Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus

We have recently used a green fluorescent protein (GFP) fusion to the gammab protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The gammab protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAbeta but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, betab (TGB1), betac (TGB3), and betad (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.     

Triple gene block protein interactions involved in movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

Cell-cell communication in the leaves of Commelina cyanea and other plants

Abstract. The fluorescent probes 6–carboxyfluorescein and lucifer yellow CH which do not pass the plasmalemma have been used to examine cell-to-cell communication in the leaf of Commelina cyanea. Dye movement from cell-to-cell occurs in epidermal, spongy and palisade mesophyll, and vascular cells. Dye movement between these tissues was also found. Hence, the epidermis, spongy and palisade mesophyll cells, and vascular tissue are all linked in a continuous symplast. However, dye injected into the epidermal cells rarely moves into guard cells, indicating that these cells are relatively isolated from the surrounding cells. In the same way, guard cells in Vicia faba and the C₄ grass Anthephora pubescens also appeared to be isolated from epidermal cells. Thus guard cell isolation from cell-to-cell communication appears to be a common phenomenon. Hence, the ion fluxes required for guard cell function must occur via the apoplast.     

Cell-to-cell movement of Potato virus X: the role of p12 and p8 encoded by the second and third open reading frames of the triple gene block

Potato virus X (PVX) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (CP) for cell-to-cell movement. When each of these proteins was co-expressed with a cytosolic green fluorescent protein (GFP) in the epidermal cells of Nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed GFP, indicating an ability to alter plasmodesmal permeability. p25, p12, and CP, expressed transiently in the initially infected cells, transcomplemented the corresponding movement-defective mutants to spread through two or more cell boundaries. Thus, these proteins probably move from cell to cell with the genomic RNA. In contrast, p8 only functioned intracellularly and was not absolutely required for cell-to-cell movement. Since overexpression of p12 overcame the p8 deficiency, p8 appears to facilitate the functioning of p12, presumably by mediating its intracellular trafficking. Considering the likelihood that p12 and p8 are membrane proteins, it is suggested that intercellular as well as intracellular movement of PVX involves a membrane-mediated process.     

Tracking viral movement in plants by means of chlorophyll fluorescence imaging

Many techniques have been applied to understand viral cell-to-cell movement in host plants, but little progress has been made in understanding viral vascular transport mechanisms. We propose the use of chlorophyll fluorescence imaging techniques, not only to diagnose the viral infection, but also to follow the movement of the virus through the vascular system and its subsequent spread into the leaves. In Nicotiana benthamiana plants, imaging of chlorophyll fluorescence parameters such as Ф_PSII and NPQ proved useful to follow infections with Pepper mild mottle virus. The results demonstrate a correlation between changes in the chlorophyll fluorescence parameters and the viral distribution analyzed by tissue printing.     

Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function

Rice dwarf virus (RDV) is a member of the genus Phytoreovirus, which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either beta-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana. Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum, a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana. A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum. By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.     

Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.Plants use macromolecular trafficking pathways through plasmodesmata (PD) as a means to regulate developmental processes and physiological functions, and they also rely on these channels as avenues to communicate and mount defense responses to pathogen challenge (2, 37, 55). Local and systemic plant virus invasion depends on the abilities of viruses to use these pathways to spread from initially infected cells to the vascular tissue and distal regions of the plant. To this end, viruses infecting plants have evolved movement proteins (MPs) that coopt host trafficking pathways to target virus genomes to the PD and to facilitate the cell-to-cell transit of infectious entities (4, 13, 36, 48, 55). Virus MPs vary in size, number, and genome organization, but they share a number of functional characteristics including localization to PD, an ability to increase the size exclusion limits of PD, and RNA binding activities (3, 7, 8, 24, 27, 58).Viruses containing triple gene block (TGB) MPs have been the subjects of a number of investigations (4, 6, 39, 53, 54). Interestingly, viruses with a range of diverse genome structures encode MPs in a TGB, but these proteins fall into two major TGB classes that have substantial differences in protein structure and variations in their physical, functional, and cellular interactions (19, 30, 39, 45, 48). For example, the hordeivirus-like TGB1 proteins contain substantial N-terminal extensions that are lacking in the potexvirus-like TGB1 proteins, but the two classes of proteins share a conserved helicase domain at their C termini (39). The available evidence also indicates that hordeivirus-like and potexvirus-like TGB1 proteins share common biochemical features, including RNA binding abilities (3, 13, 23, 35, 44, 56), RNA helicase activities (22), associated NTPase activities (3, 13, 23, 33, 35, 44), and the capacity to form homologous interactions (29, 30, 45). However, the potexvirus-like TGB1 proteins localize at the CW when expressed autonomously and also facilitate increases in PD size exclusion limits, whereas the hordeivirus-like TGB1 proteins lack both these activities (39, 53). Major differences are also evident in the organizations of the potexvirus-like and hordeivirus-like TGB3 proteins, which share no discernible relatedness, differ in the numbers of their transmembrane domains, and indeed appear to have a polyphyletic origin (39).In both TGB classes, the movement strategy employs the coordinated actions of all three proteins. However, the coat protein is dispensable for one or more phases of movement of benyvirus, hordeivirus, pecluvirus, and pomovirus, encoding hordeivirus-like (class I) MPs, but is absolutely required for cell-to-cell movement of potexvirus-like (class II) MPs encoded by allexivirus, carlavirus, foveavirus, and potexvirus (6, 19, 39, 54). These variations clearly demonstrate that the two classes of TGB proteins have profound differences in their functional properties and in their associations with other virus and host proteins. Hence, comparative analyses of the functional and biological properties of the two classes of proteins in their common hosts may reveal important activities relevant to viral pathogenesis. To provide more information about the hordeivirus-like movement mechanisms, we are investigating the TGB interactions of Barley stripe mosaic virus (BSMV).BSMV is the type member of the genus Hordeivirus, which includes Poa semilatent virus (PSLV), Lychnis ringspot virus, and Anthoxanthum latent blanching virus (6, 19). Hordeiviruses have positive-sense, single-stranded RNA genomes consisting of three segments, designated α, β, and γ. The RNAβ segment encodes the coat protein, which is translated directly from genomic RNAβ (gRNAβ), and the TGB proteins, which are expressed from two subgenomic RNAs (sgRNAs), designated sgRNAβ1 and sgRNAβ2 (60). The coat protein is dispensable for the systemic movement of BSMV (41), and mutational analyses indicate that the TGB1, TGB2, and TGB3 proteins are each essential for cell-to-cell movement in monocot and dicot hosts (28). The BSMV TGB1 (58-kDa) protein is expressed from sgRNAβ1 at higher levels than the smaller hydrophobic TGB2 (14-kDa) and TGB3 (17-kDa) proteins, which are coexpressed from the bicistronic sgRNAβ2 during replication (14, 60). BSMV TGB1 has binding activity for both single-stranded and double-stranded RNAs (13) and forms nucleoprotein complexes with each of the BSMV gRNAs and sgRNAs (30). The hordeivirus-like TGB1 proteins differ from the potexvirus-like TGB1 proteins in having longer N-terminal domains with positively charged amino acids, but both classes of proteins have conserved C-terminal NTPase/helicase domains (13, 39, 49). In BSMV, mutations of conserved amino acids within the TGB1 helicase motif abrogate cell-to-cell movement and alter subcellular localization in infected protoplasts (27). Plants infected with a BSMV β-green fluorescent protein-TGB1 (β-GFP-TGB1) reporter virus also exhibited paired foci on both sides of the CW, and the plasma membranes of infected protoplasts developed punctate foci (27). TGB1 and TGB2 are also essential for plasma membrane targeting because β-GFP-TGB1 reporter derivatives that were unable to express TGB2 or TGB3 fluoresce at perinuclear membranes of protoplasts (27). Particle bombardment studies with the related hordeivirus PSLV also suggested that the expression of TGB3 is required to shift the localization of TGB2 from the endoplasmic reticulum (ER) to the peripheral membranes (50), and transgenically expressed PSLV TGB3 appears to be associated with PD due to its colocalization with callose markers (17).We have recently shown that TGB2 and TGB3 interact physically and have identified single amino acids in each protein that are required for these interactions (19, 30). TGB3 also interacts with TGB1, and we have proposed that these interactions facilitate the transport of ribonucleoprotein (RNP) complexes to the PD (30). However, the effects of TGB protein interactions on subcellular localization have not been defined. Moreover, because of possible convergent evolution of the hordeivirus-like and potexvirus-like TGB-containing viruses (39), the mechanisms of action resulting in transport may differ among different genera or even among different virus species within a genus. To obtain more refined information about these processes, we have now expressed fluorescent TGB fusion proteins transiently in Nicotiana benthamiana leaf cells by Agrobacterium tumefaciens infiltration and have assessed the subcellular localization patterns of BSMV wild-type (wt) and mutant TGB derivatives that differ in their interactions. We also have carried out reverse genetic experiments with selected BSMV TGB mutants to provide a biological context for the localization patterns appearing during ectopic Agrobacterium expression. These findings are elaborated in a model for TGB interactions required for the cell-to-cell movement of BSMV.     

