Institute of Molecular Agrobiology, 1 Research Link, the National University of Singapore, Singapore 117604
To study the intercellular trafficking potential of alfalfa mosaic virus movement protein (AlMV MP) and the cellular structures that may assist in this activity, the AlMV MP gene was fused with the green fluorescent protein (GFP) gene and delivered into epidermal cells of onion bulb scales and Nicotiana tabacum (BY-2) suspension culture cells by biolistic gene bombardment. Its subcellular distribution as well as intracellular and intercellular trafficking in epidermal cells of onion bulb scales and in insect cells were systematically studied. Cells expressing the MP-GFP showed a fluorescent filamentous network in the cell cortex and fluorescent irregular bodies scattered in the cytoplasm and present in the cell wall. By 44 h post transfection with the MP-GFP construct, 56% of fluorescent sites consisted of clustered cells, whereas the fluorescent sites from cells bombarded with the free GFP construct consisted of isolated cells. Time course experiments demonstrated that the MP-GFP was able to move into adjacent cells from a primarily transfected cell. Optical serial sectioning studies suggested that the MP-GFP could target to the plasmodesmata, and move through them into neighboring cells. Furthermore, detailed fluorescence microscopy analyses of both tobacco and onion cells showed that the MP-GFP co-localized with the endoplasmic reticulum (ER). Subcellular fractionation of insect cells and immunoblotting analysis revealed that the MP-GFP behaves as an integral membrane protein. These results indicate that the MP-GFP is associated with the ER and suggest that the ER association may be important for intracellular and intercellular movement of the AlMV MP.