Plant viruses rely on long-distance transport to establish systemic infection within hosts, and cell-to-cell movement is often a prerequisite for vascular entry and subsequent systemic spread. Therefore, interference with virus movement represents an attractive antiviral strategy. Here, we report a series of perillaldehyde-derived compounds bearing an α-aminophosphonate moiety, evaluate their anti-phytoviral activities, and elucidate the mechanism of a representative lead. A total of 38 target derivatives were designed and synthesized by introducing an aminophosphonate pharmacophore onto the perillaldehyde scaffold. Using the half-leaf lesion assay, compound Y14 was identified as a potent inhibitor against Potato virus Y (PVY), exhibiting an inactivation half-maximal effective concentration (EC50) of 122.40 ± 9.34 μg mL-1, outperforming the commercial antiviral dufulin (140.40 ± 6.75 μg mL-1) and ribavirin (238.90 ± 10.58 μg mL-1). Activity-based protein profiling, molecular docking, microscale thermophoresis, together with immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR) analyses, collectively suggested that Ser207 in the PVY coat protein (CP) is a key residue for compound Y14 engagement. Notably, the CPS207A mutation markedly reduced the binding affinity of compound Y14 and attenuated PVY systemic infection in planta. Confocal imaging showed that the CPS207A recombinant PVY produced predominantly single-cell fluorescence, indicating impaired cell-to-cell movement. The co-immunoprecipitation results further suggested that this inhibition may arise from disrupted interaction with the host factor NtCPIP. This study identifies compound Y14 as an antiviral lead that targets PVY CP and functionally compromises viral intercellular spread, thereby suppressing systemic infection. These findings provide a mechanistically distinctive chemical for PVY management. © 2026 Society of Chemical Industry.

Plant viruses disrupt host metabolic pathways to facilitate infection. Sucrose metabolism may modulate viral resistance, yet the mechanism remains unclear. We used label-free proteomics to investigate sucrose phosphate synthase (SPS) activity in Nicotiana benthamiana during potato virus Y (PVY) infection. We identified 464 differentially expressed proteins enriched in SPS activity, amino acid metabolism, and carbohydrate biosynthesis. Functional analysis demonstrated that PVY infection significantly upregulated NbSPS3 expression and enhanced SPS activity and sucrose accumulation, which reduced viral accumulation. Silencing NbSPS3 compromised sucrose synthesis and increased PVY accumulation, confirming an antiviral defense role. NbSPS3 specifically interacted with the 14-3-3 protein NbGF14a, a positive regulator of PVY resistance. Silencing NbGF14a also enhanced susceptibility to PVY, suggesting a coordinated defense mechanism involving sucrose metabolism and 14-3-3 signaling. NbSPS3-mediated sucrose biosynthesis was revealed as a critical metabolic defense strategy against PVY, offering new insights into plant-virus interactions and potential targets for engineering crop resistance.

Food insecurity is a prominent global issue. With a predicted global population of 9 billion by 2050, food production must double at a minimum to accommodate these growing numbers. One approach to combat food insecurity is targeting plant pathogens that affect crop quality and yield, resulting in an overall increase in edible food production. Plant pathogen management has previously utilized the RNA interference (RNAi) mechanism for remediation; however, its widespread use has technical limitations. In this study, silica nanoparticles (SiO2 NPs) were utilized as nanocarriers of therapeutic double-stranded ribonucleic acid (dsRNA) to enhance dsRNA delivery into plant cells, thereby activating the RNAi system and suppressing the occurrence of potato virus Y (PVY). This highly mutable pathogen causes several adverse effects in potato and other crop plants. Fast-dissolving silica (FDS) nanoparticles, mesoporous silica nanoparticles (MSNs), and ultraporous mesostructured silica nanoparticles (UMNs) with negative and positive surface charges were synthesized. After thorough characterization, nine distinct SiO2 NP formulations were loaded with dsRNA, with UMNs showing the best loading capacity. Due to the negatively charged nature of dsRNA, positively charged UMNs were favored and employed in further application experiments. Gel electrophoresis indicated that dsRNA loaded into/onto these UMNs was released over several days. Fifteen days after inoculation, greenhouse experiments with tobacco plants demonstrated that dsRNA-loaded UMNs effectively suppressed PVY. In a field study, dsRNA loaded into/onto UMNs showed a 0% disease incidence, an improvement compared to dsRNA or nanoparticle application alone. These findings reveal that UMNs are an efficient nanocarrier for delivering dsRNA against PVY, thereby increasing crop health and yield. A techno-economic analysis was performed to evaluate the economic viability of this nanomaterial for industrial commercialization.

