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PubMed1995

Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells.

Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.

Actins↗

PubMed2006

Females and males of root-parasitic cyst nematodes induce different symplasmic connections between their syncytial feeding cells and the phloem in Arabidopsis thaliana.

Root syncytia induced by the beet cyst nematode Heterodera schachtii were thought to be symplasmically isolated. A recent study with mobile and immobile GFP constructs expressed in transgenic Arabidopsis plants under the control of pAtSUC2 showed that only mobile GFP could be detected in syncytia and suggested the existence of plasmodesmata between syncytia and the phloem. In the present study the existence of plasmodesmata between syncytia and the phloem is proven by grafting experiments. This technique rules out the possibility that GFP accumulation in syncytia is due to GFP expression in syncytia. Mobile GFP could be followed from transgenic scions carrying a pAtSUC2-gfp fusion construct via wild-type rootstocks into nematode-induced syncytia. While GFP could be detected in all syncytia associated to female nematodes, it was never observed in syncytia of male juveniles. As no GFP-mRNA could be detected in the rootstock we postulate that GFP as protein entered syncytia of females via plasmodesmata, while the protein was excluded from syncytia of male juveniles by plasmodesmata with a lower size exclusion limit.

Animals↗

PubMed1995

Cucumber mosaic virus 3a protein potentiates cell-to-cell trafficking of CMV RNA in tobacco plants.

Contrary to a previous report, electron microscopic studies on the Fny strain of cucumber mosaic virus (CMV)-infected tobacco tissues revealed that plasmodesmata were not structurally modified during CMV infection, nor were virions ever observed in plasmodesmata connecting infected cells. To further explore the basis of CMV infection, experiments were performed on the CMV 3a ORF. The 3a protein of CMV was expressed in and purified from Escherichia coli. The purified protein was labeled with fluorescein isothiocyanate (FITC) and subsequently microinjected into mesophyll cells of mature leaves of Nicotiana tabacum cv. Turkish Samsun NN. Within a brief period (as little as 1 sec), the microinjected FITC-labeled CMV 3a protein moved into neighboring cells. Co-injection of unlabeled CMV 3a protein with 9.4-kDa fluorescein-conjugated dextran (F-dextran) resulted in extensive cell-to-cell movement (diffusion) of the F-dextran, indicating that the 3a protein can interact with and dilate plasmodesmata. Furthermore, co-injection of unlabeled 3a protein with fluorescently labeled infectious CMV RNA molecules resulted in rapid and extensive cell-to-cell transport. In contrast, a mutant form of the 3a protein was unable to traffic from cell to cell, to increase the size exclusion limit of plasmodesmata, or to potentiate cell-to-cell trafficking of CMV RNA molecules. Microinjection studies performed on transgenic tobacco plants expressing the CMV 3a protein indicated that fluorescently labeled CMV RNA moved out of the target cell into the surrounding mesophyll tissue. In addition, expression of the CMV 3a protein also potentiated the cell-to-cell movement of 9.4-kDa F-dextran. Collectively, these results provide direct experimental evidence that the CMV 3a protein functions as the movement protein of CMV. These findings are consistent with the hypothesis that CMV moves from cell-to-cell in the form of a ribonucleoprotein complex.

Base Sequence↗

PubMed1997

In situ localization of the putative movement protein (pr17) from potato leafroll luteovirus (PLRV) in infected and transgenic potato plants.

The potato leafroll virus (PLRV) 17-kDa protein (pr17), the putative movement protein for this phloem-limited luteovirus, was localized on ultrathin sections of leaves from PLRV-infected and transgenic potato plants. The transgenic plants expressed the entire viral genome from a full-length cDNA copy (PLRVfl) or only the gene encoding pr17 (ORF4) under the control of the cauliflower mosaic virus 35S promoter. Virus-infected and PLRVfl-transgenic plants developed symptoms typical of virus infection, whereas pr17-transgenic plants did not display symptoms or ultrastructural alterations. Immunogold electron microscopy using an anti-pr17-serum detected pr17 in plasmodesmata, in virus-induced vesicles, in mitochondria, and in chloroplasts of phloem cells, in PLRV-infected as well as PLRVfl-transgenic plants. In addition, in transgenic plants, pr17 was expressed in mesophyll cells (which are not infected by PLRV under natural conditions) and localized to the same sites as in phloem cells, except in plasmodesmata. In contrast, in pr17-transgenic plants the protein was never observed on organelles, but was almost exclusively associated with plasmodesmata of all leaf cell types, indicating that the targeting of pr17 to plasmodesmata is an intrinsic property of the protein. These results support the role of pr17 in PLRV movement.

