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Movement proteins (MPs) modulate the size exclusion limit of plasmodesmata—membrane‐lined channels connecting plant cells—thereby allowing cell‐to‐cell movement and systemic spread of plant viruses. The largest and arguably best‐studied group of MPs is the 30K superfamily. Its family members share little sequence similarity, with only a handful of residues being well conserved. Yet, all family members appear to adopt the same jelly‐roll protein fold structure. Lettuce big‐vein associated virus (LBVaV), a member of the Rhabdoviridae family, is closely associated with lettuce big‐vein disease (LBVD). It appears to facilitate the long‐distance movement of Mirafiori lettuce big‐vein virus (MiLBVV) in plants through an unknown mechanism. Notably, enhanced MiLBVV spread correlates with severe LBVD symptoms. Despite LBVaV having been known for decades, its proteins have not been studied in detail thus far. By using a combination of Alphafold2 structure modelling and FoldSeek structure‐based homology searches, we managed to annotate all LBVaV open reading frames (ORFs), with ORF3 clustering with the 30K superfamily. While ORF3 is the most conserved protein sequence among the LBVaV‐encoded ORFs, it shares only 5%–11% protein sequence identity with related MPs in the same genus. Microscopy studies confirmed that ORF3 locates at plasmodesmata, and in planta expression of ORF3 allowed cell‐to‐cell movement of two movement‐impaired plant viruses. Thus, the Alphafold2‐FoldSeek strategy allowed successful annotation of a plant viral genome even when viral proteins show little sequence similarity. AlphaFold2 + FoldSeek structure guided search predicts the movement protein of lettuce big‐vein associated virus despite low sequence similarity.

Background Plant viral movement protein (MP) function is decisive for virus cell-to-cell movement. Often, MPs also induce membrane alterations, which are believed to play a role for the establishment of viral replication compartments. Despite these central roles in virus infection, knowledge of the underlying molecular mechanisms by which MPs cause changes in plasmodesmata (PD) size exclusion limit and contribute to the formation of viral replication compartments remain far from being complete. Methods To further identify host processes subverted by viral MPs, we here characterized the MP of Japanese soil-borne wheat mosaic virus (JSBWMV). We used confocal fluorescence microscopy to study the subcellular localization of MP JSBWMV and to address its functionality in promoting virus cell-to-cell movement. Using the biochemical and biophysical methods co-immunoprecipitation, fluorescence lifetime imaging, microscale thermophoresis and RNA immunoprecipitation we investigate the capacity of MP JSBWMV to multimerize and to bind viral and cellular RNAs. Results MP JSBWMV localized to PD, promoted cell-to-cell movement by complementing a movement-deficient unrelated virus, formed multimers in-vivo and bound to viral RNA with high affinity. Using RNA immunoprecipitation, we identified host RNAs associated with the viral MP. Within the MP-RNA complexes we found RNAs encoding proteins with key functions in membrane modification, signaling, protein folding, and degradation. We propose that binding of MP to these RNAs during infection and regulation of their spatio-temporal translation may represent a mechanism for MPs to achieve PD and host control during replication and movement. Conclusion This study provides new insight into the complex interactions between viral MPs and host cellular processes.

The ubiquitin–proteasome system is a highly conserved machinery that plays a crucial role in plant defense against viruses. However, the number of E3 ligases targeting viral proteins remains limited. Although RING‐between‐RING (RBR)‐type E3 ligases are evolutionarily conserved across organisms, their functions in plant responses to biotic stress remain largely unknown. Herein, it is found that the triple gene block 1 (TGB1) protein of the barley stripe mosaic virus (BSMV) undergoes ubiquitination during viral infection. Immunoprecipitation combined with mass spectrometry identified an RBR‐type E3 ligase that interacted with TGB1 in vivo and in vitro. The overexpression of Ariadne‐like protein 8 (ARI8) inhibits, whereas its knockout enhances, the local and systemic spread of BSMV. ARI8 mediated the ubiquitination of TGB1, and its Cys311 residue is required for the ARI8‐mediated degradation of TGB1 and inhibition of BSMV infection. In addition to BSMV, ARI8 negatively regulates infection by other TGB‐containing viruses, including potato virus X and beet necrotic yellow vein virus. Collectively, the findings identified a new E3 ligase that targets a plant viral protein and reveals a previously uncharacterized role for RBR‐type E3 ligases in plant responses to biotic stress, providing a potential molecular target for the development of antiviral strategies in plants. The plant RING‐between‐RING (RBR)‐type E3 ligase, Ariadne‐like protein 8 (ARI8), ubiquitinates and degrades a viral movement protein, restricting virus spread. ARI8 also inhibits other triple gene block 1 (TGB1)‐containing viruses, revealing a broad‐spectrum antiviral role. This study uncovers a previously unknown function of RBR‐type E3 ligases in plant immunity and offers a potential target for antiviral crop improvement.

