Analysis of the cell-to cell and long distant movement of a plant virus expressing GFP

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An infectious full-length cDNA clone (pCLSF) of the apple chlorotic leaf spot trichovirus (ACLSV) genome was constructed under the control of the cauliflower mosaic virus 35S promoter. Mechanical inoculation of pCLSF onto Chenopodium quinoa induced systemic symptoms typical of ACLSV infection. The presence of genomic RNA and viral proteins in pCLSF-inoculated leaves was demonstrated by northern blot and immunoblot analyses, respectively. Virus particles that reacted with ACLSV antiserum were also observed by immunoelectron microscopy. These results confirmed that pCLSF could initiate the infection process in the same way as whole, wild type virions. Comparison of infection efficiency between particle bombardment and mechanical inoculation showed that doses of pCLSF for 50% infection in C.quinoa plants were about 0.02 ng /plant for bombardment and 400 ng /plant for mechanical inoculation, indicating that the bombardment procedure is approximately 10ィイD14ィエD1- fold more effective than me … More chanical inoculation.The 50KDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescence 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 tubular formation on the surface of protoplasts were analysed. The 50KP-GFP fluorescence distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and on protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from cells producing it into neighboring cells in either young or mature leaves and targeted 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 initial cells producing it than that when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. The 50KP-GFP was able to complement local spread of 50KP-deficient virus when transiently expressed 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 regions (between aa positions 287 and 475) were 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 regions resulted in the complete disruption of all these activities. Less