Potato crops in Bangladesh are significantly threatened by viral diseases, underscoring the need for comprehensive virome profiling to inform sustainable management strategies. In this study, we employed high-throughput sequencing (HTS) to characterize the viral diversity in symptomatic potato leaves collected from five major potato-growing districts. A total of 10 distinct viruses were identified, representing six families and nine genera. Among these viruses, potato virus X (PVX) was the most abundant, followed by potato virus Y (PVY) and potato virus S (PVS). Notably, potato virus V (PVV) was detected for the first time in Bangladesh and, to our knowledge, not previously reported in South Asia. Additionally, we assembled the complete genome sequence of a potato virus A (PVA) isolate from Bangladesh, which was highly similar to previously reported PVA genomes. Phylogenetic analysis revealed that the Bangladeshi PVA and PVV isolates share close evolutionary relationships with global strains, particularly those from Europe and the Americas, suggesting historical or trade-related linkages. Co-infection analysis showed a high frequency of mixed infections, with PVY, PVS, and potato leafroll virus (PLRV) being the most common combinations. The correlation analysis identified strong statistical associations among specific viruses [e.g., between potato aucuba mosaic virus (PAMV) and tomato leaf curl New Delhi virus (ToLCNDV)], indicating frequent co-occurrence patterns that warrant further investigation; however, causal mechanisms were not inferred from these data. Geospatial analysis revealed that Dinajpur and Panchagarh districts had the highest overall viral prevalence, while Thakurgaon exhibited the lowest diversity of infections. These findings highlight the complexity of the potato virome in Bangladesh, emphasizing the threat posed by the dominance of PVY and the emergence of novel viruses like PVV. This study provides a foundational framework for future research on potato virus epidemiology and paves the way for sustainable agricultural practices to mitigate the impact of viral infections on potato production in Bangladesh.

Potato virus Y (PVY), widely regarded as one of the world's most important plant viruses, seriously threatens global potato production and food security. PVY deploys its proteins to interact with key host factors, thereby enabling viral replication, accumulation, and systemic infection. PVY also exhibits high genetic diversity and frequent recombination, which promote host adaptation and immune evasion. In response, potato plants perceive viral effectors through intracellular immune receptors and activate antiviral defenses. Over the past decade, significant progress has been made in elucidating PVY-host defense and counter-defense mechanisms. Here, we summarize the molecular basis of PVY pathogenicity and highlight recent advances in PVY resistance genes (e.g., Rysto and Rychc). Finally, we integrate emerging insights from plant virology and nucleotide-binding leucine-rich repeat (NLR) biology to discuss prospective, multi-pronged strategies for PVY management.