• Plant viruses encode movement proteins (MPs) that ensure the transport of viral genomes through plasmodesmata (PD) and use cell endomembranes, mostly the endoplasmic reticulum (ER), for delivery of viral genomes to PD and formation of PD-anchored virus replication compartments. • Here, we demonstrate that the Hibiscus green spot virus BMB2 MP, an integral ER protein, induces constrictions of ER tubules, decreases the mobility of ER luminal content, and exhibits an affinity to highly curved membranes. These properties are similar to those described for reticulons, cellular proteins that induce membrane curvature to shape the ER tubules. Similar to reticulons, BMB2 adopts a W-like topology within the ER membrane. • BMB2 targets PD and increases their size exclusion limit, and these BMB2 activities correlate with the ability to induce constrictions of ER tubules. We propose that the induction of ER constrictions contributes to the BMB2-dependent increase in PD permeability and formation of the PD-associated replication compartments, therefore facilitating the virus intercellular spread. • Furthermore, we show that the ER tubule constrictions also occur in cells expressing TGB2, one of the three MPs of Potato virus X (PVX), and in PVX-infected cells, suggesting that reticulon- like MPs are employed by diverse RNA viruses.

This study highlights the regulatory role of heat shock protein 90 (HSP90) in facilitating the cell-to-cell movement of broad bean wilt virus 2 (BBWV2). HSP90 interacted with VP37, the movement protein of BBWV2, specifically at plasmodesmata (PD). This study demonstrated that the HSP90-VP37 interaction is crucial for viral cell-to-cell movement and the formation of VP37-derived tubules, which are essential structures for virus transport through the PD. The ATP-dependent chaperone activity of HSP90 is integral to this interaction, as demonstrated by the inhibition of virus movement upon treatment with geldanamycin, which disrupts the function of HSP90. These findings elucidate the molecular mechanisms underlying the cell-to-cell movement of plant viruses and highlight the role of HSP90 in viral infection. This study suggests that the chaperone activity of HSP90 may function in changing the conformational structure of VP37, thereby facilitating the assembly and function of virus-induced structures required for viral cell-to-cell movement.

Tobacco mosaic virus movement protein (TMV MP) is essential for virus spread between cells. To accomplish its task, TMV MP binds viral RNA, interacts with components of the cytoskeleton, and increases the size exclusion limit (SEL) of plasmodesmata. Plasmodesmata are gated intercellular channels that allow passage of small molecules and macromolecules, including RNA and protein, between plant cells. Moreover, plasmodesmata are diverse and those connecting different cell types appear to have unique mechanisms to regulate macromolecular trafficking, which likely contributes to the establishment of distinct cell boundaries. Consequently, TMV MP might be competent to mediate RNA transport through some but not all plasmodesmal gates. Due to a lack of viral mutants defective for movement between specific cell types, the ability of TMV MP in this regard is incompletely understood. In contrast, a number of trafficking impaired Potato spindle tuber viroid (PSTVd) mutants have been identified. PSTVd is a systemically infectious non-coding RNA that nevertheless can perform all functions required for replication as well as cell-to-cell and systemic spread. Previous studies have shown that PSTVd employs different structure and sequence elements to move between diverse cell types in host plants, and mutants defective for transport between specific cell types have been identified. Therefore, PSTVd may serve as a tool to analyze the functions of MPs of viral and cellular origin. To probe the RNA transport activity of TMV MP, transgenic plants expressing the protein were inoculated with PSTVd mutants. Remarkably, TMV MP complemented a PSTVd mutant defective for mesophyll entry but could not support two mutants impaired for phloem entry, suggesting it fails to productively interface with plasmodesmata at the phloem boundary and that additional viral and host factors may be required. Consistent with this idea, TMV co-infection, but not the combination of MP and coat protein (CP) expression, was able to complement one of the phloem entry mutants. These observations suggest that phloem loading is a critical impediment to establishing systemic infection that could involve the entire ensemble of TMV proteins. They also demonstrate a novel strategy for analysis of MPs.

