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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., (© 2024 His Majesty the King in Right of Canada. New Phytologist © 2024 New Phytologist Foundation. Reproduced with the permission of the Minister of Agricultural and Agri‐Food Canada.)

Emerging evidence suggests that the localization of viral movement proteins (MPs) to both plasmodesmata (PD) and viral replication complexes (VRCs) is the key to viral cell-to-cell movement. However, the molecular mechanism that establishes the subcellular localization of MPs is not fully understood. Here, we investigated the PD localization pathway of red clover necrotic mosaic virus (RCNMV) MP and the functional regions of MP that are crucial for MP localization to PD and VRCs. Disruption analysis of the transport pathway suggested that RCNMV MP does not rely on the ER-Golgi pathway or the cytoskeleton for the localization to the PD. Furthermore, mutagenesis analysis identified amino acid residues within the alpha helix regions responsible for localization to the PD or VRCs. These α-helix regions were also essential for efficient viral cell-to-cell movement, highlighting the importance of these dynamic localization of the MPs for viral infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pear chlorotic leaf spot-associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single-stranded, negative-sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens-mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self-interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell-to-cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N-terminal 1-89 amino acids of the P5 were responsible for the self-interaction ability and RSS activity, for which the signal peptide at positions 1-19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Viruses employ a series of diverse translational strategies to expand their coding capacity, which produces viral proteins with common domains and entangles virus-host interactions. P3N-PIPO, which is a transcriptional slippage product from the P3 cistron, is a potyviral protein dedicated to intercellular movement. Here, we show that P3N-PIPO from watermelon mosaic virus (WMV) triggers cell death when transiently expressed in Cucumis melo accession PI 414723 carrying the Wmr resistance gene. Surprisingly, expression of the P3N domain, shared by both P3N-PIPO and P3, can alone induce cell death, whereas expression of P3 fails to activate cell death in PI 414723. Confocal microscopy analysis revealed that P3N-PIPO targets plasmodesmata (PD) and P3N associates with PD, while P3 localizes in endoplasmic reticulum in melon cells. We also found that mutations in residues L35, L38, P41, and I43 of the P3N domain individually disrupt the cell death induced by P3N-PIPO, but do not affect the PD localization of P3N-PIPO. Furthermore, WMV mutants with L35A or I43A can systemically infect PI 414723 plants. These key residues guide us to discover some WMV isolates potentially breaking the Wmr resistance. Through searching the NCBI database, we discovered some WMV isolates with variations in these key sites, and one naturally occurring I43V variation enables WMV to systemically infect PI 414723 plants. Taken together, these results demonstrate that P3N-PIPO, but not P3, is the avirulence determinant recognized by Wmr, although the shared N terminal P3N domain can alone trigger cell death.IMPORTANCEThis work reveals a novel viral avirulence (Avr) gene recognized by a resistance (R) gene. This novel viral Avr gene is special because it is a transcriptional slippage product from another virus gene, which means that their encoding proteins share the common N-terminal domain but have distinct C-terminal domains. Amazingly, we found that it is the common N-terminal domain that determines the Avr-R recognition, but only one of the viral proteins can be recognized by the R protein to induce cell death. Next, we found that these two viral proteins target different subcellular compartments. In addition, we discovered some virus isolates with variations in the common N-terminal domain and one naturally occurring variation that enables the virus to overcome the resistance. These results show how viral proteins with common domains interact with a host resistance protein and provide new evidence for the arms race between plants and viruses., Competing Interests: The authors declare no conflict of interest.

Cucumber mosaic virus (CMV, Cucumovirus , Bromoviridae) is an economically significant virus infecting important horticultural and field crops. Current knowledge regarding the specific functions of its movement protein (MP) is still incomplete. In the present study, potential post-translational modification sites of its MP were assayed with mutant viruses: MP/S28A, MP/S28D, MP/S120A and MP/S120D. Ser28 was identified as an important factor in viral pathogenicity on Nicotiana tabacum cv. Xanthi, Cucumis sativus and Chenopodium murale . The subcellular localization of GFP-tagged movement proteins was determined with confocal laser-scanning microscopy. The wild type movement protein fused to green fluorescent protein (GFP) (MP-eGFP) greatly colocalized with callose at plasmodesmata, while MP/S28A-eGFP and MP/S28D-eGFP were detected as punctate spots along the cell membrane without callose colocalization. These results underline the importance of phosphorylatable amino acids in symptom formation and provide data regarding the essential factors for plasmodesmata localization of CMV MP.

Background: Plant viruses move through plasmodesmata (PD) to infect new cells. To overcome the PD barrier, plant viruses have developed specific protein(s) to guide their genomic RNAs or DNAs to path through the PD., Results: In the present study, we analyzed the function of Pepper vein yellows virus P4 protein. Our bioinformatic analysis using five commonly used algorithms showed that the P4 protein contains an transmembrane domain, encompassing the amino acid residue 117-138. The subcellular localization of P4 protein was found to target PD and form small punctates near walls. The P4 deletion mutant or the substitution mutant constructed by overlap PCR lost their function to produce punctates near the walls inside the fluorescent loci. The P4-YFP fusion was found to move from cell to cell in infiltrated leaves, and P4 could complement Cucumber mosaic virus movement protein deficiency mutant to move between cells., Conclusion: Taking together, we consider that the P4 protein is a movement protein of Pepper vein yellows virus.

