The FIT C-terminal Ser271/272 site was important for the capacity of FIT to localize to NBs. (A) Confocal images of nuclear localization of FITmSS271AA-GFP at t = 0 and t = 15 min. FITmSS271AA-GFP accumulated in NBs, but NB formation required a longer time compared with FIT-GFP. See also Videos 1 and 3. Two independent experiments. Representative images from one nucleus. (B and C) Bar diagrams shown in B are the number of NBs, and in C, the sizes of NBs of FIT-GFP and FITmSS271AA-GFP at t = 5/15 min. NB number and size were determined with the software ImageJ (National Institute of Health). FIT-GFP accumulated in more and larger NBs than FITmSS271AA-GFP. See Videos 1 and 3. FITmSS271AA-GFP lacks IDR^Ser271/272. This IDR may be relevant for FIT NB formation (Fig. S2). In B and C, bar diagrams represent the mean and standard deviation for quantification of 15 nuclei from a transformed plant (n = 15). Two experiments were conducted, and one representative result is shown. Statistical analysis was performed with the Mann–Whitney test. Different letters indicate statistically significant differences (P < 0.05). Scale bar: 2 µm. Arrowheads indicate NBs. G = GFP. Analysis was conducted in transiently transformed N. benthamiana leaf epidermis cells, following the standardized FIT NB analysis procedure.