Subcellular mapping of ToBRFV movement protein in Nicotiana glutinosa via confocal laser scanning microscopy and agroinfiltration

Movement proteins (MPs) are essential for plant virus spread, mediating cell-to-cell transport through plasmodesmal connections. A plethora of studies have been reported for molecular characterization of Tobamovirus fructirugosum (tomato brown rugose fruit virus, ToBRFV) and its interactions with the host plants, however subcellular localization of the ToBRFV-movement protein (ToBRFV-MP) has not been reported in any host to date. Here, we investigated the subcellular localization of 266 amino acid long ToBRFV-MP in Nicotiana glutinosa using a transient expression system. The expression construct comprising N-terminal cMyc-tagged MP fused to cyan fluorescent protein (ToBRFV-MP-CFP) at its C-terminus via a five-amino acid linker sequence (GGGGS), and driven by CaMV 35S promotor was cloned into pCAMBIA2300 binary vector for the expression of recombinant fusion protein, and designated as pCAM-ToBRFV-MP-CFP. This construct was introduced into Agrobacterium tumefaciens LBA4404 for in planta expression through standard agroinfiltration assay. The expression and integrity of about 59 kDa fusion protein was confirmed in Western blot analysis using anti-cMyc tag antibody. The localization of MP-CFP fusion protein to inner cell periphery was suggested by the cyan fluorescence, in a distinct punctate pattern, under confocal laser scanning microscopy. Subsequent aniline blue staining demonstrated the co-localization of MP-CFP fluorescence alongside the plasmodesmata-associated callose deposits, supporting plasmodesmal localization of movement protein. These findings demonstrated the first experimental evidence for the plasmodesmata-associated localization of the ToBRFV-MP, and was in support of its proposed role in inter- and intracellular movement. By establishing this transient expression system, we have created conditions for analyzing the behavior of viral movement proteins in diverse plant species and gained insight into their role in the trafficking of viruses through plasmodesmata.

In order to spread within the host, plant viruses need to enter the cell through the cell wall. To do that, the viruses use certain movement proteins that are capable of transporting them from one cell to another through plasmodesmata (1–3). Since the discovery of movement proteins (MPs) in tobacco mosaic virus, their role in determining the host range and viral diseases as well as their implication in establishing mechanisms of viral infection has been confirmed (4). MPs increase the size-exclusion limit of plasmodesmata and interact with a variety of cellular receptors to target viral genomes and complete virus particles to neighboring cells (5). It is noteworthy that a variety of subcellular localizations of viral MPs have been identified due to the protein’s multiple functions (6). In particular, MPs have been localized in the endoplasmic reticulum, cytoskeleton, and plasma membrane (7). However, the most common location is the cell fringe, where MPs accumulate in the form of distinctive clusters at plasmodesmata, which serve as the hallmark of their activity (8).

A serious threat to tomato cultivation on a worldwide scale is the recently detected tomato brown rugose fruit virus (ToBRFV, genus Tobamovirus, species Tobamovirus fructirugosum). This virus is highly virulent and can infect and spread rapidly in tomato plants despite the presence of resistance genes (9, 10). However, despite the fact that much research has been done to understand the epidemiology and virology of the virus (11), limited information is available regarding the movement of the ToBRFV’s MP within the plant. Given that the location of MP determines how the virus moves throughout the plant and the level of infectivity it causes, figuring out where exactly the ToBRFV-MP can be found is critical.

The use of Agrobacterium -mediated transient expression, or standard agroinfiltration method, provides a fast and reliable method for investigating how proteins are located and function in plants, particularly in Nicotiana species (12). When combined with fluorescent protein tagging and confocal laser scanning microscopy, this technique makes it possible to observe recombinant proteins in living plant cells in real time, and has become a standard approach for studying the movement of viral proteins within cells (13; 14). Furthermore, checking for co-localization with markers associated with plasmodesmata can further confirm the targeting of viral proteins and improve the interpretation of fluorescence-based studies.

