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

• Published in: The Plant cell 2003-09, Vol.15 (9), p.2058-2075
• Main Authors: Laporte, Céline, Vetter, Guillaume, Loudes, Anne-Marie, Robinson, David G., Hillmer, Stefan, Stussi-Garaud, Christiane, Ritzenthaler, Christophe
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - physiology; Antibodies; Calreticulin - pharmacology; Cell Division - physiology; Cell lines; Cell membranes; Cell walls; Cells, Cultured; Cytoskeleton - drug effects; Cytoskeleton - metabolism; Daughter cells; Epidermal cells; Golgi Apparatus - metabolism; Green Fluorescent Proteins; Immunohistochemistry; Luminescent Proteins - metabolism; Microscopy, Confocal; Microscopy, Electron; Microtubules; Microtubules - physiology; Nepovirus - growth & development; Nicotiana - metabolism; Nicotiana - ultrastructure; Nicotiana - virology; Plant cells; Plant Viral Movement Proteins; Plants, Genetically Modified; Plasmodesmata; Precipitin Tests; Protein Transport - physiology; Recombinant Fusion Proteins - metabolism; Secretory Vesicles - drug effects; Secretory Vesicles - metabolism; Transfection; Viral Proteins - genetics; Viral Proteins - metabolism; Viruses
• ISSN: 1040-4651; 1532-298X
• DOI: 10.1105/tpc.013896

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.