Plant extracellular vesicles (EVs) serve as critical mediators of intercellular communication during plant-pathogen interactions, particularly through their cargo of regulatory small RNAs, enabling the transport of miRNAs to distant tissues during biotic stress. Potato virus Y (PVY), one of the most economically damaging plant viruses globally, poses significant threats to solanaceous crop production. However, the landscape of EV-associated miRNAs and their regulatory roles in PVY infection remain largely unexplored. In this study, we isolated and characterized EV-associated particles from the apoplastic fluid of both PVY-infected and healthy tomato leaves using differential ultracentrifugation, followed by transmission electron microscopy, nanoparticle size analysis, and western blotting. High-throughput small RNA sequencing revealed 96 significantly differentially expressed miRNAs in EV-associated particles upon viral challenge. Bioinformatic prediction revealed that 80% of these dysregulated miRNAs potentially target multiple genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses demonstrated significant overrepresentation of predicted target genes in pathways associated with transcription, ta-siRNA biogenesis involved in RNA interference, protein binding, RNAi-mediated antiviral immune response, oxidative phosphorylation, mRNA surveillance pathway, and eukaryotic ribosome biogenesis. Our findings demonstrate that PVY infection selectively modulates the miRNA composition within tomato EV-associated particles. These EV-associated particles delivered miRNAs may contribute to a sophisticated antiviral defense mechanism by co-regulating host immunity. This study provides novel insights into the role of EV-associated particles mediated RNA communication in plant immunity and lays a theoretical foundation for developing innovative miRNA- and EV-based antiviral strategies for crop protection.

Integrated transcriptomic and metabolomic analyses reveal hormone-mediated crosstalk during potato virus Y and potato spindle tuber viroid co-infection.

Potato virus X (PVX) and potato virus Y (PVY) are major pathogens that threaten seed potato quality and yield. To improve the efficiency of field screening, we developed monovalent PVX, monovalent PVY, and bivalent PVX/PVY nanozyme strips using Fe3O4 nanozymes as labels in a double-antibody sandwich lateral flow immunochromatographic assay. Western blot analysis demonstrated that four monoclonal antibodies (PVX 2, PVX 6, PVY 2, and PVY 5) specifically recognized their corresponding viral coat proteins. Specificity testing showed that the nanozyme strips reacted only with the target viruses and did not cross-react with other common potato viruses, including Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS), and Potato leafroll virus (PLRV). The PVX nanozyme strip detected PVX-positive extracts diluted up to 103-fold, the PVY nanozyme strip up to 104-fold, and the bivalent strip detected PVX/PVY co-infected samples diluted up to 103-fold. In addition, detection results by strips from 12 samples of plantlets in vitro were fully consistent with RT-PCR. These nanozyme strips provide rapid, simple, specific, and sensitive methods that can be stored at ambient temperature, enabling field surveys, warehouse screening, and on-site testing and supporting early detection of potato virus diseases.

Cell-intrinsic restriction factors (CIRFs) negatively regulate plant virus infections and represent valuable resources for breeding virus-resistant crops. However, the potential for ribosomal proteins to function as CIRFs and the underlying mechanisms remains elusive. Our previous work demonstrated that the Nicotiana benthamiana chloroplastic ribosomal protein of the large subunit 1 (NbRPL1) promotes the infection of tobacco vein banding mosaic virus (TVBMV) by antagonizing the NbBeclin1-mediated degradation of the viral RNA-dependent RNA polymerase, NIb. Continuing this line of research, we explored the role of another nonchloroplastic ribosomal protein, large ribosomal protein 4 (RPL4), in TVBMV replication. We found that TVBMV NIb interacts with NbRPL4; however, the NIb proteins of 2 other related potyviruses, potato virus Y (PVY) and turnip mosaic virus (TuMV), did not interact with NbRPL4. Overexpression of NbRPL4 inhibited, whereas its downregulation promoted, TVBMV replication. NbRPL4 did not affect PVY or TuMV replication. The nuclear-cytoplasmic distribution of NbRPL4 positively correlated with its antiviral effect on TVBMV replication. NbRPL4 interfered with NbXPO1-mediated nuclear export of the NIb protein, subsequently affecting the translocation of NIb into the viral replication compartment. Our work indicates that NbRPL4 functions as a restriction factor for TVBMV by inhibiting NbXPO1-mediated nuclear export of NIb in a virus-specific manner.