Carrier Proteins↗

PubMed2000

Synchronization of mitotic activity in protoplast-derived Solanum nigrum L. microcalluses is correlated with plasmodesmal connectivity.

In protoplast-derived Solanum nigrum microcalluses, plasmodesmal connectivity and cell division behaviour of the sister cells were examined by repeated pressure-injection experiments with the fluorescent dye Lucifer Yellow (LYCH; M(r) 457) and concomitant light-microscopical long-term live observations. The studies revealed that the plasmodesmal permeability of the cultured cells differs in the distinct stages of microcallus development. There was a correlation between the symplasmic connectivity of the cells and the synchronousness of their mitotic activity. Sister cells which were symplasmically interconnected by functional plasmodesmata, permitting the diffusion of LYCH, were always found to divide synchronously. However, asynchronous mitotic divisions were exclusively observed in those sister cells whose plasmodesmata were closed to LYCH. The temporary symplasmic isolation is presumably performed by reversible gating of plasmodesmata. Repeated dye-coupling experiments on the same microcalluses showed that symplasmically interconnected sister cells may become uncoupled and vice versa, according to their division behaviour. These findings on cultured cells indicate that modulation of the symplasmic connectivity determines the synchronization of mitotic activity. Yet it remains to be proven whether this is true in planta as well. The results are discussed with respect to the possible role of plasmodesmata in exerting "supracellular control" over mitotic activity by trafficking mitosis-regulating signals.

Cell Wall↗

PubMed1975

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.

Cell Nucleus↗

PubMed2001

The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy.

The shoot apex of overwintering perennials ceases its morphogenetic activity at the end of the growing season and transforms into a bud which is dormant and freezing-tolerant. In birch (Betula pubescens) these events are triggered by short photoperiod, and involve the production of 1,3-beta-D-glucan containing sphincters on the plasmodesmata. As a result, all symplasmic pathways shut down. Here we show that breakage of bud dormancy by chilling involves restoration of the symplasmic organization of the meristem. This restoration is likely to be mediated by 1,3-beta-D-glucanase, which was present in small spherosome-like vacuoles that arose de novo during dormancy induction. During chilling these vacuoles were displaced from the bulk cytoplasm to the cortical cytoplasm where they became aligned with the plasma membrane, often associated with plasmodesmata. At this stage the enzyme also appeared outside the vacuoles. During chilling, 1,3-beta-D-glucan disappeared from the plasmodesmal channels and wall sleeves, and the plasmodesmata regained the capacity for cell-cell transport, as demonstrated by microinjection of Lucifer Yellow CH and Fluorescein-tagged gibberellic acid. Collectively, the present experiments demonstrate that restoration of the symplasmic organization of the meristem is indispensable for the release of buds from dormancy and the assumption of a proliferation-competent state, and implicate 1,3-beta-D-glucanase action at the plasmodesmata. Based on these findings we propose a model for 'dormancy cycling' which depicts the meristem as passing through three sequential states of cellular communication with characteristic sensitivities to distinct environmental cues.

Cell Communication↗

PubMed1990

Molecular characterization and biological function of the movement protein of tobacco mosaic virus in transgenic plants.

We previously demonstrated, in transgenic tobacco plants, that the role of the movement protein (MP) of tobacco mosaic virus is to facilitate the cell-to-cell spread of viral progeny during infection. An analysis of different tissues of these transgenic plants indicated that the MP accumulated in leaf, stem, and root tissue. The highest levels were detected in older leaves. The relative levels of MP in leaf tissue from transgenic plants were equivalent to, or higher than, the levels of MP in tobacco mosaic virus-infected leaf tissue. Results of subcellular fractionation of homogenates of transgenic leaf tissue showed that the MP was most abundant in the cell wall fraction of older leaves and that the protein remained at high levels in the cell wall fraction as the leaves continued to age. Significant levels of the MP were detected in a crude membrane/organelle fraction and a soluble fraction in younger leaves but decreased to low levels in older leaves. These results suggest that the MP accumulates and is stable in cell walls. We have previously shown that the MP modifies the molecular exclusion limit of plasmodesmata, which is consistent with the hypothesis that plant viruses move from cell to cell through altered plasmodesmata. We show here that the ability of the tobacco mosaic virus MP to modify the molecular exclusion limit of plasmodesmata in tobacco depends on the developmental stage of the leaf. The implications of these findings on understanding virus movement and how plasmodesmata function are discussed.

