Multi-color BiFC experiments using tobacco BY-2 suspension culture cells. In A and B, BY-2 protoplasts were transfected using electroporation and visualized using epifluorescence microscopy. In C, protoplasts were transfected by direct DNA uptake and visualized using laser scanning confocal microscopy. Labels above each set of panels indicate the various constructions introduced into the cells. Labels below each set of panels indicates the filter set/channel imaged. mCherry labels the entire cell, whereas mCherry-VirD2NLS labels only the nucleus. Note that, regardless of the tag, Impa-1 localizes to the cytoplasm and Impa-4 localizes predominantly to the nucleus, with some cytoplasmic staining. In Panel A, the blue signal outside the main imaged cell indicates autofluorescence from dead cells. DIC, differential interference contrast image.