In present study, we engineered a recombinant construct in which ToBRFV-MP was fused to cyan fluorescent protein (CFP) at its C-terminus using a flexible “GGGGS linker sequence”. This fusion construct, uplifted in Agrobacterium tumefaciens LBA4404 strain, was then transiently expressed in N. glutinosa plants by agroinfiltration. The presence of ToBRFV-MP was confirmed by SDS-PAGE and Western blot analyses, before performing the localization studies. Confocal laser scanning microscopy was used to determine the intracellular location of MP-CFP. While, aniline blue staining of callose deposits was employed to examine its association with plasmodesmata. To our knowledge, this is the first study to experimentally demonstrate the subcellular localization of ToBRFV-MP using CFP as visual marker, providing a basis for future research into its intracellular movement, interaction with host cells, and its role in viral transmission.

Plant material

Healthy seeds of N. glutinosa were grown in pots having sterile soil-peat mixture (2:1 ratio) in controlled conditions with a 16/8-hour light/dark photoperiod, 25 ± 2 °C ambient temperature, and humidity range of 50-60%. Light intensity was maintained at 100-150 µmolm − ²s − ¹, supporting optimal vegetative growth and efficient agroinfiltration-based transient expression as suggested by Norkunas et al. (15). Plants were watered at regular interval and maintained under pest-free conditions. Uniform healthy seedlings of 4–5 leaf stages (40–45 days post sowing) were used for agroinfiltration of fully expanded 2–3 leaves (16). These growth conditions and plant developmental stage are consistent with established agroinfiltration protocols and were adopted to ensure reproducible transient expression and reliable downstream analysis.

Vector construction and cloning

The coding DNA sequence of ToBRFV-MP (GenBank accession NC_028478) retrieved from the publicly available NCBI databases was used for designing and development of chimeric plasmid. The expression construct was designed as a single open reading frame comprising an N-terminal c-Myc epitope tag fused in-frame to MP coding sequence (without stop codon) of ToBRFV, a glycine-rich linker sequence (GGGGS), and a C-terminal cyan fluorescent protein (CFP) tag, all driven by a CaMV 35S promotor with a ATG initiation codon at N-terminus (17). The construct, flanked by EcoR I and BamH I restriction sites, was commercially synthesized and delivered as a lyophilized plasmid (4 µg DNA), cloned into the pCAMBIA2300, and designated as pCAM-ToBRFV-MP-CFP (18). The c-Myc epitope was incorporated to facilitate potential downstream immunodetection and biochemical analyses, including Western blotting and immunoprecipitation in future functional studies. Recombinant plasmid was transformed into chemically competent Escherichia coli DH5α cells and the positive transformants were screened by colony PCR with MP gene specific primers (MP_F: 5’-ATG GCT CTT GTT AAG GGT AAA GTC-3’; MP_R: 5’-TTA ATA CGA ATC TGA ATC GGC GAC G-3’) and confirmed by restriction digestion with EcoR I and BamH I endonucleases. Integrity and correct orientation of genes in the recombinant plasmid was confirmed by sequencing (Genei Pvt. Ltd., India).

Transformation of recombinant plasmid into Agrobacterium tumefaciens

The recombinant pCAM-ToBRFV-MP-CFP (harboring ~1.5 kb insert of fusion protein with c-Myc-MP-linker-CFP) was subsequently introduced into A. tumefaciens LBA4404 by freeze-thaw method (19) Chemically competent LBA4404 cells were transformed with the recombinant construct and allowed to recover in Yeast Extract Broth (YEB) at 28 °C for 2–3 h with gentle shaking (200 rpm). Transformed cells were plated onto YEB agar supplemented with kanamycin (50 mg/L), streptomycin (250 mg/L), and rifampicin (50 mg/L), and incubated at 28 °C for 48 h. Putative transformants were screened to ascertain the presence MP gene by colony PCR using ToBRFV-MP specific primers. Such a confirmed transformants in A. tumefaciens were subsequently used for agroinfiltration assays.

Agroinfiltration procedure

A. tumefaciens LBA4404 harboring pCAM-ToBRFV-MP-CFP was cultured overnight in YEB medium (20), supplemented with kanamycin (50 mg/L), streptomycin (250 mg/L), and rifampicin (50 mg/L) at 28 °C with 200 rpm shaking. The grown secondary culture was pellet down at 6000 x g for 10 min at 4 °C and resuspended in 10 mM MES buffer (pH 5.6) supplemented with 10 mM MgCl2 and 100 µM 3’,5’-dimethoxy-4’-hydroxyacetophenone (acetosyringone) to an OD 600nm of 0.8 and rest at room temperature for 2 h for bacterium activation. N. glutinosa seedlings of 4–6 leaf stage were used for agroinfiltration at abaxial leaf surface deploying a needleless syringe. This developmental stage is optimal for transient gene expression studies, as younger fully expanded leaves exhibit higher transformation efficiency, improved infiltration capacity, and more consistent transgene expression across tissues (15). Care was taken to apply gentle pressure to avoid tissue damage and plants were maintained on above mentioned growth conditions to monitor the transient expression (21).

Protein extraction, SDS-PAGE, and Western blot analysis

To observe the expression and verify the integrity of ToBRFV-MP-CFP, total soluble proteins were extracted from N. glutinosa leaves, 72 h post-agroinfiltration (hpi). Approximately 100 mg of agroinfiltrated leaf tissue was homogenized in protein extraction buffer [50 mM Tris-HCl (pH 7.0), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM DTT, and a protease inhibitor cocktail]. The homogenate was centrifuged at 10,000 rpm, 10 min at 4 °C to remove cellular debris, and the resulting supernatant was collected as the total soluble protein fraction. Protein concentration was determined using the Bradford assay (22).

Equal amounts of total leaf protein (30 µg per sample) were subjected to SDS-PAGE and Western blot analysis following the previously standardized procedure (23). Briefly, protein samples were denatured and separated on a 10% SDS-polyacrylamide gel. Following electrophoresis, proteins were visualized by Coomassie Brilliant Blue staining and electrotransferred onto a 0.45 µm polyvinylidene fluoride (PVDF)-membrane using a wet-transfer system (24). The membrane was blocked with 5% (w/v) non-fat skim milk prepared in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and subsequently incubated overnight at 4 °C with anti-c-Myc primary antibody (Thermo Fisher Scientific Pvt. Ltd., India). After three generous washes with TBST (10 min each), the membrane was incubated with the corresponding HRP-conjugated secondary antibody (Thermo Fisher Scientific Pvt. Ltd., India) for 1 h at room temperature. Following three additional washes with TBST, immunoreactive protein bands were detected using an enhanced chemiluminescence (ECL) detection reagent according to the manufacturer’s instructions, and images were captured using a chemiluminescence imaging system (Thermo Fisher Scientific Pvt. Ltd., India).

Aniline blue staining for plasmodesmata visualization

To examine the association of ToBRFV-MP with plasmodesmata, N. glutinosa leaves transiently expressing the MP-CFP fusion protein were collected at 72 h post-agroinfiltration and cleared in 95% ethanol until chlorophyll was completely removed and then stained with 0.05% (w/v) aniline blue prepared in 0.07 M K 2 HPO 4 buffer (pH ~9.5) for 60 min in the dark at room temperature. Following staining, leaf tissues were mounted on glass slides in fresh staining solution and immediately subjected to confocal laser scanning microscopy following the protocol described elsewhere (25).

Confocal laser scanning microscopy

Subcellular localization of ToBRFV-MP-CFP fusion protein was analyzed using a Leica confocal laser scanning microscope (Leica Microsystems, India). Tissue sections of about 0.5 to 1.0 cm² in size were cut with the help of a cork borer from infiltrated areas of N. glutinosa leaves and placed on glass slides in 20% glycerol. CFP fluorescence was excited using 440 nm light, with emission detected in the 470 to 500 nm range. Aniline blue fluorescence was excited with a 405nm diode laser, and emission was detected at 450–530 nm to visualize callose deposits associated with plasmodesmata. Chlorophyll fluorescence was excited at 633nm and detected at more than 650nm emission when required. To prevent crosstalk between different channels, each image was acquired in sequential scanning mode. The spatial overlap between MP-CFP signals and aniline blue-stained plasmodesmata was assessed by creating merged composite images. To ensure reproducibility, all localization experiments were performed with three independent biological repeats, each of them representing a set of transformed N. glutinosa plants. Moreover, several leaf regions and many epidermal cells per one replicate were analyzed to obtain sufficient data. The comparative analysis across treatments was ensured by keeping all confocal acquisition settings, specifically laser excitation intensity, detector gain, pinhole aperture, objective lens selection, and frame parameters, constant throughout. Representative images in the manuscript were chosen from observations consistently reproducible across all independent biological replicates.