ABSTRACT Aphid species that test positive for plant viruses in reverse transcription‐PCR (RT‐PCR) analysis are not necessarily vectors. This is because virus RNA can also be detected in non‐vector aphid species using this method. Potato virus Y (PVY) has been previously detected using RT‐PCR from three common weed‐infesting alate aphid species, Aphis fukii Shinji, Aphis oenotherae Oestlund, and Capitophorus hippophaes javanicus Hille Ris Lambers, trapped in potato fields in Hokkaido, northern Japan. However, these three taxa are not known as PVY vectors, and it remains unclear whether they acquired PVY from their respective host plants or other common weeds. Therefore, we conducted two experiments: (i) transmission experiments of these aphids using Myzus persicae (Sulzer), a species widely regarded as the most efficient PVY vector, as a reference species, and (ii) virus susceptibility experiments of typical host plant species of the three aphid species and other common weed species in and around potato fields in Hokkaido. The transmission experiments showed that the PVY‐transmission efficiency of A . fukii and A. oenotherae was not significantly different from that of M. persicae . In contrast, C. hippophaes javanicus did not transmit PVY. The estimated maximum transmission efficiency by one aptera was 7.5%. Mechanical inoculations and field sampling revealed that the tested weed species were not susceptible to PVY. These findings suggest that A . fukii and A. oenotherae serve as common and efficient PVY vectors in potato fields in Hokkaido, despite their individual transmission efficiency being < 7.5%. They likely acquire PVY from infected potato plants and/or unidentified reservoirs. These novel insights can advance our understanding of PVY epidemiology in Japan and contribute to the development of more effective vector‐based control strategies against PVY.

Despite their great agronomic interest and widespread occurrence in germplasm resources, the quantitative resistance and tolerance of plants to their parasites have rarely been studied in terms of durability potential. Using experimental evolution under controlled conditions for 9 months, we compared the evolution of potato virus Y (PVY) (Potyvirus yituberosi) virulence, measured by the effect of viral infection on plant fresh weight, and replicative fitness, measured by systemic viral load, in five pepper (Capsicum annuum) lines contrasting in their levels of quantitative resistance and tolerance. PVY evolutionary trajectories differed between pepper lines. Three lines revealed either an increase in PVY replicative fitness or an increase or decrease in PVY virulence. Two other lines did not reveal any significant change in PVY replicative fitness or virulence. The tolerance level of three pepper lines also differed significantly when measured with initial and evolved PVY populations, often associated with changes in PVY virulence. PVY evolutionary trajectories were partly explained by parameters linked to plant resistance operating at different stages of infection (inoculation, colonization of inoculated leaves and systemic infection). This study provides information on the durability potential of quantitative resistance and tolerance to PVY in pepper.

Identification of pesticide targets is of great significance for the development of new pesticides. The new compound GLY-15, containing a pyrimidine heterocycle and a moroxydine skeleton structure, has good anti-TMV activity, but the underlying molecular targets and mechanism of action remain elusive. Here, host malate dehydrogenase (MDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and tobacco mosaic virus (TMV) coat protein (CP) were identified as potential targets of GLY-15 using activity-based protein profiling (ABPP) and drug affinity responsive target stability (DARTS), and their interactions with GLY-15 were validated by microscale thermophoresis (MST) and pull-down analysis. Functional analyses demonstrate that MDH silencing significantly reduces TMV accumulation, while transient overexpression of MDH results in elevated viral infection. Meanwhile, yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) analysis uncover that MDH interacts with CP, and their interaction is effectively inhibited by GLY-15. Site-directed mutagenesis identifies E225 as a critical residue for both GLY-15/MDH binding and MDH/CP interaction. Further investigations reveal that GLY-15 functions as an MDH inhibitor and affects its interaction with CP. Meanwhile, we showed that GLY-15 targeting MDH indicates broad antiviral activity against pepper mild mottle virus (PMMoV) and potato virus Y (PVY). This investigation systematically reveals novel insights into the anti-TMV mechanisms of GLY-15, establishing a valuable theoretical basis for antiviral target discovery and plant disease resistance breeding.

