title: Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata.
abstract: The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV, genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies (mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that PSLV TGBp3 and GFP-TGBp3 had a tendency to form large protein complexes of an unknown nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal cells in transgenic plants demonstrated that GFP-TGBp3 localized to cell wall-associated punctate bodies, which often formed pairs of opposing discrete structures that co-localized with callose, indicating their association with the plasmodesmata-enriched cell wall fields. After mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static, most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm.
authors.affiliation: Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
authors.forename: E N
authors.initials: EN
authors.lastname: Gorshkova
authors.affiliation: M. M. Shemyakin & Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Str., Moscow 117997, Russia.
authors.forename: T N
authors.initials: TN
authors.lastname: Erokhina
authors.affiliation: Institute of Microbiology, Russian Academy of Sciences, 7 Prospect 60 Let Oktyabrya, Moscow 117811, Russia.
authors.forename: T A
authors.initials: TA
authors.lastname: Stroganova
authors.affiliation: Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
authors.forename: N E
authors.initials: NE
authors.lastname: Yelina
authors.affiliation: Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
authors.forename: A A
authors.initials: AA
authors.lastname: Zamyatnin
authors.affiliation: Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
authors.forename: N O
authors.initials: NO
authors.lastname: Kalinina
authors.affiliation: Institute of Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for Agriculture and Forestry, Messeweg 11/12, D-38104 Braunschweig, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Schiemann
authors.affiliation: Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
authors.forename: A G
authors.initials: AG
authors.lastname: Solovyev
authors.affiliation: Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
authors.forename: S Yu
authors.initials: SY
authors.lastname: Morozov
mesh_terms: D015153:Blotting, Western; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D053830:Plant Viral Movement Proteins; D030821:Plants, Genetically Modified; D031425:Plasmodesmata / Q000378:metabolism; D020365:RNA Helicases / Q000032:analysis* / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D053830:Plant Viral Movement Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C462509:TGBp1 protein, Poa semilatent virus; D020365:RNA Helicases