Capsid Proteins↗

PubMed1994

Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.

Plasmodesmata are cytoplasmic bridges between plant cells thought to generally allow only the passage of small molecules and metabolites. However, large structures such as plant viruses also move from cell to cell via plasmodesmata. In tobacco mosaic virus (TMV) infection a viral movement protein (TMV-MP) mediates viral spread. Here, a microinjection assay is used to monitor the dynamics of TMV-MP function directly in wild-type plants. The results indicate that TMV-MP interacts with an endogenous plant pathway increasing plasmodesmal size exclusion limit to permit passage of 20-kDa dextrans. Furthermore, TMV-MP influences plasmodesmal size exclusion limit several cells distant from the injection site, indicating either that TMV-MP itself crosses plasmodesmata or that TMV-MP induces a diffusable signal capable of dilating microchannels of plasmodesmata. The region of TMV-MP responsible for increasing plasmodesmal size exclusion limit was mapped to the carboxyl-terminal part of the 268-amino acid residue protein between amino acid residues 126 and 224.

Biological Assay↗

PubMed2000

Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein.

Cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata, is mediated by a specialized viral movement protein (MP). In vivo studies using transgenic tobacco plants showed that MP is phosphorylated at its C-terminus at amino acid residues Ser258, Thr261 and Ser265. When MP phosphorylation was mimicked by negatively charged amino acid substitutions, MP lost its ability to gate plasmodesmata. This effect on MP-plasmodesmata interactions was specific because other activities of MP, such as RNA binding and interaction with pectin methylesterases, were not affected. Furthermore, TMV encoding the MP mutant mimicking phosphorylation was unable to spread from cell to cell in inoculated tobacco plants. The regulatory effect of MP phosphorylation on plasmodesmal permeability was host dependent, occurring in tobacco but not in a more promiscuous Nicotiana benthamiana host. Thus, phosphorylation may represent a regulatory mechanism for controlling the TMV MP-plasmodesmata interactions in a host-dependent fashion.

Amino Acids↗

PubMed2005

Effects of calreticulin on viral cell-to-cell movement.

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.

Amino Acid Sequence↗

PubMed1988

Intercellular communication-filling in the gaps.

Coordination and synchrony of a variety of cellular activities in tissues of plants and animals occur as a consequence of the transfer of low molecular weight biosynthetic and signaling molecules through specialized structures (plasmodesmata in plant cells and gap junctions in mammalian cells) that form aqueous channels between contacting cells. Investigations with rat liver demonstrated that cell-cell communication is mediated by a 32 kilodalton polypeptide that forms a hexameric pore structure in the plasma membrane. Following association with the same structure in a contiguous cell, a trans-double membrane channel is created that has been termed a gap junction. In plant tissue, long tubelike structures called plasmodesmata are suggested to serve a similar cell-cell linking function between cytoplasmic compartments. Although morphologically distinct, dynamic observations suggest similarities in transport properties between gap junctions and plasmodesmata. Recent work now provides evidence that these functional similarities may reflect a more profound identity between the paradigm animal gap junction polypeptide (32 kilodalton rat liver polypeptide) and an immunologically homologous protein localized to plant plasma membrane/cell wall fractions that may be a component of plasmodesmata.

Journal Article↗

PubMed1998

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.

Journal Article↗

PubMed1999

Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.

