Extended Data Figure 7. Nuclear localization of LIMYB.

a, Colocalization of LIMYB with the nuclear marker gene Glycine max (Gm)NAC81. N. benthamiana leaves were co-infiltrated with A. tumefaciens carrying a 35S∷LIMYB–GFP construct and a 35S∷YFP–GmNAC81 construct. Forty-eight hours after infiltration, the subcellular localizations of the fluorescent fusion proteins were examined by confocal microscopy. The figure shows representative confocal images from two independent experiments. b, Confocal fluorescence image of transiently expressed GFP (left) or LIMYB–GFP (right) in epidermal cells of tobacco leaves. Scale bars, 10 μm. The figure shows representative confocal images from two independent experiments. c, Immunoblotting of transiently expressed LIMYB–GFP in epidermal cells of tobacco leaves. Total protein was extracted from agro-infiltrated N. benthamiana leaves containing the 35S∷GFP (left lanes) or 35S∷LIMYB–GFP (right lanes) constructs and immunoblotted with an anti-GFP monoclonal antibody to examine the stability of the fusion protein. The positions of molecular mass are shown in kDa. d, Confocal fluorescence image of transiently expressed GFP–LIMYB in epidermal cells of tobacco leaves. Scale bars, 10 μm. The figure shows representative confocal images from four independent experiments. e, Confocal fluorescence image of transiently expressed LIMYB– GFP in epidermal cells of tobacco leaves. Scale bars, 10 μm. The figure shows representative confocal images from four independent experiments. f, g, Confocal fluorescence image of root cells stably transformed with YFP– LIMYB or LIMYB–GFP. Root tips from transgenic seedlings expressing YFP–LIMYB (f) or LIMYB–GFP (g) were directly examined under a laser confocal microscope. Scale bars, 20 μm. The figures show representative confocal images from three biological replicas. Neither the fusion of YFP to the LIMYB N terminus nor GFP to its C terminus altered the nuclear localization of LIMYB in either agro-inoculated N. tabacum leaves or stably transformed Arabidopsis roots. h, i, Confocal fluorescent image of LIMYB fused to GFP or mCherry under the control of its own promoter. The figures show representative confocal images from two independent experiments. The fluorescence was also concentrated in the nucleus of transfected cells by expression of LIMYB–GFP or LIMYB–mCherry fusions under the control of the LIMYB endogenous promoter. Scale bars, 20 μm. Collectively, these results indicate that LIMYB was localized in the nucleus.