Plant RNA interference (RNAi) is a fundamental antiviral defense that relies on coordinated activities of DICER-like endonucleases (DCLs), Argonaute proteins (AGOs) and RNA-dependent RNA polymerases (RDRs). Over the past decades, studies using model and crop species have uncovered complex and often redundant roles for DCLs and RDRs in generating and amplifying virus-derived small interfering RNAs (vsiRNAs), in addition to connections with transcriptional gene silencing (TGS) and epigenetic defenses against DNA viruses. Concurrently, plant viruses have evolved diverse counterstrategies—proteinaceous RNA silencing suppressors (RSSs), exoribonuclease (XRN)-resistant noncoding RNAs, and indirect manipulation of host pathways—to evade RNAi. Driven by the co-evolutionary arms race, plants have developed sophisticated counter-countermeasures that modulate or overcome viral anti-RNAi activity. Accumulated evidence suggests that plants encode host factor genes that are activated to degrade or sequester viral components such as RSSs against viral infection. On the other hand, plants have also evolved endogenous host modulators of antiviral RNAi that can either reinforce the antiviral response or be co-opted by viruses to antagonize it, representing a furious dynamic molecular battling mechanism. Here, we review recent advances in the molecular functions of DCLs and RDRs across species, summarize newly discovered viral counter-defenses (including RNA-based suppressors), and discuss host counter-countermeasures. We research key areas—such as the roles of RDRγ-class proteins, RTL1 (RNase three-like 1)-mediated competition with DCLs, and the mechanistic impact of viral noncoding RNAs—and outline translational opportunities for improving virus resistance in crops through breeding, biotechnological approaches, and RNA-based applications.

Tomato brown rugose fruit virus (ToBRFV; Tobamovirus fructirugosum) is a recently discovered new species in the genus Tobamovirus that causes an ongoing pandemic and threatens tomato production worldwide. In this study, we determined the complete genome sequences of three ToBRFV isolates collected from greenhouse tomato plants in Canada in 2019, analyzed their possible phylogenetic relationships, and developed corresponding full-length infectious cDNA clones. Using the ToBRFV infectious clone, we generated gene-specific mutants via site-directed mutagenesis, followed by an infection assay. We confirmed that both the movement protein and the coat protein (CP) are indispensable for ToBRFV long-distance movement. Moreover, we found that alanine substitution of amino acid D89 or R114, both of which are highly conserved among tobamoviral CPs, compromised ToBRFV systemic infection. Confocal and electron microscopy analyses further revealed that either D89A or R114A substitution disrupted CP self-interactions and virion assembly. Additionally, we demonstrated that the ToBRFV 126-kDa replicase has RNA silencing suppression activity. Taken together, our data contribute to a better understanding of ToBRFV-encoded proteins at the molecular level, and the ToBRFV full-length infectious cDNA clones developed from this study are a useful tool to facilitate ToBRFV research. [Formula: see text] © 2026 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cherry leaf roll virus (CLRV; Nepovirus avii) is a major threat to cherry production. CLRV has been extensively documented in the western states and provinces of the USA and Canada. Symptoms include upward rolling of leaf margins, decline, and unmarketable fruit. CLRV was detected using RT-PCR based approaches, and validated using ELISA and RNAseq. From the RNAseq data, complete genomic sequences of two isolates of CLRV were determined. Both isolates were 95.2-95.8 and 94.7-94.8% identical to RNA1 and RNA2 from the Olm1 isolate from Washington, USA, respectively. Both Ontario isolates had polymorphisms that would putatively result in elimination of the first translation start codon. CLRV can cause serious disease, which can be further exacerbated with co-infection of other viruses. This is the first report of CLRV in Ontario. Further monitoring and elimination of CLRV in the region will help promote healthy production systems.