Comparison of barley stripe mosaic virus strains

BSMV (barley stripe mosaic virus) particles were obtained in a pure state from infected host plant tissues of Hordeum vulgare. The three genomic parities (alpha, beta and gamma) were amplified by PCR using specific primers for each particle; each was cloned. Partial sequence of the alpha, beta and gamma segments was determined for the Egyptian isolate of barley stripe mosaic virus (BSMV AE1). Alignment of nucleotide sequences with that of other known strains of the virus, BSMV type strains (CV17, ND18 and China), and the generation of phylogenetic trees was performed. A low level of homology was detected comparing 467 bp of the a and 643 bp of the segments to that of the other strains, and thus BSMV alpha and beta segments were in separate clusters. However, 1154 bp of the gamma segments of BSMV AE1 showed a high level of homology especially to strain BSMV ND18, as they both formed a distinct cluster. Northern blotting of pure BSMV AE1 virus and H. vulgare-infected tissue were compared using an alpha ND18 specific probe. Western blotting using antibodies specific for the coat protein (CP) and the triple gene block 1 (TGB1) protein, which are both encoded by the beta ND18 segment, still indicated a high level of similarity between proteins produced by BSMV ND18 and AE1. We suggest that the BSMV AE1 isolate is a distinct strain of BSMV which reflects the genetic evolutionary divergence among BSMV strains and members of the Hordeivirus group.     

Flavonoids are differentially taken up and transported long distances in Arabidopsis

Flavonoids are synthesized in response to developmental and environmental signals and perform many functions in plants. Arabidopsis (Arabidopsis thaliana) roots grown in complete darkness do not accumulate flavonoids since the expression of genes encoding enzymes of flavonoid biosynthesis is light dependent. Yet, flavonoids accumulate in root tips of plants with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain, and localized aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating uptake and movement systems. This was confirmed by time-course fluorescence experiments and high-performance liquid chromatography. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application to midroot or cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol, and dihydroquercetin were taken up at the root tip, midroot, or cotyledons and traveled long distances via cell-to-cell movement to distal tissues, followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Using ATP-binding cassette (ABC) transporter and H(+)-ATPase inhibitors suggested that a multidrug resistance-associated protein ABCC transporter facilitated flavonoid movement away from the application site.     

