C. Stussi-Garaud

Reevaluation of the Effects of Brefeldin A on Plant Cells Using Tobacco Bright Yellow 2 Cells Expressing Golgi-Targeted Green Fluorescent Protein and COPI Antisera

Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells by inhibiting vesicle formation at the Golgi apparatus. Although this toxin has been used in many studies, its effects on plant cells are still shrouded in controversy. We have reinvestigated the early responses of plant cells to BFA with novel tools, namely, tobacco Bright Yellow 2 (BY-2) suspension-cultured cells expressing an in vivo green fluorescent protein–Golgi marker, electron microscopy of high-pressure frozen/freeze-substituted cells, and antisera against Atγ-COP, a component of COPI coats, and AtArf1, the GTPase necessary for COPI coat assembly. The first effect of 10 μg/mL BFA on BY-2 cells was to induce in <5 min the complete loss of vesicle-forming Atγ-COP from Golgi cisternae. During the subsequent 15 to 20 min, this block in Golgi-based vesicle formation led to a series of sequential changes in Golgi architecture, the loss of distinct Golgi stacks, and the formation of an endoplasmic reticulum (ER)–Golgi hybrid compartment with stacked domains. These secondary effects appear to depend in part on stabilizing intercisternal filaments and include the continued maturation of cis- and medial cisternae into trans-Golgi cisternae, as predicted by the cisternal progression model, the shedding of trans-Golgi network cisternae, the fusion of individual Golgi cisternae with the ER, and the formation of large ER-Golgi hybrid stacks. Prolonged exposure of the BY-2 cells to BFA led to the transformation of the ER-Golgi hybrid compartment into a sponge-like structure that does not resemble normal ER. Thus, although the initial effects of BFA on plant cells are the same as those described for mammalian cells, the secondary and tertiary effects have drastically different morphological manifestations. These results indicate that, despite a number of similarities in the trafficking machinery with other eukaryotes, there are fundamental differences in the functional architecture and properties of the plant Golgi apparatus that are the cause for the unique responses of the plant secretory pathway to BFA.

Involvement of the Secretory Pathway and the Cytoskeleton in Intracellular Targeting and Tubule Assembly of Grapevine fanleaf virus Movement Protein in Tobacco BY-2 Cells

Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP). The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown. To study intracellular GFLV MP trafficking, a green fluorescent protein–MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter. We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules. During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction. In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation. However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent. Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein. These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized.

Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes

ABSTRACT Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear “viral compartment.” We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

Nucleic acid-binding properties of the alfalfa mosaic virus movement protein produced in yeast.

The movement protein of alfalfa mosaic virus (P3) was purified from yeasts transformed with an expression vector containing the P3 gene. Its nucleic acid-binding properties were tested by electrophoretic retardation, nitrocellulose retention, and RNA-protein cross-linking. The recombinant protein had a higher affinity for single-stranded RNA and DNA than for double-stranded nucleic acids. Each nucleic acid molecule bound several protein molecules without sequence specificity. The binding was 80% inhibited by 0.2 M NaCl. These properties are qualitatively similar, but not strictly identical, to those of two other viral movement proteins, the 30-kDa protein of tobacco mosaic virus and the gene I product of cauliflower mosaic virus.

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.

The Fate of the Transport Protein of Tobacco Mosaic Virus in Systemic and Hypersensitive Tobacco Hosts

Summary The transport protein of tobacco mosaic virus (TMV) (M r 30000, 30K non-structural protein) was detected on Western blots, using an antiserum to a synthetic C-terminal nonapeptide. Accumulation of this protein in subcellular fractions of inoculated leaves was measured during TMV infection of Nicotiana tabacum cv. Samsun and cv. Samsun NN. In cv. Samsun, a systemic host, the 30K protein appeared transiently in a crude membrane fraction but accumulated more stably in cell walls. In cv. Samsun NN, which is a hypersensitive host giving only localized infection, the early accumulation (up to 40 h; before any necrosis was visible) was the same as in cv. Samsun. However, as soon as necrosis was visible, the amount of 30K detected in the cell wall fraction decreased sharply and coat protein synthesis stopped. This drop in the amount of 30K protein is most easily interpreted as a side-effect of the hypersensitive reaction and may explain why TMV infection becomes localized in leaves of cv. Samsun NN.

Detection by immunogold labelling of P75 readthrough protein near an extremity of beet necrotic yellow vein virus particles

SummaryRNA 2 of beet necrotic yellow vein virus carries the cistron for the 21 kd coat protein at its 5′-extremity. During translation, the coat protein cistron termination codon is suppressed about 10% of the time so that translation continues into the adjacent open reading frame to produce a 75 kd species, known as P75, which contains the coat protein sequence at its N-terminus. Immunoblotting experiments with a P75-specific antiserum showed that P75 is present in only trace amounts in purified virus preparations. Electron microscopic visualization of immunogold-labelled virions in crude tissue extracts has provided evidence for an association between P75 and at least a fraction of the BNYVV particles, with P75 being predominantly located near one end of the rod-shaped virions. This finding is discussed in the context of the current model for the role of P75 in virus assembly and vector transmission.

Role of the beet western yellows virus readthrough protein in virus movement in Nicotiana clevelandii

Luteoviruses such as beet western yellows polerovirus (BWYV) are confined to and multiply within the phloem compartment of their hosts. The readthrough domain (RTD) of the minor BWYV capsid protein P74 is required for efficient virus accumulation in Nicotiana clevelandii. Experiments were carried out to determine if the low virus titres observed following agro-inoculation of whole plants with certain RTD mutants are due to a defect in virus multiplication in the nucleate cells of the phloem compartment or to inefficient virus movement to new infection sites. Immuno-localization of wild-type and an RTD-null mutant virus in thin sections of petioles and in phloem cells of leaf lamina, as well as electron microscopy observations, were all consistent with the conclusion that the RTD is not essential for efficient virus multiplication in the nucleate phloem cells but intervenes in virus movement to increase the rate at which new infection foci are established and expand.

In situ localization of cacao swollen shoot virus in agroinfected Theobroma cacao

SummaryCacao swollen shoot virus (CSSV) is a small non-enveloped bacilliform virus with a double-stranded DNA genome. A very restricted host range and difficulties in transmitting the virus, either mechanically or via its natural vector, have hindered the study of cacao swollen shoot disease. As an alternative to the particle-bombardment method previously reported, we investigated another approach to infect Theobroma cacao. A greater-than-unit length copy (1.2) of the CSSV DNA genome was cloned into the Agrobacterium binary vector pBin19 and was transferred into young plants via Agrobacterium tumefaciens. Typical leaf symptoms and stem swelling were observed seven and eleven weeks post inoculation, respectively. Viral DNA, CSSV coat protein and virions were detected in leaves with symptoms. Agroinfected plants were used to study the in situ localization of CSSV and its histopathologic effects in planta. In both leaves and petioles, virions were only seen in the cytoplasm of phloem companion cells and of a few xylem parenchyma cells. Light microscopy showed that stem swelling results from a proliferation of the xylem, phloem and cortex cells.

Binding of RNA by the alfalfa mosaic virus movement protein is biphasic

The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single‐stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated ‘primary complexes’) were detected by a nitrocellulose‐retention assay within 1 min of mixing, both At 4 and 22°C. In contrast, an incubation of 30 min at 22°C was necessary to obtain electrophoretically retarded complexes (‘stabilized complexes’), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4°C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a biomolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.