title: Intracellular distribution, cell-to-cell trafficking and tubule-inducing activity of the 50 kDa movement protein of Apple chlorotic leaf spot virus fused to green fluorescent protein.
abstract: The 50 kDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescent protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis and tubule formation on the surface of protoplasts were analysed. The 50KP-GFP fluorescence was distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from the cells that produced it into neighbouring cells in both young and mature leaves and targetted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from the initial cells that produced it than when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. 50KP-GFP was able to complement local spread of 50KP-deficient virus when expressed transiently in leaf epidermis of C. quinoa. Expression of 50KP-GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal region (aa 287-457) was not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus or tubule formation on protoplasts. In contrast, deletions in the N-terminal region resulted in the complete disruption of all these activities.
authors.affiliation: Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan1.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Satoh
authors.affiliation: Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan1.
authors.forename: Hironori
authors.initials: H
authors.lastname: Matsuda
authors.affiliation: Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan1.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Kawamura
authors.affiliation: Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan1.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Isogai
authors.affiliation: Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan1.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Yoshikawa
authors.affiliation: Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan1.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Takahashi
mesh_terms: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis*; D008970:Molecular Weight; D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins / Q000032:analysis; D019685:Rosales / Q000821:virology*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000032:analysis* / Q000737:chemistry / Q000502:physiology
chemical_list: D008164:Luminescent Proteins; D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins