N's Nuclear Localization, but Not p50′s, Is Required for Function

<div><p>(A) Subcellular localization of N with NES. gN-Citrine tagged with a NES is excluded from plant cell nuclei and is found only in the cytoplasm (column 2). gN-Citrine carrying a mutated NES accumulates in the nuclei of plant cells (column 3), and its distribution is similar to that of gN-Citrine (column 1). Citrine fluorescence was imaged with 514-nm laser line of a 15-mW argon laser. Scale bar represents 20 μm.</p> <p>(B) HR assay for function of nuclear N. gN-Citrine expressed alone does not cause HR (column 1, top), but causes HR in the presence of p50-U1 (column 1, bottom). Nuclear-excluded gN-Citrine-NES does not cause HR in the absence (column 2, top) or presence (column 2, bottom) of p50-U1. gN-Citrine-NES^mut causes HR only in the presence of p50-U1 (column 3).</p> <p>(C) Defining N's domain required for nuclear localization. NΔTIR-Citrine (column 1), NΔNBS-Citrine (column 2), and NΔLRR2–14-Citrine (column 3) are cytoplasmic and nuclear. There are reduced levels of NΔLRR2–14-Citrine in the nucleus, but the visibility of the nucleolus confirms its presence (column 3). Scale bars represent 20 μm.</p> <p>(D) Subcellular localization of p50-U1 with NES. p50-U1-Ceruelan tagged with a NES is excluded from nuclei and is found only in the cytoplasm (column 2). p50-U1-Ceruelan-NES^mut accumulates in nuclei (column 3), and its pattern of distribution is identical to that of p50-U1-Ceruelan (column 1). The 458-nm laser line of a 15-mW argon laser with appropriate emission filters was used to image Cerulean fluorescence. Scale bars represent 20 μm.</p>