Three NB markers and speckle components became localized in FIT NBs and colocalized fully with FIT (designated type III), suggesting that FIT NBs have speckle function. Confocal images showing localization of FIT-GFP and NB markers (type III) upon coexpression in the nucleus at t = 0 and t = 5 min. (A–C) Coexpression of FIT-GFP with A, UAP56H2-mRFP, B, P15H1-mRFP, and C, PININ-mRFP. All three type III NB markers were homogeneously distributed and colocalized with FIT-GFP in the nucleus at t = 0, while they colocalized with FIT-GFP in FIT NBs at t = 5 min. UAP56H2-mRFP and P15H1-mRFP showed homogeneous localization in the single expression at both t = 0 and t = 5 min (compare with Fig. S3, E and F), while PININ-mRFP localized mainly in one large and several small NBs upon single expression at t = 0 and t = 5 min (compare with Fig. S3 G). Hence, these three markers adopted the localization of FIT-GFP upon coexpression and suggest that FIT NBs have a speckle function. Scale bar: 2 µm. Arrowheads indicate colocalization within NBs. G = GFP; R = mRFP. Fluorescence protein analysis was conducted in transiently transformed N. benthamiana leaf epidermis cells, following the standardized FIT NB analysis procedure. In all examined cells, the proteins showed full colocalization. Representative images from four to seven independent experiments are shown. Contrast of images in A at t = 5 was enhanced for better assessment. For data with type I markers (no colocalization) and type II markers (partial colocalization) see Fig. S3, A and B; and Fig. 5.