Physical connections between higher plant cell walls and the plasma membrane have been identified visually, but the molecules involved in the contact are unknown. We describe here an Arabidopsis thaliana protein kinase, designated Wak1 for wall-associated kinase, whose predicted extracytoplasmic domain contains several epidermal growth factor repeats and identity with a viral movement protein. Wak1 fractionates with insoluble material when plant tissue is ground in a variety of buffers and detergents, suggesting a tight association with the plant extracellular matrix. Immunocytochemistry confirms that Wak1 is associated with the cell wall. Enzymatic digestion of the cell wall allows the release of Wak1 from the insoluble cell wall fraction, and protease experiments indicate that Wak1 likely has a cytoplasmic kinase domain, and the EGF containing domain is extracellular. Wak1 is found in all vegetative tissues of Arabidopsis, and has relatives in other angiosperms, but not Chlamydomonas. We suggest that Wak1 is a good candidate for a physical continuum between the cell wall and the cytoplasm, and since the kinase is cytoplasmic, it also has the potential to mediate signals to the cytoplasm from the cell wall.
A series of deletion mutants of tobacco mosaic virus movement protein (TMV-MP) was used to identify domains of the protein necessary for membrane association. A membrane fraction was isolated from tobacco BY-2 protoplasts infected with wild-type and mutant TMV that produce MP carrying a 3 aa deletion. Deletions that affected membrane association were clustered around the two major hydrophobic regions of MP that are predicted to be transmembrane. Deletions in other hydrophobic regions also reduced membrane association. In addition, a non-functional mutant of MP, in which one of the known phosphorylation sites was eliminated, was not associated with cellular membranes, while a functional second site revertant restored membrane association. This indicates that MP function requires interaction with membrane; however, membrane association was not sufficient for function. Results are consistent with the hypothesis that TMV-MP is an integral or tightly associated membrane protein that includes two hydrophobic transmembrane domains. INTRODUCTIONCell-to-cell movement of plant viruses requires one or more virus-encoded proteins that enable infectious entities to traverse the plasmodesmata (Pd). Pd are structural channels that provide cytoplasmic and endoplasmic reticulum (ER) continuity between adjacent cells (Staehelin, 1997;Oparka et al., 1999;Lazarowitz & Beachy, 1999). Infection by Tobacco mosaic virus (TMV) leads to production of a virusencoded 30 kDa movement protein (MP) that is required for cell-to-cell movement of the complex that contains viral RNA. MP induces a transient modification of Pd, resulting in an increase in size exclusion limit (SEL) of the Pd from 0?5 to over 10 kDa (Deom et al., 1987;Oparka et al., 1997).Virus replication complexes (VRCs) of many different viruses are associated with one or more cellular organelles, including lysosomal or endosomal membranes (Froshauer et al., 1988; Kujala et al., 2001), peroxisomes (Bleve-Zacheo et al., 1997), mitochondrial outer membranes (Miller et al., 2001), chloroplast envelope (Prod'homme et al., 2003) or vacuolar membranes (van der Heijden et al., 2001; Hasegawa et al., 2003). In the case of TMV, the VRCs assemble on ER membranes. VCRs contain viral RNA, viral replicase and MP plus many unidentified host proteins, including elements of the cell cytoskeleton such as microtubules and actin filaments (Mas & Beachy, 1999;Lazarowitz & Beachy, 1999;Beachy & Heinlein, 2000;Asurmendi et al., 2004). More recently, real-time imaging of TMV infection that monitored the accumulation of MPgreen fluorescent protein (GFP) fusion protein provided evidence that TMV infection spreads from cell to cell as intact VRCs (Kawakami et al., 2004). Because of the intimate association of MP with ER and its role in forming VRCs on ER, it is important to characterize the association of MP with ER. The hypothesis that MP contains two transmembrane domains that might anchor the protein (Berna, 1995) was supported by recent studies using purified recombinant MP (Brill et al., 2000(Brill ...
A previously unknown cytoskeletal structure, now named the plasmalemmal reticulum (Gens et al. 2000, Protoplasma 212, 115–134), was found in cultured BY-2 tobacco cells during a search for a force-focusing mechanism that might enhance signal transduction by the cells’ mechanosensory Ca2+-selective cation channels (MCaCs). This polyhedral structure, which links cell wall, plasma membrane, and internal cytoplasm, prominently contains arabinogalactan protein (AGP). To check for reticulum-promoted Ca2+ elevation, the AGP-binding reagent (β-d-glucosyl)3 Yariv phenylglycoside has been applied to BY-2 cells expressing a free cameleon Ca2+ reporter. Ca2+ elevation was substantial and prolonged. Moreover it occurred in the nucleus as well as the cytoplasm. Cells treated with non-binding mannosyl Yariv reagent could not be discriminated from untreated controls or those treated with carrier solution alone. Supply of the MCaC inhibiter Gd3+ just before treatment with Yariv reagent prevented Ca2+ rise. These data strongly support the hypothesis that the plasmalemmal reticulum controls MCaC activity. The massive inward spread of Ca2+ suggested that entry of the ion through the channels initiated a wave of release from the ER, and YCX in the ER showed Ca2+ levels consistent with this premise. Cytosolic and nuclear Ca2+ often pulsed in control cells in near synchrony and at rates ranging from zero to five cycles per ∼20-min recording. (Pulsation was over-ridden by the applied amounts of glucosyl Yariv compound.) Suggestively but very crudely, oscillation rate was assessed as possibly correlating with stage of cell cycle. Because cell Ca2+ was lowered and pulsation was eliminated by Gd3+, MCaCs appear to participate in these endogenous fluctuations. The extent to which pulsing plays regulatory roles in relatively undifferentiated types of cells should be evaluated.
The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.
Arabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.
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