Subcellular localization and immunodetection of movement proteins of olive latent virus 1.

Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was observed in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata.

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles.

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.

Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis.

Genotoxic stress activates complex cellular responses allowing for the repair of DNA damage and proper cell recovery. Although plants are exposed constantly to increasing solar UV irradiation, the signaling cascades activated by genotoxic environments are largely unknown. We have identified an Arabidopsis mutant (mkp1) hypersensitive to genotoxic stress treatments (UV-C and methyl methanesulphonate) due to disruption of a gene that encodes an Arabidopsis homolog of mitogen-activated protein kinase phosphatase (AtMKP1). Growth of the mkp1 mutant under standard conditions is indistinguishable from wild type, indicating a stress-specific function of AtMKP1. MAP kinase phosphatases (MKPs), the potent inactivators of MAP kinases, are considered important regulators of MAP kinase signaling. Although biochemical data from mammalian cell cultures suggests an involvement of MKPs in cellular stress responses, there is no in vivo genetic support for this view in any multicellular organism. The genetic and biochemical data presented here imply a central role for a MAP kinase cascade in genotoxic stress signaling in plants and indicate AtMKP1 to be a crucial regulator of the MAP kinase activity in vivo, determining the outcome of the cellular reaction and the level of genotoxic resistance.

Subcellular localization and in vivo identification of the putative movement protein of olive latent virus 2.

The gene encoding the 36.5 kDa ('36K') nonstructural protein located on RNA3 of olive latent virus 2 (OLV-2) was cloned, expressed with the Escherichia coli pGEX-2T system and the purified protein used to raise a polyclonal antiserum. Immunoblot analysis of OLV-2-infected Nicotiana benthamiana plants showed that the 36K protein accumulated in the early stages of infection and was associated with a subcellular fraction enriched in cytoplasmic membranes. In infected cells there were tubular structures, some containing virus-like particles, scattered in the cytoplasm or protruding from or penetrating the cell wall at the plasmodesmata. Immunogold labelling localized the 36K protein in the plasmodesmata of OLV-2-infected cells and showed it to be associated with virus-containing tubules. Leaf trichome cells of N. tabacum plants, transformed with a 36K-green fluorescent protein (GFP) fusion construct, revealed localized fluorescence in the cell walls, possibly due to association of the fusion protein with plasmodesmata. When the same 36K-GFP fusion protein was expressed in N. tabacum protoplasts, long tubular fluorescent structures protruded from the protoplast surface, suggesting that the 36K protein is responsible for tubule induction. The conclusion is drawn that this protein is likely to be the OLV-2 movement protein, mediating cell-to-cell virus movement, and that movement is by a tubule-guided mechanism.

Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-terminal propeptide-mediated sorting pathway.

The green fluorescent protein (GFP) from Aequorea victoria can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C-terminal endoplasmic reticulum (ER)-retention motif of mGFP5-ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18-24 h) depending on the cell type. Most chloroplast-rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast-poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non-acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.