Plasmodesmata (PD) are essential for intercellular trafficking of molecules required for plant life, from small molecules like sugars and ions to macromolecules including proteins and RNA molecules that act as signals to regulate plant development and defense. As obligate intracellular pathogens, plant viruses have evolved to manipulate this communication system to facilitate the initial cell-to-cell and eventual systemic spread in their plant hosts. There has been considerable interest in how viruses manipulate the PD that connect the protoplasts of neighboring cells, and viruses have yielded invaluable tools for probing the structure and function of PD. With recent advances in biochemistry and imaging, we have gained new insights into the composition and structure of PD in the presence and absence of viruses. Here, we first discuss viral strategies for manipulating PD for their intercellular movement and examine how this has shed light on our understanding of native PD function. We then address the controversial role of the cytoskeleton in trafficking to and through PD. Finally, we address how viruses could alter PD structure and consider possible mechanisms of the phenomenon described as 'gating'. This discussion supports the significance of virus research in elucidating the properties of PD, these persistently enigmatic plant organelles.

Pepino mosaic virus (PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor ( Sl TXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. Sl TXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamily closest to phosducin-like protein-3. In PepMV-infected and healthy Nicotiana benthamiana plants, Nb TXND9 mRNA levels were comparable, and expression levels remained stable in both local and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α- Sl TXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27⁻35 kDa, in the 1000 and 30,000 g pellets, and the soluble fraction of extracts of healthy and PepMV-infected N. benthamiana leaves, but not in the cell wall. These bands likely consist of the homologous protein Nb TXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labelling of ultrathin sections of PepMV-infected N. benthamiana leaves using α- Sl TXND9 IgG revealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress.

Key Message: Citrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle. The genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.

Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement. Consistent with this hypothesis, we show that the MP of CPsV (MP CPsV ) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MP CPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MP CPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34K CPsV ), but not the 20-kDa fragment (20K CPsV ), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MP CPsV (and also the 34K CPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20K CPsV retains the protease activity and is able to cleave a cleavage-deficient MP CPsV in trans Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MP CPsV by an aspartic protease activity, which removes the 20K CPsV protease and thereby releases the 34K CPsV protein for PDLP1-dependent tubule formation at PD. IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34K CPsV and 20K CPsV , that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control., (Copyright © 2018 American Society for Microbiology.)

Plant viruses cross the barrier of the plant cell wall by moving through intercellular channels, termed plasmodesmata, to invade their hosts. They accomplish this by encoding movement proteins (MPs), which act to alter plasmodesmal gating. How MPs target to plasmodesmata is not well understood. Our recent characterization of the first plasmodesmal localization signal (PLS) identified in a viral MP, namely, the MP encoded by the Tobamovirus Tobacco mosaic virus (TMV), now provides the opportunity to identify host proteins that recognize this PLS and may be important for its plasmodesmal targeting. One such candidate protein is Arabidopsis synaptotagmin A (SYTA), which is required to form endoplasmic reticulum (ER)-plasma membrane contact sites and regulates the MP-mediated trafficking of begomoviruses, tobamoviruses, and potyviruses. In particular, SYTA interacts with, and regulates the cell-to-cell transport of, both TMV MP and the MP encoded by the Tobamovirus Turnip vein clearing virus (TVCV). Using in planta bimolecular fluorescence complementation (BiFC) and yeast two-hybrid assays, we show here that the TMV PLS interacted with SYTA. This PLS sequence was both necessary and sufficient for interaction with SYTA, and the plasmodesmal targeting activity of the TMV PLS was substantially reduced in an Arabidopsis syta knockdown line. Our findings show that SYTA is one host factor that can recognize the TMV PLS and suggest that this interaction may stabilize the association of TMV MP with plasmodesmata. IMPORTANCE Plant viruses use their movement proteins (MPs) to move through host intercellular connections, plasmodesmata. Perhaps one of the most intriguing, yet least studied, aspects of this transport is the MP signal sequences and their host recognition factors. Recently, we have described the plasmodesmal localization signal (PLS) of the Tobacco mosaic virus (TMV) MP. Here, we identified the Arabidopsis synaptotagmin A (SYTA) as a host factor that recognizes TMV MP PLS and promotes its association with the plasmodesmal membrane. The significance of these findings is two-fold: (i) we identified the TMV MP association with the cell membrane at plasmodesmata as an important PLS-dependent step in plasmodesmal targeting, and (ii) we identified the plant SYTA protein that specifically recognizes PLS as a host factor involved in this step., (Copyright © 2018 Yuan et al.)

The nonstructural protein pc6 encoded by rice grassy stunt virus (RGSV) plays a significant role in viral cell-to-cell movement, presumably by transport through plasmodesmata (PD). We confirmed the association of pc6 with PD, and also elucidated the mechanisms of protein targeting to PD. Several inhibitor treatments showed conclusively that pc6 is targeted to PD via the ER-to-Golgi secretory system and actin filaments. In addition, VIII-1 myosin was also found to be involved in pc6 PD targeting. Deletion mutants demonstrated that C-terminal amino acid residues 209-229 (transmembrane domain 2; TM2) are essential for pc6 to move through PD.

Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD.

Hibiscus green spot virus (HGSV) is a recently discovered and so far poorly characterized bacilliform plant virus with a positive-stranded RNA genome consisting of three RNA species. Here, we demonstrate that the proteins encoded by the ORF2 and ORF3 in HGSV RNA2 are necessary and sufficient to mediate cell-to-cell movement of transport-deficient Potato virus X in Nicotiana benthamiana. These two genes represent a specialized transport module called a 'binary movement block' (BMB), and ORF2 and ORF3 are termed BMB1 and BMB2 genes. In agroinfiltrated epidermal cells of N. benthamiana, green fluorescent protein (GFP)-BMB1 fusion protein was distributed diffusely in the cytoplasm and the nucleus. However, in the presence of BMB2, GFP-BMB1 was directed to cell wall-adjacent elongated bodies at the cell periphery, to cell wall-embedded punctate structures co-localizing with callose deposits at plasmodesmata, and to cells adjacent to the initially transformed cell. Thus, BMB2 can mediate the transport of BMB1 to and through plasmodesmata. In general, our observations support the idea that cell-to-cell trafficking of movement proteins involves an initial delivery to membrane compartments adjacent to plasmodesmata, subsequent entry of the plasmodesmata cavity and, finally, transport to adjacent cells. This process, as an alternative to tubule-based transport, has most likely evolved independently in triple gene block (TGB), double gene block (DGB), BMB and the single gene-coded transport system., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

The tomato Tobacco mosaic virus resistance-2 2 ( Tm-2 2 ) gene encodes a coiled-coil-nucleotide binding site-Leu-rich repeat protein lacking a conventional plasma membrane (PM) localization motif. Tm-2 2 confers plant extreme resistance against tobamoviruses including Tobacco mosaic virus (TMV) by recognizing the avirulence (Avr) viral movement protein (MP). However, the subcellular compartment where Tm-2 2 functions is unclear. Here, we demonstrate that Tm-2 2 interacts with TMV MP to form a protein complex at the PM We show that both inactive and active Tm-2 2 proteins are localized to the PM When restricted to PM by fusing Tm-2 2 to the S -acylated PM association motif, the Tm-2 2 fusion protein can still induce a hypersensitive response cell death, consistent with its activation at the PM Through analyses of viral MP mutants, we find that the plasmodesmata (PD) localization of the Avr protein MP is not required for Tm-2 2 function. These results suggest that Tm-2 2 -mediated resistance takes place on PM without requirement of its Avr protein to be located to PD., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The movement of some plant viruses are accomplished by three proteins encoded by a triple gene block (TGB). The second protein (TGBp2) in the block is a transmembrane protein. This study was aimed to unravel the mechanism underlying the relatively inefficient cell-to-cell movement of Bamboo mosaic virus (BaMV) caused by amino acid substitutions for the three Cys residues, Cys-109, Cys-112 and Cys-119, at the C-terminal tail of TGBp2. Results from confocal microscopy revealed that substitutions of the three Cys residues of TGBp2, especially Cys-109 and Cys-112, would reduce the efficiency of TGBp2- and TGBp3-dependent PD localization of TGBp1. Moreover, there is an additive effect of the substitutions on reducing the efficiency of PD localization of TGBp1. These results indicate that the Cys residues in the C-terminal tail region of TGBp2 participate in the TGBp2- and TGBp3-dependent PD localization of TGBp1, and thus influence the cell-to-cell movement capability of BaMV., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The movement protein NSm of Tomato spotted wilt tospovirus (TSWV) plays pivotal roles in viral intercellular trafficking. Recently, the TSWV NSm was also identified as an avirulence (Avr) determinant during the Sw-5b-mediated hypersensitive response (HR). However, whether the cell-to-cell movement of NSm is coupled to its function in HR induction remains obscure. Here, we showed that the NSm mutants defective in targeting plasmodesmata and cell-to-cell movement were still capable of inducing Sw-5b-mediated HR. In addition, introduction of a single amino-acid substitution, C118Y or T120N, identified previously from TSWV resistance-breaking isolates, into the movement-defective NSm mutants resulted in the failure of HR induction. Collectively, our results showed that the intercellular trafficking of NSm is uncoupled from its function in HR induction. These findings shed light on the evolutionary mechanism of R-Avr recognition and may be used to explain why this uncoupled phenomenon can be observed in many different viruses.

Cowpea mosaic virus forms tubules constructed from the movement protein (MP) in plasmodesmata (PD) to achieve cell-to-cell movement of its virions. Similar tubules, delineated by the plasma membrane (PM), are formed protruding from the surface of infected protoplasts. These PM-tubule complexes were isolated from protoplasts by immunoprecipitation and analysed for their protein content by tandem mass spectrometry to identify host proteins with affinity for the movement tubule. Seven host proteins were abundantly present in the PM-tubule complex, including molecular chaperonins and an AAA protein. Members of both protein families have been implicated in establishment of systemic infection. The potential role of these proteins in tubule-guided cell-cell transport is discussed.

