synergy in influencing illness severity, host adaptation, and viral evolution (82). Although multi-omics methods, RNAi-based tactics, and CRISPR/Cas systems have shown strong potential, their practical application at the field level and sustainable disease management remains limited. Long-term biosafety, viral escape mutations, regulatory restrictions, and genotype-dependent efficacy continue to pose major translational application challenges. Therefore, future research should focus on developing integrative strategies combining comparative genomics, functional virology, AI-assisted breeding, high-throughput phenotyping, and multi-environment validation of resistance sources.

7 Conclusion

ToLCNDV has become a highly destructive and adaptable virus that affects vegetable production, especially across diverse agricultural ecosystems. Fortunately, significant progress has been made in understanding how ToLCNDV functions at the molecular level, including how the host defends itself against ToLCNDV through RNA silencing, epigenetic regulation, and resistance-gene expression. Wild tomato and cucurbit species have been identified as sources of resistance to ToLCNDV, and mapping of major loci and quantitative traits has helped accelerate the development of disease-resistant plants using molecular breeding techniques. However, the most common type of resistance is partial or strain specific and will generally fail or break down if the population of ToLCNDV infecting them undergoes high selection pressure for many generations. Therefore, long-term solutions for controlling ToLCNDV must be developed through improved and multidimensional approaches. In order to manage ToLCNDV in the future, integrated approaches combining host resistance, vector management, clean seed programs, rapid diagnosis capabilities, surveillance, and improved agronomic practices will be essential. Additionally, new technologies in molecular biology will provide new opportunities to enhance resistance to ToLCNDV and predict disease outbreaks. In conclusion, the implementation of scientific knowledge discovered at the molecular level into successful control of ToLCNDV will require a sustained commitment to continuous vigilance against a rapidly evolving viral threat.

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Footnotes

Edited by: Ravi Ranjan Kumar, Bihar Agricultural University, India

Reviewed by: Vinod Kumar, Bihar Agricultural University, India

Kumari Rajani, Bihar Agricultural University, India