Potyvirus genomes are expressed as a single large open reading frame, which is translated into a polyprotein that is post-translationally cleaved by three virus-encoded proteases into 10 functional proteins. Several of these potyviral proteins, including nuclear inclusion protein b (NIb), are multifunctional. Here, using the classic GFP silencing in Nicotiana benthamiana gfp-transgenic plants, we show that potato virus Y (PVY) NIb, in addition to its canonical role as the viral RNA-dependent RNA polymerase (RdRP), functions as a suppressor of RNA silencing. Mutational analyses reveal a previously unreported NIb nuclear localization signal (NLS) consisting of a triple-lysine motif. NIb suppression of RNA silencing activity was lost when the NLS was mutated, suggesting that nuclear localization is required for NIb suppression of RNA silencing activity. Analysis of sequenced GFP siRNAs revealed three reproducible hotspot regions at ≈175 nt, ≈320-330 nt, and a broader 3'-proximal region spanning ≈560-700 nt that contains multiple local maxima. These data show differences in the positional distribution of siRNAs between samples expressing NIb and those expressing NIbDel3×2, the NIb null mutant that does not suppress RNA silencing. However, the positional distribution of GFP-derived small RNAs across the transgene differed modestly between NIb and NIbDel3×2, while both treatments showed the same three reproducible hotspot regions. Furthermore, NIb was found to interact with four key RNA silencing pathway proteins-AGO4, HSP70, HSP90, and SGS3. Except for HSP90, each of these proteins showed degradation products that were absent in NIb mutants that did not suppress RNA silencing. These findings support a role for NIb in countering host defense during virus infection.

Potato virus Y (PVY) and potato leafroll virus (PLRV) are the most damaging viruses for potato production worldwide. Mixed infections not only result in additive detrimental effects on plant growth and tuber yield but also complicate the development of durable and broad-spectrum viral resistance. Heterologous protection against PVY can be achieved through the expression of the coat protein (CP) of lettuce mosaic virus (LMV) (CPLMV), conferring resistance via a capsid protein-mediated mechanism. On the other hand, we have previously demonstrated that transgenic lines expressing the PLRV ORF2 (RepPLRV) exhibit resistance to different PLRV isolates. In this study, potato transgenic lines of cv. Kennebec expressing CPLMV and RepPLRV were developed to confer dual virus resistance. Transgenic and non-transgenic control plants were molecularly and phenotypically characterized in greenhouse and field conditions. Across multiple growing seasons, two selected transgenic lines consistently displayed robust resistance to both major viruses, without exhibiting yield penalties or noticeable phenotypic alterations. These results constitute a significant advancement, demonstrating that dual resistance to PVY and PLRV can be achieved while preserving the original agronomic performance of the cultivar. This breakthrough not only contributes to long-term crop productivity but also provides a more sustainable strategy for managing viral diseases in potato production.

Abstract Whilst molecular methods for plant virus detection are usually based on PCR amplification, loop mediated isothermal amplification (LAMP) is a rapid and sensitive molecular diagnostic tool increasingly used for plant virus detection, particularly in field conditions and resource-limited laboratories. In this study, a novel LAMP primer set (ID: 334) was developed for the specific detection of Potato virus Y (PVY), a globally significant pathogen responsible for yield losses of up to 80% in potatoes. Primer set 334 was designed based on conserved regions identified across 30 complete PVY genomes. It was evaluated against 33 PVY isolates representing 5 strain groups. The assay demonstrated 100% inclusivity, successfully detecting all isolates, including both recombinant and non-recombinant strains. Specificity testing confirmed no cross amplification with related potyviruses (Potato virus A (PVA) and Potato virus V (PVV)) or unrelated potato viruses (Potato virus X (PVX), Potato leafroll virus (PLRV), and Potato mop-top virus (PMTV)), further validating the exclusiveness of the assay. Moreover, the assay was validated on multiple host matrices, including leaves and tuber tissues of potato. Compared to a previously published LAMP assay, primer set 334 exhibited 2.6-fold greater sensitivity, detecting PVY at four orders of magnitude lower concentrations, with performance comparable to qPCR but with faster time-to-result. This improved LAMP assay provides a rapid, specific, and sensitive tool for PVY detection in leaves and tubers, supporting both laboratory, field, and post-harvest diagnostics.