The movement protein VP37 of broad bean wilt virus 2 (BBWV 2) forms tubules in the plasmodesmata (PD) for the transport of virions between cells. This paper reports a mutual association between the BBWV 2 VP37-tubule complex and PD at the cytological level as determined by transmission electron microscopy. The generation of VP37-tubules within different PD leads to a different occurrence frequency as well as different morphology lines of virus-like particles. In addition, the frequency of VP37-tubules was different between PD found at different cellular interfaces, as well as between single-lined PD and branched PD. VP37-tubule generation also induced structural alterations of PD as well as modifications to the cell wall (CW) in the vicinity of the PD. A structural comparison using three-dimensional (3D) electron tomography (ET), determined that desmotubule structures found in the center of normal PD were absent in PD containing VP37-tubules. Using gold labeling, modification of the CW by callose deposition and cellulose reduction was observable on PD containing VP37-tubule. These cytological observations provide evidence of a mutual association of MP-derived tubules and PD in a natural host, improving our fundamental understanding of interactions between viral MP and PD that result in intercellular movement of virus particles.

Citrus tristeza virus (CTV), one of the most economically important viruses, produces a unique protein, p33, which is encoded only in the genomes of isolates of CTV. Recently, we demonstrated that membrane association of the p33 protein confers virus ability to extend its host range. In this work we show that p33 shares characteristics of viral movement proteins. Upon expression in a host cell, the protein localizes to plasmodesmata and displays the ability to form extracellular tubules. Furthermore, p33 appears to traffic via the cellular secretory pathway and the actin network to plasmodesmata locations and is likely being recycled through the endocytic pathway. Finally, our study reveals that p33 colocalizes with a putative movement protein of CTV, the p6 protein. These results suggest a potential role of p33 as a noncanonical viral movement protein, which mediates virus translocation in the specific hosts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The multifunctional cylindrical inclusion (CI) protein of potyviruses contains ATP binding and RNA helicase activities. As part of the viral replication complex, it assists viral genome replication, possibly by binding to RNA and unwinding the RNA duplex. It also functions in viral cell-to-cell movement, likely via the formation of conical structures at plasmodesmata (PD) and the interaction with coat protein (CP)., Methods: To further understand the role of CI in the viral infection process, we employed the alanine-scanning mutagenesis approach to mutate CI in the infectious full-length cDNA clone of Turnip mosaic virus (TuMV) tagged by green fluorescent protein. A total of 40 double-substitutions were made at the clustered charged residues. The effect of these mutations on viral genome amplification was determined using a protoplast inoculation assay. All the mutants were also introduced into Nicotiana benthamiana plants to assess their cell-to-cell and long-distance movement. Three cell-to-cell movement-abolished mutants were randomly selected to determine if their mutated CI protein targets PD and interacts with CP by confocal microscopy., Results: Twenty CI mutants were replication-defective (5 abolished and 15 reduced), one produced an elevated level of viral genome in comparison with the parental virus, and the remaining 19 retained the same replication level as the parental virus. The replication-defective mutations were predominately located in the helicase domains and C-terminal region. All 15 replication-reduced mutants showed delayed or abolished cell-to-cell movement. Nine of 20 replication-competent mutants contained infection within single cells. Five of them distributed mutations within the N-terminal 100 amino acids. Most of replication-defective or cell-to-cell movement-abolished mutants failed to infect plants systemically. Analysis of three randomly selected replication-competent yet cell-to-cell movement-abolished mutants revealed that the mutated CI failed to form regular punctate structures at PD and/or to interact with CP., Conclusions: The helicase domain and C-terminal region of TuMV CI are essential for viral genome replication, and the N-terminal sequence modulates viral cell-to-cell movement. TuMV CI plays both interlinked and distinct roles in replication and intercellular movement. The ability of CI to target PD and interact with CP is associated with its functional role in viral cell-to-cell movement.

Plant viruses exploit the host machinery for targeting the viral genome-movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER-GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER-GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130-138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm interacts with NP via its N-terminal unfolded region and the NSm-NP complex could in turn interact with the ER membrane via the C-terminal coiled coil domain of NSm to form vesicles that are targeted to PD and there by assist the cell to cell movement of the viral genome complex., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Phloem vasculature is the route that most plant viruses use to spread widely around the plant. In addition, phloem sap transports signals that trigger systemic defense responses to infection. We investigated the proteome-level changes that occur in phloem sap during virus infection using the 2D-DIGE technique. Total proteins were extracted from phloem exudates of healthy and Melon necrotic spot virus infected melon plants and analyzed by 2D-DIGE. A total of 1046 spots were detected but only 25 had significant changes in abundance. After mass spectrometry, 19 different proteins corresponding to 22 spots were further identified (13 of them up-accumulated and 9 down-accumulated). Most of them were involved in controlling redox balance and cell death. Only two of the differentially altered proteins had never been described to be present in the phloem before: a carboxylesterase and the fumarylacetoacetate hydrolase 1, both considered negative regulators of cell death. RT-PCR analysis of phloem sap RNAs revealed that the transcripts corresponding to some of the identified protein could be also loaded into the sieve elements. The impact of these proteins in the host response against viral infections and the potential involvement in regulating development, growth and stress response in melon plants is discussed., Biological Significance: Despite the importance of phloem as an integrative pathway for resource distribution, signaling and plant virus transport little is known about the modifications induced by these pathogens in phloem sap proteome. Only one previous study has actually examined the phloem sap proteome during viral infection using conventional two-dimensional electrophoresis. Since the major limitation of this technique has been its low sensitivity, the authors only identified five phloem proteins with altered abundance. To circumvent this issue we use two-dimensional difference in-gel electrophoresis (2D DIGE) technique, which combined with DeCyder Differential Analysis Software allows a more accurate and sensitive quantitative analysis than with conventional 2D PAGE. We identified 19 different proteins which accumulation in phloem sap was altered during a compatible plant virus infection including redox and hypersensitivity response-related proteins. Therefore, this work would help to understand the basic processes that occur in phloem during plant-virus interaction., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

