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In JoVE (2)
- A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment
- Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
Other Publications (8)
Articles by Shoko Ueki in JoVE
A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment
Shoko Ueki1, Benjamin L. Meyers1, Farzana Yasmin2, Vitaly Citovsky2
1Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 2Bio-Medical Engineering Department, NED University of Engineering and Technology
Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.
Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
Shoko Ueki, Benoît Lacroix, Vitaly Citovsky
Department of Biochemistry and Cell Biology, State University of New York
Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.
Other articles by Shoko Ueki on PubMed
Nature Cell Biology. Jul, 2002 | Pubmed ID: 12055637
Systemic movement is central to plant viral infection. Exposure of tobacco plants to low levels of cadmium ions blocks the systemic spread of turnip vein-clearing tobamovirus (TVCV). We identified a tobacco glycine-rich protein, cdiGRP, specifically induced by low concentrations of cadmium and expressed in the cell walls of plant vascular tissues. Constitutive cdiGRP expression inhibited systemic transport of TVCV, whereas suppression of cdiGRP production allowed TVCV movement in the presence of cadmium. cdiGRP exerted its inhibitory effect on TVCV transport by enhancing callose deposits in the vasculature. So cdiGRP may function to control plant viral systemic movement.
Proceedings of the National Academy of Sciences of the United States of America. Feb, 2005 | Pubmed ID: 15684080
Identification of an Interactor of Cadmium Ion-induced Glycine-rich Protein Involved in Regulation of Callose Levels in Plant Vasculature
Proceedings of the National Academy of Sciences of the United States of America. Aug, 2005 | Pubmed ID: 16103368
Cadmium-induced glycine-rich protein (cdiGRP) is a cell wall-associated factor that increases callose levels in plant vasculature. To better understand the cdiGRP/callose regulation system, we identified a tobacco protein, GrIP (cdiGRP-interacting protein, GrIP), that associates with cdiGRP and localizes at the plant cell wall. Constitutive overexpression of GrIP enhanced the accumulation of the cdiGRP protein and callose in vasculature-associated cells with or without treatment with cadmium ions. That GrIP gene expression was not affected by cadmium ions indicated that GrIP does not directly modulate the callose levels induced by the treatment. Instead, GrIP most likely functions by further elevating the accumulated amount of cdiGRP, the expression of which is up-regulated by the cadmium ions. Interestingly, the levels of cdiGRP mRNA were not affected by constitutive expression of GrIP, demonstrating that the enhancement in cdiGRP protein accumulation by GrIP overexpression occurs posttranslationally. Collectively, these observations suggest that GrIP interacts with cdiGRP and increases its level of accumulation; in turn, the elevated amounts of cdiGRP induce callose deposits in the plant cell walls. Therefore, GrIP and cdiGRP represent sequentially acting factors in a biochemical pathway that regulates callose accumulation in the plant vasculature.
A Human Lung Carcinoma Cell Line Supports Efficient Measles Virus Growth and Syncytium Formation Via a SLAM- and CD46-independent Mechanism
Journal of Virology. Nov, 2007 | Pubmed ID: 17715217
Measles virus (MV) propagates mainly in lymphoid organs throughout the body and produces syncytia by using signaling lymphocyte activation molecule (SLAM) as a receptor. MV also spreads in SLAM-negative epithelial tissues by unknown mechanisms. Ubiquitously expressed CD46 functions as another receptor for vaccine strains of MV but not for wild-type strains. We here show that MV grows and produces syncytia efficiently in a human lung adenocarcinoma cell line via a SLAM- and CD46-independent mechanism using a novel receptor-binding site on the hemagglutinin protein. This infection model could advance our understanding of MV infection of SLAM-negative epithelial cells and tissues.
Nature Protocols. 2009 | Pubmed ID: 19131958
Transient gene expression is an indispensable tool for studying functions of gene products. In the case of plants, transient introduction of genes by Agrobacterium infiltration is a method of choice for many species. However, this technique does not work efficiently in Arabidopsis leaf tissue, the most widely used model system for basic plant biology research. Here we present an optimized protocol for biolistic delivery of plasmid DNA into the epidermis of Arabidopsis leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol yields efficient and reproducible transient expression of diverse genes and is exemplified here for use in a functional assay of a transcription repressor and for the subcellular localization and cell-to-cell movement of plant viral movement protein. This protocol is suitable for studies of biological function and subcellular localization of the gene product of interest directly in planta by utilizing different types of activity-based assays. Using this procedure, the data are obtained after 2-4 d of work.