Image processing and quantitative analysis

Confocal micrographs were acquired and processed using Leica LAS X software. The only adjustments made to images after capture were linear transformations of brightness and contrast applied to the whole image; no attempt was made to correct specific areas to enhance fluorescence or apparent localization. For quantification, confocal image stacks (.lif files) were imported into Fiji/ImageJ (26, 27) using the Bio-Formats plugin to retain spatial information and pixel calibration. Numbers of MP-CFP puncta per cell, plus relative fluorescence intensities at cell edges compared to the cytoplasm, were then determined for multiple cells from three biological replicates.

To assess peripheral enrichment of CFP signals, a straight line was drawn across adjacent cell boundaries, extending from the cytoplasm of one cell through the fluorescent cell periphery into the cytoplasm of the neighboring cell. Fluorescence intensity profiles were generated using the Plot Profile function of Fiji/ImageJ. Peripheral enrichment was calculated as the ratio of the maximum fluorescence intensity at the cell periphery to the mean fluorescence intensity of the adjacent cytoplasmic regions along the same line. Measurements were obtained from several epidermal cells across three independent images of MP-CFP-expressing tissue (n = 9 lines profiles) and two images of empty-vector (pCAMBIA2300) control tissue (n = 6 line profiles). Data are presented as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). Differences between groups were evaluated using an unpaired two-tailed Welch’s t-test, and values of P < 0.05 were considered statistically significant.

Design, construction, and validation of ToBRFV-MP expression cassette

The expression construct was designed for transient expression of ToBRFV-MP and its subcellular localization analysis. The MP coding sequence of 798 bp (lacking its native stop codon) was synthesized to generate an in-frame fusion construct comprising of an N-terminal c-Myc epitope tag (EQKLISEEDL), the 266 amino acid long MP coding sequence, a flexible “GGGGS” linker, and a C-terminal CFP reporter, driven by the CaMV 35S promoter (Figure 1A). The incorporated c-Myc epitope tag enabled downstream immunodetection and biochemical characterization, whereas CFP reporter facilitated live-cell imaging via confocal microscopy. The plasmid harboring engineered pCAM-ToBRFV-MP-CFP cassette was screened by PCR using MP gene specific primers and validated by restriction digestion with Eco RI and Bam HI, releasing expected insert of ~1.5 kb (Supplementary Figure S1). Sequencing of pCAM-ToBRFV-MP-CFP corroborated well with the structural integrity of construct, showing precise in-frame alignment of c-Myc tag, ToBRFV-MP coding region, GGGGS flexible linker, and CFP reporter sequence, with no evidence of frameshift mutation. This verified plasmid was further used to conduct transient expression assays in N. glutinosa.

Validation of MP-CFP fusion protein expression by SDS-PAGE and Western blot analysis

The total leaf protein, isolated from the agroinfiltrated zone, was separated by SDS-PAGE and subjected to Western blot analysis for MP-CFP fusion protein expression. Immunoblot analysis using anti-c-Myc polyclonal antibody revealed the presence of a single immunoreactive band of approximately 59 kDa, corresponding to the expected molecular weight of the MP-CFP fusion protein (Supplementary Figure S2). Whereas, no such band were observed in empty pCAMBIA2300 and buffer infiltrated leaves. Also, the absence of any lower-molecular-weight immunoreactive bands was indicative of any protein degradation or cleavage.

Subcellular localization of ToBRFV-MP

Representative agroinfiltration sites on abaxial leaf surface of N. glutinosa confirmed uniform permeation of Agrobacterium- suspension into the host tissues, remaining phenotypically indistinguishable from the non-infiltrated leaves (Figures 1B–E). At 72 hpi, macroscopical appearance of treated leaves revealed no chlorosis, tissue damage, or necrosis (Figures 2F–H). This indicates transient expression was successfully established without compromising the structural integrity of leaf.

Confocal laser scanning microscopy of epidermal cells expressing the MP-CFP fusion revealed a distinct punctate fluorescence profile that restricted to the cell periphery (Figure 3). These punctate focal points were consistently maintained along cellular boundaries across numerous epidermal cells (n = 3). Whereas, no CFP signal was observed in leaves infiltrated either with empty pCAMBIA2300 backbone or buffer alone under identical acquisition settings (negative controls), verifying that the peripheral fluorescence stemmed exclusively from the expression of the recombinant MP-CFP fusion (Supplementary Figure S3). Besides this, weak diffused fluorescence signals were occasionally captured within the cortical cytoplasm, hinting at a transient intracellular trafficking of MP-CFP complex, as the movement protein journeys toward the plasma membrane.

