Apple chlorotic leaf spot virus 50 kDa protein is targeted to plasmodesmata and accumulates in sieve elements in transgenic plant leaves

Yoshikawa, Nobuyuki; Oogake, S.; Terada, Mai; Miyabayashi, Shigeaki; Ikeda, Yoshiharu; Takahashi, Tsuyoshi; Ogawa, K.

doi:10.1007/s007050050660

Cited by 21 publications

(9 citation statements)

References 32 publications

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“…A limited number of microscopic and physiological techniques have been used to provide more evidence for plasmodesmal association, in addition to observing GFP fusion proteins. Co‐localization with the polysaccharide callose, which is deposited at the necks of plasmodesmata (Radford et al , 1998), is a commonly used technique, involving either fluorescence immunolocalization (Blackman et al , 1998; Baluska et al , 1999; Yoshikawa et al , 1999; Erhardt et al , 2000) or staining with aniline blue (Gorshkova et al , 2003; Sagi et al , 2005; Levy et al , 2007). Ultimately, immunogold transmission electron microscopy with antibodies against either the protein of interest (Epel et al , 1996; Blackman et al , 1998; Grieco et al , 1999; Yoshikawa et al , 1999) or its GFP tag (Medina‐Escobar et al , 2003) is the most definitive technique available for confirming plasmodesmal association.…”

Section: Resultsmentioning

confidence: 99%

Reflection across plant cell boundaries in confocal laser scanning microscopy

Liu

Kuhlmey

Smith

et al. 2008

Journal of Microscopy

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SummaryThe fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFPtagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finitedifference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal

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Section: Resultsmentioning

confidence: 99%

Reflection across plant cell boundaries in confocal laser scanning microscopy

Liu

Kuhlmey

Smith

et al. 2008

Journal of Microscopy

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“…(i) The amino acid sequence of 50KP has some similarity to MPs of other plant viruses and the protein was detected in the cell wall fraction from infected tissues (Sato et al , 1993 , 1995 ). (ii) Immunoelectron microscopy with an antiserum against 50KP showed that the protein is localized to plasmodesmata in infected Chenopodium quinoa cells (Yoshikawa et al , 1999 ). (iii) In transgenic plants expressing 50KP fused to GFP, the fluorescence was associated with plasmodesmata and accumulated in sieve elements (Yoshikawa et al , 1999 ).…”

Section: Introductionmentioning

confidence: 99%

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

Satoh

Matsuda

Kawamura

et al. 2000

Journal of General Virology

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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.

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“…The MP has been assigned to the '30K superfamily' by comparative sequence analyses (Mushegian & Koonin, 1993). Immunoelectron microscopy with antiserum against the MP showed that the protein is localized to plasmodesmata within virus-infected cells without the formation of tubules; virions were not observed in cell-wall plasmodesmata (Sato et al, 1995;Yoshikawa et al, 1999), although tubular structures protruded from the cell surface when MP fused to green fluorescent protein (MP-GFP) was expressed transiently in protoplasts (Satoh et al, 2000).…”

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confidence: 99%

Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein

Isogai

Yoshikawa

2005

Journal of General Virology

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The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin-UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82-126 and 127-287 potentially contain two independently active, single-stranded nucleic acid-binding domains.

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Apple chlorotic leaf spot virus 50 kDa protein is targeted to plasmodesmata and accumulates in sieve elements in transgenic plant leaves

Cited by 21 publications

References 32 publications

Reflection across plant cell boundaries in confocal laser scanning microscopy

Reflection across plant cell boundaries in confocal laser scanning microscopy

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

Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein

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