ANK, a Host Cytoplasmic Receptor for the Tobacco Mosaic Virus Cell-to-cell Movement Protein, Facilitates Intercellular Transport Through Plasmodesmata
PLoS Pathogens. 2010 | Pubmed ID: 21124937
Plasmodesma (PD) is a channel structure that spans the cell wall and provides symplastic connection between adjacent cells. Various macromolecules are known to be transported through PD in a highly regulated manner, and plant viruses utilize their movement proteins (MPs) to gate the PD to spread cell-to-cell. The mechanism by which MP modifies PD to enable intercelluar traffic remains obscure, due to the lack of knowledge about the host factors that mediate the process. Here, we describe the functional interaction between Tobacco mosaic virus (TMV) MP and a plant factor, an ankyrin repeat containing protein (ANK), during the viral cell-to-cell movement. We utilized a reverse genetics approach to gain insight into the possible involvement of ANK in viral movement. To this end, ANK overexpressor and suppressor lines were generated, and the movement of MP was tested. MP movement was facilitated in the ANK-overexpressing plants, and reduced in the ANK-suppressing plants, demonstrating that ANK is a host factor that facilitates MP cell-to-cell movement. Also, the TMV local infection was largely delayed in the ANK-suppressing lines, while enhanced in the ANK-overexpressing lines, showing that ANK is crucially involved in the infection process. Importantly, MP interacted with ANK at PD. Finally, simultaneous expression of MP and ANK markedly decreased the PD levels of callose, β-1,3-glucan, which is known to act as a molecular sphincter for PD. Thus, the MP-ANK interaction results in the downregulation of callose and increased cell-to-cell movement of the viral protein. These findings suggest that ANK represents a host cellular receptor exploited by MP to aid viral movement by gating PD through relaxation of their callose sphincters.
To Gate, or Not to Gate: Regulatory Mechanisms for Intercellular Protein Transport and Virus Movement in Plants
Molecular Plant. Sep, 2011 | Pubmed ID: 21746703
Cell-to-cell signal transduction is vital for orchestrating the whole-body physiology of multi-cellular organisms, and many endogenous macromolecules, proteins, and nucleic acids function as such transported signals. In plants, many of these molecules are transported through plasmodesmata (Pd), the cell wall-spanning channel structures that interconnect plant cells. Furthermore, Pd also act as conduits for cell-to-cell movement of most plant viruses that have evolved to pirate these channels to spread the infection. Pd transport is presumed to be highly selective, and only a limited repertoire of molecules is transported through these channels. Recent studies have begun to unravel mechanisms that actively regulate the opening of the Pd channel to allow traffic. This macromolecular transport between cells comprises two consecutive steps: intracellular targeting to Pd and translocation through the channel to the adjacent cell. Here, we review the current knowledge of molecular species that are transported though Pd and the mechanisms that control this traffic. Generally, Pd traffic can occur by passive diffusion through the trans-Pd cytoplasm or through the membrane/lumen of the trans-Pd ER, or by active transport that includes protein-protein interactions. It is this latter mode of Pd transport that is involved in intercellular traffic of most signal molecules and is regulated by distinct and sometimes interdependent mechanisms, which represent the focus of this article.
Protoplasma. Jan, 2011 | Pubmed ID: 21116665
The turnover of callose (β-1,3-glucan) within cell walls is an essential process affecting many developmental, physiological and stress related processes in plants. The deposition and degradation of callose at the neck region of plasmodesmata (Pd) is one of the cellular control mechanisms regulating Pd permeability during both abiotic and biotic stresses. Callose accumulation at Pd is controlled by callose synthases (CalS; EC 126.96.36.199), endogenous enzymes mediating callose synthesis, and by β-1,3-glucanases (BG; EC 188.8.131.52), hydrolytic enzymes which specifically degrade callose. Transcriptional and posttranslational regulation of some CalSs and BGs are strongly controlled by stress signaling, such as that resulting from pathogen invasion. We review the role of Pd-associated callose in the regulation of intercellular communication during developmental, physiological, and stress response processes. Special emphasis is placed on the involvement of Pd-callose in viral pathogenicity. Callose accumulation at Pd restricts virus movement in both compatible and incompatible interactions, while its degradation promotes pathogen spread. Hence, studies on mechanisms of callose turnover at Pd during viral cell-to-cell spread are of importance for our understanding of host mechanisms exploited by viruses in order to successfully spread within the infected plant.