The tomato Tm-22 gene is a highly effective, and durable resistance gene in agriculture that has protected tomato production against viruses of the Tobamovirus genus, such as tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) for over 60 years. This dominant R gene, originally sourced from wild tomato species (Solanum peruvianum), acts by recognizing the viral movement protein (MP) and triggering an immune response, often resulting in extreme resistance (ER). However, this durable protection is challenged by a recently emerged new tobamovirus named tomato brown rugose fruit virus (ToBRFV, Tobamovirus fructirugosum). ToBRFV-encoded MP is responsible for ER breakdown. Here, we present evidence that while ToBRFV can evade Tm-22-mediated ER, Nicotiana benthamiana and tomato plants carrying Tm-22 still remain partially resistant to ToBRFV. We show that ToBRFV MP is recognized by and interacts with Tm-22 to trigger an attenuated hypersensitive response. Moreover, we discover that overexpression of Tm-22 can enhance resistance to ToBRFV. These findings demonstrate the practical value of Tm-22 in ongoing resistance breeding programs and open a potential avenue to restore Tm-22 immunity through upregulation of Tm-22 expression.

The vast majority of known viruses belong to the positive-sense single-stranded RNA (+ssRNA) class. Tobamoviruses are among the most destructive plant viruses and threaten global food security. It is generally accepted that +ssRNA viruses including tobamoviruses encode proteins solely on their positive strand (+RNA). Here, we identified additional open-reading frames (ORFs) in the negative strand of tobamoviruses, named reverse ORFs (rORFs). Using cucumber green mottle mosaic virus (CGMMV) as a model, we detected the corresponding peptides of rORFs by mass spectrometry analysis and confirmed the translation of rORFs by ribosome profiling. Furthermore, we demonstrated that these rORFs may be translated from an internal ribosome entry site. Mutation of rORF1 and rORF2 significantly reduced the virulence of CGMMV, whereas ectopic expression of rORF1 and rORF2 could rescue the pathogenicity of the mutants. While the rORF2 protein localizes at the cell membrane and in the nucleolus, rORF1 colocalizes with peroxisomes, where it interacts with the viral 126-kD replication protein. Additionally, we screened peroxisomal rORF1-interacting proteins using artificial intelligence tools and found that PEX3 mediated rORF1 targeting to peroxisomes. This study reveals that the tobamoviral proteome is larger than previously thought, and sheds light on peroxisomes as novel virulence targets important for virus infectivity.

RNA interference (RNAi, also known as RNA silencing) is one of the most important plant defense responses against viral invasion. Although major components of the RNAi pathway, steps leading to viral small interfering RNA biogenesis, and viral counterdefense strategies via RNAi suppressors have been well studied, the broader roles of RNAi in viral infection and seed transmission remain less thoroughly characterized. In particular, the increasing complexity of RNAi-associated mechanisms and their integration with other biological processes have not been comprehensively summarized. Increasing numbers of studies have identified non-canonical RNAi pathways, novel host factors involved in RNAi, and the possibility of small RNAs acting across kingdoms to modulate plant–virus–vector tritrophic interactions. In this review, we provide an overview of the roles of RNAi in plant viral infections and describe recent advances, with emphasis on the discoveries of novel positive and negative RNAi regulators, potential signaling pathways upstream and downstream of antiviral RNAi, and the prospects and challenges of double-stranded RNA applications, either expressed from transgenes or supplied exogenously via spraying. We also discuss how these findings reshape current views on antiviral RNAi, highlight remaining knowledge gaps, and examine how these advances influence plant–virus co-evolution while informing strategies for managing plant virus diseases and reducing their impact.

Tomato brown rugose fruit virus (ToBRFV) is a recently emerged viral pathogen in the Tobamovirus genus first observed in 2014 in the Middle East that has since spread worldwide, causing significant losses in greenhouse tomato production. ToBRFV is easily mechanically transmitted and can escape the durable Tm-22 resistance gene, facilitating its global spread. Seed companies have identified novel sources of resistance and introduced these resistance traits into commercial cultivars. The identity, number, and mechanisms of these putative novel resistance genes are largely unknown but could be exerting selective pressures on ToBRFV. Here, we report 15 new ToBRFV genomic sequences from Canadian greenhouse production systems in susceptible and novel resistant or tolerant cultivars collected since 2023. We combined these sequences with five other Canadian ToBRFV genomes previously deposited in Genbank and a further five consensus sequences derived from metagenomic-based wastewater monitoring sequence data and conducted phylogenetic analysis. Most Canadian sequences grouped together when compared with 332 publicly available international sequences, but several isolates appeared distantly related, suggesting multiple introductions to Canadian production systems. High sequence identity between samples suggest movement of ToBRFV between independent greenhouses, highlighting areas where biosecurity can be improved. Several novel non-synonymous polymorphisms identified in the p126 and movement protein (MP) open reading frames (ORFs) were unique to Canadian sequences and associated with infection of novel resistant tomato cultivars. Many polymorphisms in the p126 ORF are located in a region of the protein associated with Tm-1 resistance-breaking isolates of tomato mosaic virus and ToBRFV, but have not been previously reported. Four novel polymorphisms in MP were also identified and do not appear to be associated with sites previously identified as interacting with Tm-22 and could be related to other unknown resistance genes. Together, these results confirm the difficulties in preventing the transmission of ToBRFV, identify putative adaptations to novel and existing resistance genes, and emphasize the urgent need for the cloning and characterization of these new sources of resistance to ToBRFV.

It is with profound sadness and a deep sense of loss that we mourn the passing of Professor Andrew Otis Jackson on 6 July 2025 [...]

Potyviridae is the largest family of plant-infecting RNA viruses. All members of the family (potyvirids) have single-stranded positive-sense RNA genomes, with polyprotein processing as the expression strategy. The 5'-proximal regions of all potyvirids, except bymoviruses, encode two types of leader proteases: the serine protease P1 and the cysteine protease HCPro. However, their arrangement and sequence composition vary greatly among genera or even species. The leader proteases play multiple important roles in different potyvirid-host combinations, including RNA silencing suppression and virus transmission. Here, we report that viruses in the genus Arepavirus, which encode two HCPro leader proteases in tandem (HCPro1-HCPro2), can naturally lose the coding sequences for these two proteins during infection. Notably, this loss is associated with a shift in foliage symptoms from severe necrosis to mild chlorosis or even asymptomatic infections. Further analysis revealed that the deleted region is flanked by two short repeated sequences in the parental isolates, suggesting that recombination during virus replication likely drives this genomic deletion. Reverse genetic approaches confirmed that the loss of leader proteases weakens RNA silencing suppression and other critical functions. A field survey of areca palm trees displaying varied symptom severity identified a transitional stage in which full-length viruses and deletion mutants coexist in the same tree. Based on these findings, we propose a scenario in which full-length isolates drive robust infections and facilitate plant-to-plant transmission, eventually giving rise to leader protease-less variants that mitigate excessive damage to host trees, allowing long-term coexistence with the perennial host. To our knowledge, this is the first report of potyvirid self-attenuation via coding sequence loss.

Mitogen-activated protein kinase (MAPK) cascades play vital roles in plant responses to biotic and abiotic stresses; however, their regulation during viral infection and the mechanisms by which viruses counteract these defenses remain poorly understood. Here, we report that the Arabidopsis thaliana atypical dual-specificity phosphatase (DSP) DSP4 negatively regulates plant immunity against turnip mosaic virus (TuMV), a member of the Potyviridae family. Subcellular localization, fractionation, and mutagenesis revealed that DSP4 is anchored to the cellular membrane via its C-terminus. Notably, only the membrane-bound form of DSP4 interacts with and dephosphorylates the MAPKs, MPK6 and MPK3, which redundantly restrict TuMV infection. Furthermore, TuMV P3 protein binds to DSP4, maintaining it on the membrane to dephosphorylate MPKs, whereas DSP4 is typically released from the membrane during immune priming. These findings unveil a molecular mechanism wherein TuMV P3 exploits this membrane-associated phosphatase to dampen MAPK-mediated immunity and promote virus infection.

The chemical modifications of DNA and proteins are powerful mechanisms for regulating molecular and biological functions, influencing a wide array of signaling pathways in eukaryotes. Recent advancements in epitranscriptomics have shown that RNA modifications play crucial roles in diverse biological processes. Since their discovery in the 1970s, scientists have sought to decipher, identify, and elucidate the functions of these modifications across biological systems. Over the past decade, mounting evidence has demonstrated the importance of RNA modification pathways in plants, prompting significant efforts to decipher their physiological relevance. With the advent of high-resolution mapping techniques for RNA modifications and the gradual uncovering of their biological roles, our understanding of this additional layer of regulation is beginning to take shape. In this review, we summarize recent findings on the major RNA modifications identified in plants, with an emphasis on N6methyladenosine (m6A), the most extensively studied modification. We discuss the functional significance of the effector components involved in m6A modification and its diverse roles in plant biotic interactions, including plant-virus, plant-bacterium, plant-fungus, and plant-insect relationships. Furthermore, we highlight new technological developments driving research progress in this field

Potyvirids are the largest group of plant RNA viruses. Pelota, a core component of RNA quality controls (RQC), promotes the degradation of potyvirids' genomic RNA by recognizing a specific G1-2A6-7 motif. Here we demonstrate that the viral RNA-dependent RNA polymerase, NIb, acts as a SUMOylation decoy to effectively reduce Pelota SUMOylation by competing with SCE1 to inhibit Pelota-mediated RQC. TuMV NIb is comprised of two functional SUMO interacting motif (SIM): SIM2 and SIM3. The former is identified as the key site for NIb's SUMOylation by SUMO3, whereas the latter is responsible for the interaction with SCE1. These two SIMs are conserved among the majority of potyvirids-encoded NIbs. Thus, virus protein-mediated SUMOylation decoy strategy to suppress host defense may be a common feature in plant virus pathosystems. These findings highlight a dynamic interplay between plant defense mechanism and viral counter-strategy by orchestrating the post-translational modifications of virus and host defense components.

Commercially cultivated Prunus species are commonly grown in adjacent or mixed orchards and can be infected with unique or commonly shared viruses. Apple (Malus domestica), another member of the Rosacea and distantly related to Prunus, can share the same growing regions and common pathogens. Pollen can be a major route for virus transmission, and analysis of the pollen virome in tree fruit orchards can provide insights into these virus pathogen complexes from mixed production sites. Commercial honey bee (Apis mellifera) pollination is essential for improved fruit sets and yields in tree fruit production systems. To better understand the pollen-associated virome in tree fruits, metagenomics-based detection of plant viruses was employed on bee and pollen samples collected at four time points during the peak bloom period of apricot, cherry, peach, and apple trees at one orchard site. Twenty-one unique viruses were detected in samples collected during tree fruit blooms, including prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) (Genus Ilarvirus, family Bromoviridae), Secoviridae family members tomato ringspot virus (genus Nepovirus), tobacco ringspot virus (genus Nepovirus), prunus virus F (genus Fabavirus), and Betaflexiviridae family member cherry virus A (CVA; genus Capillovirus). Viruses were also identified in composite leaf and flower samples to compare the pollen virome with the virome associated with vegetative tissues. At all four time points, a greater diversity of viruses was detected in the bee and pollen samples. Finally, the nucleotide sequence diversity of the coat protein regions of CVA, PDV, and PNRSV was profiled from this site, demonstrating a wide range of sequence diversity in pollen samples from this site. These results demonstrate the benefits of area-wide monitoring through bee pollination activities and provide new insights into the diversity of viruses in tree fruit pollination ecosystems.

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana , and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.

Infection by positive-strand RNA viruses induces extensive remodeling of the host endomembrane system in favor of viral replication and movement. The integral membrane protein 6K2 of potyviruses induces the formation of membranous virus replication vesicles at the endoplasmic reticulum exit site (ERES). The intracellular trafficking of 6K2-induced vesicles along with microfilaments requires the vesicular transport pathway, actomyosin motility system, and possibly post-Golgi compartments such as endosomes as well. Recent studies have shown that endocytosis is essential for the intracellular movement of potyviruses from the site of viral genome replication/assembly site to plasmodesmata (PD) to enter neighboring cells. In this chapter, we describe a detailed protocol of how to use endomembrane trafficking pathway-specific chemical inhibitors and organelle-selective fluorescence dye to study the trafficking of potyviral proteins and potyvirus-induced vesicles and to unravel the role of endocytosis and the endocytic pathway in potyvirus infection in Nicotiana benthamiana plants.

IntroductionHoney bee (Apis mellifera) pollination is widely used in tree fruit production systems to improve fruit set and yield. Many plant viruses can be associated with pollen or transmitted through pollination, and can be detected through bee pollination activities. Honey bees visit multiple plants and flowers in one foraging trip, essentially sampling small amounts of pollen from a wide area. Here we report metagenomics-based area-wide monitoring of plant viruses in cherry (Prunus avium) and apple (Malus domestica) orchards in Creston Valley, British Columbia, Canada, through bee-mediated pollen sampling.MethodsPlant viruses were identified in total RNA extracted from bee and pollen samples, and compared with profiles from double stranded RNA extracted from leaf and flower tissues. CVA, PDV, PNRSV, and PVF coat protein nucleotide sequences were aligned and compared for phylogenetic analysis.ResultsA wide array of plant viruses were identified in both systems, with cherry virus A (CVA), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and prunus virus F (PVF) most commonly detected. Citrus concave gum associated virus and apple stem grooving virus were only identified in samples collected during apple bloom, demonstrating changing viral profiles from the same site over time. Different profiles of viruses were identified in bee and pollen samples compared to leaf and flower samples reflective of pollen transmission affinity of individual viruses. Phylogenetic and pairwise analysis of the coat protein regions of the four most commonly detected viruses showed unique patterns of nucleotide sequence diversity, which could have implications in their evolution and management approaches. Coat protein sequences of CVA and PVF were broadly diverse with multiple distinct phylogroups identified, while PNRSV and PDV were more conserved.ConclusionThe pollen virome in fruit production systems is incredibly diverse, with CVA, PDV, PNRSV, and PVF widely prevalent in this region. Bee-mediated monitoring in agricultural systems is a powerful approach to study viral diversity and can be used to guide more targeted management approaches.

Telosma mosaic virus (TelMV, Potyvirus, Potyviridae ) is an emerging viral pathogen causing a major global threat to passion fruit plantations. However, an efficient strategy for controlling such viruses is not yet available. Cross protection is a phenomenon in which pre-infection of a plant with one virus prevents or delays superinfection with the same or closely related virus. HC-Pro is the potyviral encoded multifunctional protein involved in several steps of viral infection, including multiplication, movement, transmission and RNA silencing suppression. The main hypothesis we tested in this study was whether it is possible to generate attenuated viral strains capable of conferring protection against severe TelMV infection by manipulating the HC-Pro gene. By introducing point mutation into the potyviral conserved motif FRNK of HC-Pro, we have successfully obtained three highly attenuated mutants of TelMV (R181K, R181D and R181E, respectively) that can systemically infect passion fruit plants without any noticeable symptoms. Importantly, these mutants confer complete protection against subsequent infection of severe recombinant virus TelMV-GFP, evidenced by no detection of viral RNA or protein of the superinfection virus in the systemic leaves of passion fruit plants in both early and late stages. Lastly, we demonstrated that the HC-Pros harbored by the highly attenuated mutants exhibited reduced RNA silencing suppression activity in Nicotiana benthamiana leaves. Altogether, this study provides the first demonstration of the generation of highly attenuated strains for TelMV and highlights key amino acid residue involved in complete cross protection against TelMV, opening a new avenue to fight TelMV in the field.### Competing Interest StatementThe authors have declared no competing interest.

Pathogenesis-related (PR) proteins are induced by abiotic and biotic stresses and generally considered as part of the plant defense mechanism. However, it remains yet largely unclear if and how they are involved in virus infection. Our recent quantitative, comparative proteomic study identified three PR-5 family proteins that are significantly differentially accumulated in the plasmodesmata (PD)-enriched fraction isolated from Nicotiana benthamiana leaves infected by turnip mosaic virus (TuMV). In this study, we employed the TuMV-Arabidopsis pathosystem to characterize the involvement of two Arabidopsis orthologs, AtOSM34 and AtOLP of the three N. benthamiana PR-5-like proteins. We show that AtOSM34 and AtOLP are PD-localized proteins and their expression is up- and downregulated in response to TuMV infection, respectively. Deficiency or overexpression of AtOLP does not affect viral RNA accumulation. Knockdown of AtOSM34 inhibits TuMV infection, whereas its overexpression promotes viral infection. We further demonstrate that AtOSM34 functions as a proviral factor through diminishing PD callose deposition to promote viral intercellular movement, targeting the viral replication complex to enhance viral replication, and suppressing the ROS-mediated antiviral response. Taken together, these data suggest that TuMV has evolved the ability to selectively upregulate and subvert AtOSM34, a PR-5 family protein to assist its infection.