Subcellular, sequence-specific detection of RNA in vivo is a powerful tool to study the macromolecular transport that occurs through plasmodesmata. The RNA-binding domain of Pumilio proteins can be engineered to bind RNA sequences of choice and fused to fluorescent proteins for RNA imaging. This chapter describes the construction of a Pumilio-based imaging system to track the RNA of Tobacco mosaic virus in vivo, and practical aspects of RNA live-cell imaging.

It is generally accepted that in order to establish a systemic infection in a plant, viruses move from the initially infected cell to the vascular tissues by cell-to-cell movement through plasmodesmata (PD), and load into the vascular conducting tubes (i.e. phloem sieve elements and xylem vessel elements) for long-distance movement. The viral unit in these movements can be a virion or a yet-to-be-defined ribonucleic protein (RNP) complex. Using live-cell imaging, our laboratory has previously demonstrated that membrane-bound replication complexes move cell-to-cell during turnip mosaic virus (TuMV) infection. Our recent study shows that these membrane-bound replication complexes end up in the vascular conducting tubes, which is likely the case for potato virus X (PVX) also. The presence of TuMV-induced membrane complexes in xylem vessels suggests that viral components could also be found in other apoplastic regions of the plant, such as the intercellular space. This possibility may have implications regarding how we approach the study of plant innate immune responses against viruses.

Plant viruses utilize plasmodesmata (PD), unique membrane-lined cytoplasmic nanobridges in plants, to spread infection cell-to-cell and long-distance. Such invasion involves a range of regulatory mechanisms to target and modify PD. Exciting discoveries in this field suggest that these mechanisms are executed by the interaction between plant cellular components and viral movement proteins (MPs) or other virus-encoded factors. Striking working analogies exist among endogenous non-cell-autonomous proteins and viral MPs, in which not only do they all use PD to traffic, but also they exploit same regulatory components to exert their functions. Thus, this review discusses on the viral strategies to move via PD and the PD-regulatory mechanisms involved in viral pathogenesis.

The symplastic communication network established by plasmodesmata (PD) and connected phloem provides an essential pathway for spatiotemporal intercellular signaling in plant development but is also exploited by viruses for moving their genomes between cells in order to infect plants systemically. Virus movement depends on virus-encoded movement proteins (MPs) that target PD and therefore represent important keys to the cellular mechanisms underlying the intercellular trafficking of viruses and other macromolecules. Viruses and their MPs have evolved different mechanisms for intracellular transport and interaction with PD. Some viruses move from cell to cell by interacting with cellular mechanisms that control the size exclusion limit of PD whereas other viruses alter the PD architecture through assembly of specialized transport structures within the channel. Some viruses move between cells in the form of assembled virus particles whereas other viruses may interact with nucleic acid transport mechanisms to move their genomes in a non-encapsidated form. Moreover, whereas several viruses rely on the secretory pathway to target PD, other viruses interact with the cortical endoplasmic reticulum and associated cytoskeleton to spread infection. This chapter provides an introduction into viruses and their role in studying the diverse cellular mechanisms involved in intercellular PD-mediated macromolecular trafficking.

The trafficking and asymmetric distribution of cytoplasmic RNA is a fundamental process during development and signaling across phyla. Plants support the intercellular trafficking of RNA molecules such as gene transcripts, small RNAs, and viral RNA genomes by targeting these RNA molecules to plasmodesmata (PD). Intercellular transport of RNA molecules through PD has fundamental implications in the cell-to-cell and systemic signaling during plant development and in the systemic spread of viral disease. Recent advances in time-lapse microscopy allow researchers to approach dynamic biological processes at the molecular level in living cells and tissues. These advances include the ability to label RNA molecules in vivo and thus to monitor their distribution and trafficking. In a broadly used RNA labeling approach, the MS2 method, the RNA of interest is tagged with a specific stem-loop (SL) RNA sequence derived from the origin of assembly region of the bacteriophage MS2 genome that binds to the bacteriophage coat protein (CP) and which, if fused to a fluorescent protein, allows the visualization of the tagged RNA by fluorescence microscopy. Here we describe a protocol for the in vivo visualization of transiently expressed SL-tagged RNA and discuss key aspects to study RNA localization and trafficking to and through plasmodesmata in Nicotiana benthamiana plants.