Co-localization of ToBRFV-MP and aniline blue with plasmodesmata

To ascertain the subcellular localization of ToBRFV-MP, leaf tissue expressing the MP-CFP fusion protein were stained with 0.05% aniline blue (which dominantly labels callose deposits that are associated with plasmodesmata). Confocal laser scanning microscopy revealed substantial spatial overlap between punctate MP-CFP fluorescence and aniline blue- labeled callose deposits along the cell periphery (Figure 2, Supplementary Figure S4). Merged fluorescence images demonstrated that the majority of peripheral MP-CFP puncta coincided with aniline blue-positive sites, providing experimental evidence that the ToBRFV-MP is associated with plasmodesmata only. No detectable fluorescence was observed in leaf tissue infiltrated with empty pCAMBIA2300 or buffer alone, under identical imaging conditions. As expected, these control samples exhibited only aniline blue fluorescence corresponding to endogenous callose deposits, confirming that the CFP fluorescence detected in experimental samples resulted specifically from expression of the recombinant MP-CFP fusion protein.

Quantitative analysis of MP-CFP fluorescence distribution

In addition to the qualitative confocal observations, fluorescence distribution was quantified using Fiji/ImageJ (Figure 4). Line profile plots for adjacent epidermal cells showed that fluorescence intensity peaks confined to the cell periphery in MP-CFP-expressing cells, with signal intensity dropping to near-baseline levels within the cytoplasm. conversely, the empty vector did not show similar enrichment. Quantitative metrics indicate that the membrane-to-cytoplasm fluorescence intensity ratio was substantially higher in cells expressing MP-CFP than in empty-vector controls (18.83 ± 5.77 vs. 3.08 ± 1.63; mean ± SD), reflecting an approximate sixfold enrichment at the cellular boundary. An unpaired two-tailed t-test with Welch’s correction confirmed that this difference was highly significant (-t = 7.74, df = 9.8, P < 0.0001; n = 9, and n = 6 line profiles, respectively). This finding supports our initial qualitative analysis as the ToBRFV-MP protein demonstrates its enrichment on cell periphery over its cytoplasmic distribution in most cases.

MPs are indispensable for intercellular spread of plant viruses, where they facilitate the transport of viral protein-complexes including genome through plasmodesmata connections by modifying the plasmodesmata size exclusion limit and interacting with host cellular machinery (1, 2, 6, 28). Although, MP of tobacco mosaic virus (TMV) has been extensively investigated and shown to localize to plasmodesmata (4, 29, 30), comparable experimental information for the recently emerging ToBRFV is still lacking, and therefore we demonstrated the MP-plasmodesmata interaction for another virus of genus Tobamovirus. Previous studies on ToBRFV have primarily focused on its genome organization, host range, pathogenicity, resistance-breaking ability, and epidemiology (9–11, 31), whereas the intracellular localization of its MP has not been experimentally characterized. In the present study, we established a transient agroinfiltration-based expression system in N. glutinosa and, for the first time, experimentally demonstrated that the ToBRFV-MP-CFP fusion protein exhibits a distinct punctate distribution predominantly at the cell periphery (32). Co-localization of this complex with the aniline blue-stained callose deposits further supported the association of MP with the plasmodesmata. This localization pattern is consistent with the conserved role of MPs of genus Tobamovirus es in mediating the symplastic transport and supports the proposed function of ToBRFV-MP in facilitating cell-to-cell viral movement (30, 33–35).

Plasmodesmata-targeted MPs are known to interact extensively with host cellular components during intracellular trafficking (5, 36). Studies on Tobamovirus es have demonstrated that MPs utilize the cortical endoplasmic reticulum, cytoskeleton, plasma membrane, and vesicle trafficking pathways before accumulating at plasmodesmata (6, 7). In the present study, the predominant punctate fluorescence observed along the cell periphery is consistent with plasmodesmatal localization, whereas the occasional weak diffuse cytoplasmic fluorescence may represent transient intracellular trafficking of the MPs before its accumulation at plasmodesmata (37). Identification of the host components involved in these trafficking events will improve our understanding of ToBRFV movement and may reveal novel targets for engineering durable virus resistance.