β-1,3-Glucanases: Plasmodesmal Gate Keepers for Intercellular Communication

Plasmodesmata (Pd), coaxial membranous channels that connect adjacent plant cells, are not static, but show a dynamic nature and can be opened or closed. These controlled changes in Pd conductivity regulate plant symplasmic permeability and play a role both in development and defense processes. One of the mechanisms shown to produce these changes is the deposition and hydrolysis of callose by β-1-3-synthase and glucanase, respectively. Recently we have identified the first β-1,3-glucanase Arabidopsis enzyme that is associated to the macromolecular Pd complex, termed AtBG_pap. When fused to GFP, this previously identified GPI-anchored protein localizes to the ER and the plasma membrane where it appears in a punctuate pattern that colocalizes with callose present around Pd. In T-DNA insertion mutants that do not transcribe AtBG_pap, GFP cell-to-cell movement between epidermal cells is reduced and callose levels around Pd are elevated. In this addenda we review the plant developmental processes of symplasmic regulation that have been shown to include callose deposition and β-1,3-glucanase activity, and suggest a role for AtBG_pap in these processes. Additionally, based on the ability of viral movement proteins (MPs) to interact with ankyrin repeat proteins, and together with our recent findings showing the involvement of viral particles in callose degradation, we also purpose a new model for the ability of viruses to overcome Pd-callose deposition, and mediate their cell-to-cell movement.Key Words: plasmodesmata, cell-cell communication, callose, β-1,3-glucanase, movement protein, ankyrin repeats     

A new cell-to-cell transport model for Potexviruses

In the last five years, we have gained significant insight into the role of the Potexvirus proteins in virus movement and RNA silencing. Potexviruses require three movement proteins, named triple gene block (TGB)p1, TGBp2, and TGBp3, and the viral coat protein (CP) to facilitate viral cell-to-cell and vascular transport. TGBp1 is a multifunctional protein that has RNA helicase activity, promotes translation of viral RNAs, increases plasmodesmal size exclusion limits, and suppresses RNA silencing. TGBp2 and TGBp3 are membrane-binding proteins. CP is required for genome encapsidation and forms ribonucleoprotein complexes along with TGBp1 and viral RNA. This review considers the functions of the TGB proteins, how they interact with each other and CP, and how silencing suppression might be linked to viral transport. A new model of the mechanism for Potexvirus transport is proposed.     

P42 movement protein of Beet necrotic yellow vein virus is targeted by the movement proteins P13 and P15 to punctate bodies associated with plasmodesmata

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins--P42, P13, and P15--organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.     

Transient coexpression of individual genes encoded by the triple gene block of potato mop-top virus reveals requirements for TGBp1 trafficking

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the "triple gene block" (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBpl to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.     

Cell-to-cell movement of three genera (+) ss RNA plant viruses

The current investigations of three genera plant virus cell-to-cell movement were presented. Viruses reveal different local transport strategies, but all of them are the results of virus factors–host components interactions. The Tobacco mosaic virus (TMV) does not require capsid protein for translocation through plasmodesmata but 30 K movement protein participates in this process. It was found direct or indirect TMV movement proteins host partners in Tobamovirus movement like: pectin methylesterase, movement protein binding 2C, chaperones or cytoskeleton components and endoplasmatic reticulum membranes. The Potex- and Potyvirus cell-to-cell movement is closely related to replication network. The PVX capsid protein and triple gene block protein system are responsible for efficient local transport. Potyviruses move through the plasmodesmata by involving viral encoded proteins but not specific movement proteins. While the Potyvirus is the biggest known plant virus genus, host components participating in or regulating directly its plasmodesmata-movement are still not clear.     

The Ability of PVX p25 to Form RL Structures in Plant Cells Is Necessary for Its Function in Movement, but Not for Its Suppression of RNA Silencing

The p25 triple gene block protein of Potato virus X (PVX) is multifunctional, participating in viral movement and acting as a suppressor of RNA silencing. The cell-to-cell movement of PVX is known to depend on the suppression function of p25. GFP-fused p25 accumulates in rod-like (RL) structures with intense fluorescence in cells. By monitoring the location of fluorescence at different times, we have now shown that the RL structure is composed of filaments. P25 mutants without the conditional ability to recover movement function could not form RL structures while the mutants that had the ability did form the structure, suggesting that the ability of p25 to form RL structures is necessary for its function in cell-to-cell movement, but not for its suppressor function. Moreover, chemical inhibition of microfilaments in cells destroyed the formation of the complete RL structure. Additionally, TGBp2 and TGBp3 were recruited into the RL structure, suggesting a relationship between the TGBps in virus movement.     

Two plant-viral movement proteins traffic in the endocytic recycling pathway

Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed.     

Reciprocal phosphorylation and glycosylation recognition motifs control NCAPP1 interaction with pumpkin phloem proteins and their cell-to-cell movement

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.