In this study, we investigated the fine regulation of cell-to-cell movement of Bamboo mosaic virus (BaMV). We report that the coat protein (CP) of BaMV is phosphorylated in planta at position serine 241 (S241), in a process involving Nicotiana benthamiana casein kinase 2α (NbCK2α). BaMV CP and NbCK2α colocalize at the plasmodesmata, suggesting that phosphorylation of BaMV may be involved in its movement. S241 was mutated to examine the effects of temporal and spatial dysregulation of phosphorylation on i) the interactions between CP and viral RNA and ii) the regulation of cell-to-cell movement. Replacement of S241 with alanine did not affect RNA binding affinity but moderately impaired cell-to-cell movement. A negative charge at position 241 reduced the ability of CP to bind RNA and severely interfered with cell-to-cell movement. Deletion of residues 240 to 242 increased the affinity of CP to viral RNA and dramatically impaired cell-to-cell movement. A threonine at position 241 changed the binding preference of CP toward genomic RNA and inhibited cell-to-cell movement. Together, these results reveal a fine regulatory mechanism for the cell-to-cell movement of BaMV, which involves the modulation of RNA binding affinity through appropriate phosphorylation of CP by NbCK2α.

Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

The causal agents of Citrus leprosis are viruses; however, extant diagnostic methods to identify them have failed to detect known viruses in orange, mandarin, lime and bitter orange trees with severe leprosis symptoms in Mexico, an important citrus producer. Using high throughput sequencing, a virus associated with citrus leprosis was identified, belonging to the proposed Dichorhavirus genus. The virus was termed Citrus Necrotic Spot Virus (CNSV) and contains two negative-strand RNA components; virions accumulate in the cytoplasm and are associated with plasmodesmata-channels interconnecting neighboring cells-suggesting a mode of spread within the plant. The present study provides insights into the nature of this pathogen and the corresponding plant response, which is likely similar to other pathogens that do not spread systemically in plants.

The Triple Gene Block 1 (TGBp1) protein encoded by the Potato virus X is a multifunctional protein that acts as a suppressor of RNA silencing or facilitates the passage of virus from cell to cell by promoting the plasmodesmata opening. We previously showed that the membrane raft protein StRemorin1.3 is able to impair PVX infection. Here, we show that overexpressed StRemorin1.3 does not impair the silencing suppressor activity of TGBp1, but affects its ability to increase plasmodesmata permeability. A similar effect on plasmodesmata permeability was observed with other movement proteins, suggesting that REM is a general regulator of plasmodesmal size exclusion limit. These results add to our knowledge of the mechanisms underlying the StREM1.3 role in virus infection., (Copyright © 2014. Published by Elsevier B.V.)

The pc4 protein encoded by rice stripe virus (RSV) is a viral movement protein (MP). A transmembrane (TM) domain spanning AAs 106-123 of pc4 was identified and shown to be essential for localization of pc4 to plasmodesmata (PD) (but not to chloroplasts) and for its ability to recover the movement of movement-deficient PVX. Analysis of alanine-scanning mutants showed that M116A and G120A had a similar localization to wild type pc4, being localized at PD and chloroplasts, but all other mutants were only localized at chloroplasts and not at PD. Mutants that could not be localized at PD had little (G123A) or no ability to recover PVX-GFPΔp25 movement, indicating that PD localization is crucial for the function of pc4 as a movement protein. Strangely, mutants M116A and G120A localized at PD and retained the ability to bind single-stranded RNA but did not support PVX-GFPΔp25 movement, indicating that properties other than PD localization and nucleotide binding ability may be needed for the function of pc4 as a movement protein., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A unique feature shared by all plant viruses of the Potyviridae family is the induction of characteristic pinwheel-shaped inclusion bodies in the cytoplasm of infected cells. These cylindrical inclusions are composed of the viral-encoded cylindrical inclusion helicase (CI protein). Its helicase activity was characterized and its involvement in replication demonstrated through different reverse genetics approaches. In addition to replication, the CI protein is also involved in cell-to-cell and long-distance movements, possibly through interactions with the recently discovered viral P3N-PIPO protein. Studies over the past two decades demonstrate that the CI protein is present in several cellular compartments interacting with viral and plant protein partners likely involved in its various roles in different steps of viral infection. Furthermore, the CI protein acts as an avirulence factor in gene-for-gene interactions with dominant-resistance host genes and as a recessive-resistance overcoming factor. Although a significant amount of data concerning the potential functions and subcellular localization of this protein has been published, no synthetic review is available on this important multifunctional protein. In this review, we compile and integrate all information relevant to the current understanding of this viral protein structure and function and present a mode of action for CI, combining replication and movement.

After replication in the cytoplasm, viruses spread from the infected cell into the neighboring cells through plasmodesmata, membranous channels embedded by the cell wall. As obligate parasites, viruses have acquired the ability to utilize host factors that unwillingly cooperate for the viral infection process. For example, the viral movement proteins (MP) interacts with the host pectin methylesterase (PME) and both proteins cooperate to sustain the viral spread. However, how and where PMEs interact with MPs and how the PME/MP complexes favor the viral translocation is not well understood. Recently, we demonstrated that the overexpression of PME inhibitors (PMEIs) in tobacco and Arabidopsis plants limits the movement of Tobacco mosaic virus and Turnip vein clearing virus and reduces plant susceptibility to these viruses. Here we discuss how overexpression of PMEI may reduce tobamovirus spreading.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form β-strands, namely β1 and β2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the β1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within β1 without preventing the membrane association or movement function of P4.