Macromolecular trafficking within the sieve element-companion cell complex, phloem unloading, and post-phloem transport were studied using the jellyfish green fluorescent protein (GFP). The GFP gene was expressed in Arabidopsis and tobacco under the control of the AtSUC2 promoter. In wild-type Arabidopsis plants, this promoter regulates expression of the companion cell-specific AtSUC2 sucrose-H+ symporter gene. Analyses of the AtSUC2 promoter-GFP plants demonstrated that the 27-kD GFP protein can traffic through plasmodesmata from companion cells into sieve elements and migrate within the phloem. With the stream of assimilates, the GFP is partitioned between different sinks, such as petals, root tips, anthers, funiculi, or young rosette leaves. Eventually, the GFP can be unloaded symplastically from the phloem into sink tissues, such as the seed coat, the anther connective tissue, cells of the root tip, and sink leaf mesophyll cells. In all of these tissues, the GFP can traffic cell to cell by symplastic post-phloem transport. The presented data show that plasmodesmata of the sieve element-companion cell complex, as well as plasmodesmata into and within the analyzed sinks, allow trafficking of the 27-kD nonphloem GFP protein. The data also show that the size exclusion limit of plasmodesmata can change during organ development. The results are also discussed in terms of the phloem mobility of assimilates and of small, low molecular weight companion cell proteins.

Arabidopsis↗

PubMed2005

Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins.

Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK). A 34-kD protein, isolated from a plasmodesmal preparation, exhibits calcium-independent kinase activity and displays substrate specificity in that it recognizes a subset of viral and endogenous non-cell-autonomous proteins. This PAPK specifically phosphorylates the C-terminal residues of tobacco mosaic virus movement protein (TMV MP); this posttranslational modification has been shown to affect MP function. Molecular analysis of purified protein established that tobacco (Nicotiana tabacum) PAPK is a member of the casein kinase I family. Subcellular localization studies identified a possible Arabidopsis thaliana PAPK homolog, PAPK1. TMV MP and PAPK1 are colocalized within cross-walls in a pattern consistent with targeting to plasmodesmata. Moreover, Arabidopsis PAPK1 also phosphorylates TMV MP in vitro at its C terminus. These results strongly suggest that Arabidopsis PAPK1 is a close homolog of tobacco PAPK. Thus, PAPK1 represents a novel plant protein kinase that is targeted to plasmodesmata and may play a regulatory role in macromolecular trafficking between plant cells.

Arabidopsis↗

PubMed2006

[Ultrastructural observation related to cell-to-cell movement and long-distance systemic transport on the hosts infected with BBWV 2].

The alteration of ultrastructure in Pisum sativum and Vicia faba leaf cells infected with B935 isolate of BBWV 2 were investigated by electron microscopy, immunogold-labeling technique. The results showed that the membranous proliferation, virus-formed crystals and tubular structures were found in leaf cells of two hosts. At early stages of infection, the tubules containing virus-like particles associate with plasmodesmata in mesophyll cell. Immunogold particles anti-BBWV 2 were localized to the plasmodesmata modified by tubules passing through them. The membranous proliferation and virus-formed tubules were also found in the parenchyma cells, companion cells and transfer cells of vascular bundle. Some virus-like particles located within sieve tube can be labeled immunogold particles anti-BBWV 2. These suggest that BBWV 2, similar CPMV, produce tubules extending into the plasmodesmata. Virions assembled in the cytoplasm are escorted to the tubular structures through interactions with their MP and are then transported to the adjacent cell. Many 160 nm in diameter virus-formed tubules in the cytoplasm, as a special aggregate, not directly relate to cell-to-cell movement; Intact virions are long-distance sustemic transported possibly through sieve elements.

Biological Transport↗

PubMed1990

The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein.

The P30 protein of tobacco mosaic virus (TMV) is required for cell to cell movement of viral RNA, which presumably occurs through plant intercellular connections, the plasmodesmata. The mechanism by which P30 mediates transfer of TMV RNA molecules through plasmodesmata channels is unknown. We have identified P30 as an RNA and single-stranded (ss) DNA binding protein. Binding of purified P30 to ss nucleic acids is strong, highly cooperative, and sequence nonspecific with a minimal binding site of 4-7 nucleotides per P30 monomer. In-frame deletions across P30 were used to localize the ss nucleic acid binding domain to within amino acid residues 65-86 of the protein. We propose that binding of P30 to TMV RNA creates an unfolded protein-RNA complex that functions as an intermediate in virus cell to cell movement through plasmodesmata.

Chromosome Deletion↗

PubMed1999

A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein.

Plant virus-encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall-associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell-to-cell movement of tobacco mosaic virus.

Base Sequence↗