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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.

Due to their capability of eliciting a form of posttranscriptional gene silencing (termed virus-induced gene silencing or VIGS), plant viruses are increasingly used as reverse-genetics tools for functional characterization of plant genes. RNA viruses have been shown to trigger silencing in a variety of host plants, including members of Solanacae and Arabidopsis (Arabidopsis thaliana). Several factors affect the silencing response, including host range and viral tropism within the plant. The work presented here demonstrates that a modified tobacco rattle virus (TRV) vector retaining the helper protein 2b, required for transmission by a specific vector nematode, not only invades and replicates extensively in whole plants, including meristems, but also triggers a pervasive systemic VIGS response in the roots of Nicotiana benthamiana, Arabidopsis, and tomato (Lycopersicon esculentum). This sustained VIGS response was exemplified by the silencing of genes involved in root development (IRT1, TTG1 [transparent testa glabra], RHL1 [root hairless1], and beta-tubulin), lateral root-meristem function (RML1 [root meristemless1]), and nematode resistance (Mi). Roots of silenced plants exhibit reduced levels of target mRNA and phenocopy previously described mutant alleles. The TRV-2b vector displays increased infectivity and meristem invasion, both key requirements for efficient VIGS-based functional characterization of genes in root tissues. Our data suggest that the TRV helper protein 2b may have an essential role in the host regulatory mechanisms that control TRV invasion.

This review covers recent advances in imaging techniques that were presented during the 11th International Workshop on Plant Membrane Biology, Cambridge, August 1998. Cell biology has been revolutionized by the arrival of green fluorescent protein (GFP) and GFP is now routinely used as a cell-lineage marker in plants, to localize proteins to subcellular compartments or as a tag to follow dynamics in endomembrane compartments. More recent developments include modification of GFP to form physiological sensors for calcium and pH. These provide a transgenic approach in parallel with conventional chemical dyes to track signalling events or follow membrane recycling pathways. Confocal microscopy has become a routine technique to visualize these fluorescent probes, particularly in intact tissues. Multi-photon microscopy may push the capability of imaging techniques further, allowing imaging at even greater depths and long-(red)-excitation of UV fluorochromes. The luminescent calcium indicator, aequorin, has been much more extensively used in plants than the transgenic fluorescent calcium indicators, and photon-counting imaging systems can now record calcium transients at video-rate in intact plants. Digital imaging, whether camera, confocal or multi-photon, provides quantitative data, but correct interpretation requires rigorous analysis of noisy and partially correlated imaging data. Statistical analysis by Bayesian inference may well become the most appropriate technique to handle such images, but has only recently been applied to ratio imaging in plants. In addition to generating images, light can also be used to manipulate intracellular events using photolysis of caged probes. The control of the location, timing and amplitude of the release allows exquisitely subtle manipulation of signalling networks. More aggressive pulses of UV laser-light provide an equally powerful tool either to ablate whole cells completely for developmental studies or to punch-out tiny holes in the cell wall to give access to the plasma membrane for patch-clamping and electrophysiological investigation of cells in situ.

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

Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX. The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread

The fluorescent dye Lucifer Yellow CH (LYCH) was introduced directly into the symplast of potato (Solanum tuberosum L.) tuber storage parenchyma by microinjection and also into the apoplast through cuts made in the stolon cortex. Microinjected LYCH moved away rapidly from a single storage cell and spread radially via the symplast. When the microinjected tissue was subsequently fixed in glutaraldehyde and sectioned the dye was seen clearly to be localised in the cytoplasm but not in the vacuole. In comparison, when LYCH was introduced into cuts made in the stolon cortex the dye entered the tuber by the xylem and subsequently spread apoplastically. No movement of dye was observed in the phloem. In glutaraldehyde-fixed tissues, in which LYCH was introduced to the apoplast, the dye was found within xylem vessels, in the cell walls and in intercellular spaces. Wall regions, possibly associated with plasmodesmata, became stained by the dye as it moved through the apoplast. Three hours after introduction of the dye to the stolon, intense desposits of LYCH were found in the vacuoles of all cells in the tuber, many aligned along the tonoplast. Differentiating vascular parenchyma elements contained large amounts of dye within enlarging vacuoles. However, with the exception of plasmolysed and-or damaged cells, LYCH was absent from the cytoplasm following its introduction to the apoplast. Since LYCH is unable to cross the plasmalemma it is suggested that the most likely pathway from the cell wall to the vacuole was by endocytosis, the dye being transported across the cytoplasm in membrane-bound vesicles. Clathrin-coated vesicles were abundant in the storage cells, providing a possible endocytotic pathway for dye movement. The significance of these observations is discussed in relation to the movement of LYCH in plant tissues and to the movement of solutes within and between storage cells of the tuber.

A transient plasmolysis/deplasmolysis (plasmolytic cycle) of onion epidermal cells has been shown to induce the formation of fluid-phase endocytic vesicles. Plasmolysis in the presence of the membrane-impermeant fluorescent probes Lucifer Yellow CH (LYCH) and Cascade Blue hydrazide resulted in the uptake of these probes by fluid-phase endocytosis. Following deplasmolysis, many of the dye-containing vesicles left their parietal positions within the cell and underwent vigorous streaming in the cytoplasm. Vesicles were observed to move within transvacuolar strands and their movements were recorded over several hours by video-microscopy. Within 2 h of deplasmolysis several of the larger endocytic vesicles had clustered around the nuclear membrane, apparently lodged in the narrow zone of cytoplasm surrounding the nucleus. In further experiments LYCH was endocytically loaded into the cells during the first plasmolytic cycle and Cascade Blue subsequently loaded during a second plasmolytic cycle. This resulted in the introduction of two populations of endocytic vesicles into the cells, each containing a different probe. Both sets of vesicles underwent cytoplasmic streaming. The data are discussed in the light of previous observations of fluid-phase endocytosis in plant cells.

The movement of water in the dorsal region of the developing rice caryopsis was studied using solutions of the heavy metals lanthanum and uranium. In the electron microscope electron-opaque deposits were confined to the cell walls of the pigment strand indicating that this is the main route for the water which enters and leaves the caryopsis during grain filling. The pathway of assimilates into the developing caryopsis was examined using isolated caryopses which had taken up solutions of fluorescent dyes and also by autoradiography of caryopses which had transported 14C-labelled assimilates in vivo. The results show that assimilates unloaded from the phloem move through the pigment strand and circumferentially via cells of the nucellus before entering the endosperm. A scheme is presented for the interrelations of water and assimilate transport during grain filling

The molecular weight exclusion limit of plasmodesmata in subveinal epidermal cells of Nicotiana clevelandii (Gray) leaves was estimated by microinjection and fluorescence microscopy using fluorescein isothiocyanate-peptide conjugates, carboxyfluorescein and Lucifer Yellow CH. The largest fluorochrome which moved symplastically between cells had a molecular weight of 749, although movement did not appear to depend purely on molecular weight parameters. Systemic infection of plants by tobacco rattle tobravirus, tomato black ring nepovirus or potato Y potyvirus did not alter the limits of plasmodesmatal conductance of the fluorochromes. However, carrot mottle umbravirus and groundnut rosette umbravirus diminished the symplastic mobility of some fluorescent tracers. These results imply that intercellular movement of these viruses does not involve a long-lasting increase in the plasmodesmatal molecular size exclusion limit.