β-1,3-Glucanases (BG) have been implicated in enhancing virus spread by degrading callose at plasmodesmata (Pd). Here, we investigate the role of Arabidopsis BG in tobamovirus spread. During Turnip vein clearing virus infection, the transcription of two pathogenesis-related (PR)-BG AtBG2 and AtBG3 increased but that of Pd-associated BG AtBG_pap did not change. In transgenic plants, AtBG2 was retained in the endoplasmic reticulum (ER) network and was not secreted. As a stress response mediated by salicylic acid, AtBG2 was secreted and appeared as a free extracellular protein localized in the entire apoplast but did not accumulate at Pd sites. At the leading edge of Tobacco mosaic virus spread, AtBG2 co-localized with the viral movement protein in the ER-derived bodies, similarly to other ER proteins, but was not secreted to the cell wall. In atbg2 mutants, callose levels at Pd and virus spread were unaffected. Likewise, AtBG2 overexpression had no effect on virus spread. However, in atbg_pap mutants, callose at Pd was increased and virus spread was reduced. Our results demonstrate that the constitutive Pd-associated BG but not the stress-regulated extracellular PR-BG are directly involved in regulation of callose at Pd and cell-to-cell transport in Arabidopsis, including the spread of viruses.

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum-derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5' end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.

Rice black-streaked dwarf virus (RBSDV), a plant-infecting reovirus (genus Fijivirus), generally induces virus-containing tubules in infected cells. The nonstructural protein P7-1, encoded by the first open reading frame of segment 7, is involved in forming the structural matrix of these tubules. In experiments to investigate the subcellular localization of P7-1 in Nicotiana benthamiana epidermal cells, fluorescence of P7-1:eGFP was observed in the nucleus, cytoplasm and cell periphery, and in punctate points along the cell wall of plasmolyzed cells. Co-localization with plasmodesmata-located protein 1 showed that P7-1 formed the punctate points at plasmodesmata. Mutational analysis demonstrated that transmembrane domain 1 and adjacent residues were necessary and sufficient for P7-1 to form punctate structures at the cell wall in the plasmolyzed cells. Chemical drug and protein inhibitor treatments indicated that P7-1 utilized the ER-to-Golgi secretory pathway and the actomyosin motility system for its intracellular transport. The plasmodesmatal localization of RBSDV P7-1 is therefore dependent on the secretory pathway and the actomyosin motility system.

Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.

Plant virus infection spreads from cell-to-cell within the host with the aid of viral movement proteins (MPs) that transport infectious genomes through intercellular pores called plasmodesmata (PD). MPs are able to accomplish RNA trafficking independent of virus infection. However, although dispensable for replication, they often associate with or assist in the formation of viral replication complexes. Quantitative analyses of genetic bottlenecks during infection, as well as considerations of transport specificity, suggest that intricate links between replication and movement may facilitate efficient delivery of plant viruses through PD during early infection, at a stage when viral genomes are still rare., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing virus (RTYV), has been postulated to encode a cell-to-cell movement protein (MP). Trans-complementation experiments using a movement-defective tomato mosaic virus and the P3 protein of RTYV, encoded by gene 3, facilitated intercellular transport of the mutant virus. In transient-expression experiments with the GFP-fused P3 protein in epidermal leaf cells of Nicotiana benthamiana, the P3 protein was associated with the nucleus and plasmodesmata. Immunogold-labelling studies of thin sections of RTYV-infected rice plants using an antiserum against Escherichia coli-expressed His(6)-tagged P3 protein indicated that the P3 protein was located in cell walls and on virus particles. In Western blots using antisera against E. coli-expressed P3 protein and purified RTYV, the P3 protein was detected in purified RTYV, whilst antiserum against purified RTYV reacted with the E. coli-expressed P3 protein. After immunogold labelling of crude sap from RTYV-infected rice leaves, the P3 protein, as well as the N protein, was detected on the ribonucleocapsid core that emerged from partially disrupted virus particles. These results provide evidence that the P3 protein of RTYV, which functions as a viral MP, is a viral structural protein and seems to be associated with the ribonucleocapsid core of virus particles.

Objective: Expression and subcellular location of NSm protein of Tomato spotted wilt virus were studied using plant and insect cells., Methods: First, the NSm gene, located on the ambisense M RNA segment of tomato spotted wilt virus, was cloned into the pCHF3 vector which includes a GFP gene. Agrobacterium-mediated transient expression from N. benthamiana leaves was used to study the location of NSm in plant cells. Second, to test whether plant-specific components were involved in tubule formation, the NSm gene was also expressed in a heterologous expression system, i. e., insect cells. T. ni (Tn) cells were infected with a recombinant baculovirus expressing the NSm gene., Results: NSm-GFP fusion proteins diffused in the tobacco epidermal cells and were located at the edge of the cell walls. These proteins can also form discontinuous green fluorescent spots at the plasmodesmata, which were sometimes present in pairs between two neighboring cells. However, GFP proteins expressed alone distributed evenly around the cell wall and in the nucleus. In the entomic Tn cells, NSm proteins formed a large number of tubular structures extending from the surface., Conclusion: These findings suggest that NSm protein target the plasmodesmata specifically in plant cells, and they also could form tubular structures on the surface when expressed in entomic Tn cells.