Transient agroinfiltration proved to be a rapid and reproducible system for investigating the subcellular localization of ToBRFV-MP (12, 13). The localization pattern was consistently reproduced across independent biological replicates, demonstrating the robustness of the expression system. Furthermore, SDS-PAGE and Western blot analysis confirmed the expression and integrity of the recombinant c-Myc-MP-CFP fusion protein, indicating that the fluorescence observed during confocal microscopy originated from the intact fusion protein rather than degradation products. Quantitative image analysis using Fiji/ImageJ further strengthened the qualitative confocal observations by demonstrating significant enrichment of fluorescence at the cell periphery relative to the cytoplasm, thereby providing objective support for the observed localization pattern (26, 27, 38).

Co-localization with aniline blue although provided strong evidence supporting the association of ToBRFV-MP with plasmodesmata (39). This staining method labels callose deposits surrounding plasmodesmata rather than the MP itself and therefore cannot definitively distinguish plasmodesmata from closely associated structures such as the plasma membrane or adjacent endomembrane compartments (40). Future studies employing genetically encoded plasmodesmata markers such as PDLP1 or PDCB1, together with plasma membrane, cytoskeletal, and endosomal markers, will allow more precise characterization of the intracellular trafficking route of ToBRFV MP.

An additional consideration is the presence of the N-terminal c-Myc epitope tag in the fusion construct. The c-Myc tag was incorporated to facilitate downstream immunodetection and biochemical analyses, including Western blotting and protein interaction studies. Previous reports have suggested that the N-terminal region of Tobamovirus -MPs contains determinants important for plasmodesmatal targeting (41). Although the presence of an N-terminal tag could potentially influence intracellular targeting, the reproducible punctate localization together with co-localization with aniline blue indicates that the c-Myc tag did not abolish plasmodesmatal targeting under the experimental conditions employed. Nevertheless, future studies using constructs containing only a C-terminal fluorescent fusion together with functional movement assays will be valuable for confirming that the observed localization accurately reflects the native behavior of ToBRFV-MP.

The present study was designed primarily to establish a transient expression system and determine the subcellular localization of the ToBRFV-MP. Functional validation of MP-mediated intercellular transport, including macromolecular trafficking assays such as the 2×GFP movement assay, remains an important objective for future investigation. Likewise, identifying host proteins interacting with ToBRFV-MP will provide further insight into the molecular mechanisms governing viral movement, host adaptation, and pathogenicity. Overall, this study provides the first experimental evidence supporting the plasmodesmata-associated localization of the ToBRFV-MP and establishes a reliable experimental platform for investigating its intracellular trafficking and host interactions. These findings contribute to a better understanding of ToBRFV biology and provide a foundation for future studies aimed at elucidating the molecular mechanisms underlying viral movement and developing effective strategies for disease management.

This study provides the first experimental characterization of ToBRFV-MP within plant cells. By developing a reliable transient agroinfiltration system in tobacco, we successfully expressed and visualized the MP-CFP fusion protein. A Western immunoblot confirmed the structural integrity of this full-length recombinant protein, and high-resolution confocal microscopy revealed a reproducible punctate pattern along the cell periphery (plasma membrane). This peripheral accumulation was shown to target plasmodesmata through spatial co-localization with aniline blue-stained callose deposits and quantitative morphometric analysis, consistent with the established function of Tobamovirus MPs in facilitating symplastic cell-to-cell movement. Although these findings establish a solid baseline for ToBRFV-MP localization, determining its full capacity to direct macromolecular transport is a top priority. In this regard, future work should combine the functional transport assays, such as 2X-GFP intercellular trafficking experiments, with co-expression of genetically encoded markers targeting plasmodesmata, the plasma membrane, endosomes, and the cytoskeleton to map the exact intracellular pathway of the virus. Moreover, mapping the host interactome commandeered by ToBRFV-MP will provide crucial insights into the molecular networks that regulate viral spread and pathogenicity. Overall, this study enhances our understanding of ToBRFV-MP biology by offering the first experimental evidence confirming its localization at plasmodesmata and creating a useful foundation for future research focused on uncovering viral movement mechanisms and devising effective approaches to managing ToBRFV disease.