The emergence of plant viruses is a complex process influenced by viral genetic variation, host species, and environmental factors. To better predict and manage new plant diseases, it is important to understand how viruses adapt to novel hosts. In this study, we examined how two isolates of potato virus Y (PVY), PVYNb, and PVYSt, evolve when repeatedly passed through three solanaceous plants: Bentham’s tobacco (Nicotiana benthamiana), potato (Solanum tuberosum), and tomato (Solanum lycopersicum). We also tested whether switching between hosts could reduce the impact of strong population bottlenecks, which often occur in poorly suited hosts. Our findings show that benthamiana supports high viral RNA accumulation and genetically stable diversity, consistent with large effective population sizes. In contrast, potato creates strong bottlenecks, often leading to viral lineage extinction and increased mutation fixation due to genetic drift. Tomato served as an intermediate host, with outcomes varying by virus strain, and acted as a sink host for some lineages, resulting in unsuccessful infection in the next passage. PVYNb showed greater standing diversity and more lineage-specific nonsynonymous change, whereas PVYSt exhibited greater genomic stability and pervasive purifying selection across most cistrons. Only a few late-passage benthamiana lineages displayed elevated πN/πS ratios, indicating that positive selection was rare and not consistently replicated. Overall, our results show that the balance between selection and drift depends strongly on host permissiveness and demographic constraints. Importantly, alternating between permissive and restrictive hosts helped prevent lineage extinction, suggesting that heterogeneous host environments, such as those encountered in agricultural systems, may facilitate virus persistence and adaptation. This study deepens our understanding of the ecological and evolutionary forces that drive the emergence of plant viruses.

Sustainable nanotechnology, particularly chitosan (CS)-based biodegradable nanoparticles (NPs), offers an eco-friendly delivery system comparedto conventional techniques. Potato virus Y (PVY) causes severe potato yield losses and reduces tuber quality. Its control is challenging owing to nonpersistent aphid transmission, especially by Myzus persicae, the most efficient vector. Chemical insecticides lead to environmental pollution, health risks, and resistance, highlighting the need for alternatives. This study assessed pure CS, thyme essential oil (TEO)-loaded CS, CS-lecithin (LH) encapsulated TEO, and TEO alone for their ability to block PVY transmission by M. persicae. To the best of our knowledge, no prior studies have examined the CS + LH + TEO formulation's effect on aphid-mediated PVY transmission. The results clearly indicate that all NP formulations significantly reduced aphid vectored transmission of PVY to healthy plants and inhibited virus acquisition from infected sources. The CS + LH + TEO and pure TEO treatments were the most effective, with CS + LH + TEO reducing transmission by 50.0% and pure TEO suppressing acquisition by 57.5%. Moreover, these formulations, especially CS + LH + TEO, substantially enhanced antioxidant enzyme activities, increased proline levels, and upregulated expression of the defense genes PAL-1and PR-2. As a result, accumulation of post-PVY infection oxidative stress markers H₂O₂ and MDA was considerably lower in NP-treated plants, emphasizing their protective effect against membrane damage. In this study, TEO was successfully encapsulated using CS and LH. Although previous research has primarily focused on the individual effects of CS and TEO on aphids, the inhibitory effect of CS + LH + TEO NPs on PVY transmission and acquisition by aphids offers a promising avenue for developing novel integrated pest management strategies. © 2026 Society of Chemical Industry.

