SummaryWe have visualized the relationship between the endoplasmic reticulum (ER) and Golgi in leaf cells of Nicotiana clevelandii by expression of two Golgi proteins fused to green fluorescent protein (GFP). A fusion of the transmembrane domain (signal anchor sequence) of a rat sialyl transferase to GFP was targeted to the Golgi stacks. A second construct that expressed the Arabidopsis H/KDEL receptor homologue aERD2, fused to GFP, was targeted to both the Golgi apparatus and ER, allowing the relationship between these two organelles to be studied in living cells for the first time. The Golgi stacks were shown to move rapidly and extensively along the polygonal cortical ER network of leaf epidermal cells, without departing from the ER tubules. Co-localization of F-actin in the GFPexpressing cells revealed an underlying actin cytoskeleton that matched precisely the architecture of the ER network, while treatment of cells with the inhibitors cytochalasin D and N-ethylmaleimide revealed the dependency of Golgi movement on actin cables. These observations suggest that the leaf Golgi complex functions as a motile system of actin-directed stacks whose function is to pick up products from a relatively stationary ER system. Also, we demonstrate for the first time in vivo brefeldin Ainduced retrograde transport of Golgi membrane protein to the ER.

Leaves undergo a sink-source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink-source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific "macromolecular trafficking" is a general feature of simple plasmodesmata in sink leaves.

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.

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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.

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 yrions were over twice the diameter of wild-type PVX virions.

Using noninvasive imaging techniques, we compared phloem unloading of the membrane-impermeant, fluorescent solute carboxyfluorescein (CF) with that of potato virus X expressing the gene for the green fluorescent protein. Although systemic virus transport took considerably longer to occur than did CF transport, unloading of both solute and virus occurred predominantly from the class 111 vein network, a highly branched veinal system found between class II veins. The minor veins (classes IV and V) played no role in solute or virus import but were shown to be functional in xylem transport at the time of import by labeling with Texas Red dextran. After virus exit from the class 111 phloem, the minor veins eventually became infected by cell-to-cell virus movement from the mesophyll. During the sink/source transition, phloem unloading of CF was inhibited from class 111 veins before the cessation of phloem import through them, suggesting a symplastic isolation of the phloem in class 111 veins before its involvement in export. The progression of the sink/source transition for carbon was unaffected by the presence of the virus in the sink leaf. However, the virus was unable to cross the sink/source boundary for carbon that was present at the time of vira1 entry, suggesting a limited capacity for cell-to-cell virus movement into the apical (source) region of the leaf. A functional model of the sink/source transition in Nicotiana benthamiana is presented. This model provides a framework for the analysis of solute and virus movement in leaves.