Callose is a β-l,3-glucan with diverse roles in the viral pathogenesis of plants. It is widely believed that the deposition of callose and hypersensitive reaction (HR) are critical defence responses of host plants against viral infection. However, the sequence of these two events and their resistance mechanisms are unclear. By exploiting a point inoculation approach combined with aniline blue staining, immuno-electron microscopy and external sphincters staining with tannic acid, we systematically investigated the possible roles of callose deposition during viral infection in soybean. In the incompatible combination, callose deposition at the plasmodesmata (PD) was clearly visible at the sites of inoculation but viral RNA of coat protein (CP-RNA) was not detected by RT-PCR in the leaf above the inoculated one (the upper leaf). In the compatible combination, however, callose deposition at PD was not detected at the site of infection but the viral CP-RNA was detected by RT-PCR in the upper leaf. We also found that in the incompatible combination the fluorescence due to callose formation at the inoculation point disappeared following the injection of 2-deoxy-D-glucose (DDG, an inhibitor of callose synthesis). At same time, in the incompatible combination, necrosis was observed and the viral CP-RNA was detected by RT-PCR in the upper leaf and HR characteristics were evident at the inoculation sites. These results show that, during the defensive response of soybean to viral infection, callose deposition at PD is mainly responsible for restricting the movement of the virus between cells and it occurs prior to the HR response.

Potato virus X (PVX) requires three virally encoded proteins, the triple gene block (TGB), for movement between cells. TGB1 is a multifunctional protein that suppresses host gene silencing and moves from cell to cell through plasmodesmata, while TGB2 and TGB3 are membrane-spanning proteins associated with endoplasmic reticulum-derived granular vesicles. Here, we show that TGB1 organizes the PVX "X-body," a virally induced inclusion structure, by remodeling host actin and endomembranes (endoplasmic reticulum and Golgi). Within the X-body, TGB1 forms helically arranged aggregates surrounded by a reservoir of the recruited host endomembranes. The TGB2/3 proteins reside in granular vesicles within this reservoir, in the same region as nonencapsidated viral RNA, while encapsidated virions accumulate at the outer (cytoplasmic) face of the X-body, which comprises a highly organized virus "factory." TGB1 is both necessary and sufficient to remodel host actin and endomembranes and to recruit TGB2/3 to the X-body, thus emerging as the central orchestrator of the X-body. Our results indicate that the actin/endomembrane-reorganizing properties of TGB1 function to compartmentalize the viral gene products of PVX infection.

Cell-to-cell transport of plant viruses is mediated by virus-encoded movement proteins and occurs through plasmodesmata interconnecting neighboring cells in plant tissues. Three movement proteins coded by the "triple gene block" (TGB) and named TGBp1, TGBp2 and TGBp3 have distinct functions in viral transport. TGBp1 binds viral genomic RNAs to form ribonucleoprotein complexes representing the transport form of viral genome, while TGBp2 and TGBp3 are necessary for intracellular delivery of such complexes to plasmodesmata. Recently, it was revealed that overexpression of Potato virus X TGBp3 triggers the unfolded protein response mitigating the endoplasmic reticulum (ER) stress leading to cell death if this protein reaches high levels in the ER. Here we report microscopic studies of the influence of the Poa semilatent hordeivirus TGBp3 overexpressed in Nicotiana benthamiana epidermal cells by particle bombardment on cell endomembranes and demonstrate that the protein C-terminal transmembrane segment contains a determinant responsible for vesiculation and coalescence of the endoplasmic reticulum and Golgi presumably accompanying the ER stress that can be induced upon high-level TGBp3 expression.

Plant viruses are a class of plant pathogens that specialize in movement from cell to cell. As part of their arsenal for infection of plants, every virus encodes a movement protein (MP), a protein dedicated to enlarging the pore size of plasmodesmata (PD) and actively transporting the viral nucleic acid into the adjacent cell. As our knowledge of intercellular transport has increased, it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD. Just as viruses are too large to fit through an unmodified plasmodesma, they are also too large to be freely diffused through the cytoplasm of the cell. Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP, including viral proteins originally associated with replication or gene expression. In this review, we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD, in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread.

Cell-to-cell signal transduction is vital for orchestrating the whole-body physiology of multi-cellular organisms, and many endogenous macromolecules, proteins, and nucleic acids function as such transported signals. In plants, many of these molecules are transported through plasmodesmata (Pd), the cell wall-spanning channel structures that interconnect plant cells. Furthermore, Pd also act as conduits for cell-to-cell movement of most plant viruses that have evolved to pirate these channels to spread the infection. Pd transport is presumed to be highly selective, and only a limited repertoire of molecules is transported through these channels. Recent studies have begun to unravel mechanisms that actively regulate the opening of the Pd channel to allow traffic. This macromolecular transport between cells comprises two consecutive steps: intracellular targeting to Pd and translocation through the channel to the adjacent cell. Here, we review the current knowledge of molecular species that are transported though Pd and the mechanisms that control this traffic. Generally, Pd traffic can occur by passive diffusion through the trans-Pd cytoplasm or through the membrane/lumen of the trans-Pd ER, or by active transport that includes protein-protein interactions. It is this latter mode of Pd transport that is involved in intercellular traffic of most signal molecules and is regulated by distinct and sometimes interdependent mechanisms, which represent the focus of this article.