Numerous, diverse plant viruses encode movement proteins (MPs) that aid the virus movement through plasmodesmata, the plant intercellular channels. MPs are essential for virus spread and propagation in distal tissues, and several unrelated MPs have been identified. The 30K superfamily of MPs (named after the molecular mass of tobacco mosaic virus MP, the classical model of plant virology) is the largest and most diverse MP variety, represented in 16 virus families, but its evolutionary origin remained obscure. Here, we show that the core structural domain of the 30K MPs is homologous to the jelly-roll domain of the capsid proteins (CPs) of small RNA and DNA viruses, in particular, those infecting plants. The closest similarity was observed between the 30K MPs and the CPs of the viruses in the families Bromoviridae and Geminiviridae . We hypothesize that the MPs evolved via duplication or horizontal acquisition of the CP gene in a virus that infected an ancestor of vascular plants, followed by neofunctionalization of one of the paralogous CPs, potentially through the acquisition of unique N- and C-terminal regions. During the subsequent coevolution of viruses with diversifying vascular plants, the 30K MP genes underwent explosive horizontal spread among emergent RNA and DNA viruses, likely permitting viruses of insects and fungi that coinfected plants to expand their host ranges, molding the contemporary plant virome.

The size of viral genomes is limited, thus the majority of encoded proteins possess multiple functions. The main function of tobamoviral movement protein (MP) is to perform plasmodesmata gating and mediate intercellular transport of the viral RNA. MP is a remarkable example of a protein that, in addition to the initially discovered and most obvious function, carries out numerous activities that are important both for the manifestation of its key function and for successful and productive infection in general. Briefly, MP binds the viral genome, delivers it to the plasmodesmata (PD) and mediates its intercellular transfer. To implement the transport function, MP interacts with diverse cellular factors. Each of these cellular proteins has its own function, which could be different under normal conditions and upon viral infection. Here, we summarize the data available at present on the plethora of cellular factors that were identified as tobamoviral MP partners and analyze the role of these interactions in infection development.

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta . Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana . Mutating acidic residues (D/E-G) reduced droplet formation in vitro , increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.

Summary To establish systemic infection, plant viruses must replicate, and conduct intra‐ and intercellular movement and long‐distance movement, all of which require the participation of host factors. Tobamoviruses move in the form of movement protein (MP)–viral RNA complex and utilize endocytosis for intracellular movement. However, how tobamoviral MPs hijack host factors to reach the plasma membrane (PM) and then plasmodesmata (PD) is still largely unknown. Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus that mainly infects tomatoes and peppers. Here, we show that tomato RabE1a, a small Rab GTPase, interacts with ToBRFV MP and participates in the movement of ToBRFV. Knocking out RabE1a in tomatoes could inhibit infection by ToBRFV. RabE1a positively regulates MP transport to the PM, and this transport process is regulated by its nucleotide‐binding state. Furthermore, RabE1a interacts with the exocyst subunit SEC10b to jointly regulate MP transport to the PM and intracellular movement of ToBRFV. The adaptor protein AP2β interacts with MP and transports MP from the PM to the PD for intercellular movement of ToBRFV. We further find that knocking out RabE1a could also inhibit the infection of other tobamoviruses. In summary, MP exocytosis is coregulated by RabE1a and the exocyst subunit SEC10b for transport to the PM, where it then uses AP2β‐regulated endocytosis to PD. These results provide a comprehensive overview of tobamoviral MP intracellular transport and are insightful for breeding tomato plants resistant to ToBRFV and related tobamoviruses.