Tobacco leaf diseases significantly affect yield and quality, underscoring the need for rapid and non-destructive diagnostic tools. Although hyperspectral imaging (HSI) has been applied in tobacco pathology, most existing studies focus on single diseases and lack generalized, interpretable frameworks for multi-class identification. In this study, hyperspectral images of healthy leaves and four major diseases-brown spot, wildfire, Tobacco Mosaic Virus (TMV), and Potato virus Y (PVY)-were collected to construct a balanced, leaf-independent dataset. Pixels were grouped by leaf ID, and the entire dataset was strictly partitioned at the leaf level to prevent pixel-level data leakage and ensure generalization to unseen leaves. Multiple preprocessing techniques, wavelength-selection methods, and machine-learning classifiers were systematically compared. A compact ANN model integrating Savitzky-Golay preprocessing and SPA-based wavelength selection achieved the best overall performance while requiring only a small number of informative wavelengths. A Transformer model provided slightly stronger predictive capacity but depended on full-spectrum inputs and substantially higher computational cost. Pixel-level predictions enabled lesion-area-based severity estimation for the two leaf-spot diseases. SHAP analysis highlighted physiologically meaningful spectral regions associated with pigment absorption and structural variation. Overall, this study presents an efficient and interpretable HSI framework for multi-disease tobacco diagnosis, supporting the development of practical hyperspectral or multispectral systems.

N6-methyladenosine (m6A), a reversible epigenetic modification, is ubiquitously present across diverse RNA species, including viral RNA. This modification plays a pivotal role in orchestrating RNA metabolism. Nevertheless, the mechanisms by which plants utilize m6A modifications to fine-tune antiviral immunity remain largely unknown. In this study, we systematically examined the dynamic changes and biological significance of m6A modifications throughout potato virus Y (PVY) infection in host plants. Methylated RNA immunoprecipitation sequencing reveals a conserved m6A distribution pattern in Nicotiana benthamiana, predominantly enriched in 3' untranslated regions of transcripts. The nuclear transcription factor NFYA3_0 exhibits robust hypermethylation accompanied by transcriptional upregulation during PVY infection. NFYA3_0 knockdown promotes PVY accumulation and reduces global m6A modification levels in the host. NFYA3_0 positively regulates transcription of the m6A methyltransferase gene NbMTA, whose loss of function similarly compromises viral resistance while diminishing m6A abundance. Moreover, NbMTA anchors and methylates the PVY coat protein-coding region, thereby facilitating viral RNA degradation and effectively restricting infection. These findings indicate that the core antiviral mechanism of the nuclear transcription factor NFYA3_0 enables targeted degradation of viral RNA by activating methyltransferase NbMTA-mediated m6A epitranscriptome regulation. This work provides a new strategy for developing green virus-resistant crop lines based on epigenetic editing.

Potato virus Y (PVY), potato mop-top virus (PMTV), potato virus S (PVS), and tobacco rattle virus (TRV) can be difficult to identify based on visual foliar symptoms. Using tuber samples collected from seven locations and 12 cultivars during 2017 to 2019, we developed a molecular assay using customized Whatman Flinders Technology Associates Plantsaver cards (FTA cards) and a reverse-transcription PCR (RT-PCR) for efficient sample collection and nucleic acid extraction. PMTV and PVY were detected more frequently on the stem end, TRV on the rose end, and PVS was evenly detected across the stem-end to rose-end axis of the tubers. Differences were seen in virus species localization within a tuber, thereby a composite of samples taken from multiple locations on a tuber improved virus detection. Regardless of sampling location on the tuber, tissue excised from the surface to 0.5 cm deep provided the best detection for all four viruses. For PVY, PMTV, and TRV, the proportion of tubers with viruses detected from field samples was highest at 100, 150, and 175 days after harvest, respectively. However, the probability of detecting PMTV and PVY 25 days after harvest was not different than the probability of detection at their peak detection times after harvest. The probability of TRV detection was lower at 25 days after harvest than at 175 days after harvest (P < 0.05).