Fig. 5: Cytosol localization of CP-Ym1 interaction is essential for cell death triggering activity.

A Ym1-GFP alone was subcellular localized in nucleolus and cytoplasm (upper panel), Ym1-GFP together with WYMV-CP was subcellular localized only in cytoplasm (lower panel). Agrobacterium mixture containing plasmids expressing Ym1-GFP and CP-HA fusion proteins were infiltrated into N. benthamiana leaves. H2B-RFP was used as the nucleus marker. The signals were observed under a confocal microscope at 2 days post inoculation (dpi). The arrows point the merged signals within nucleus. Bars = 25 μm. The experiment was repeated three times independently with similar results. B Ym1-GFP and CP-HA extracted from the cytosol and nucleus components of N. benthamiana leaf issues were detected by Western blotting with anti-HA or anti-GFP antibodies. Cytosol-localized protein Actin and nucleus-localized protein Histone 3 were detected with their monoclonal antibodies and used as positive controls of cytosol and nucleus components, respectively. The experiment was repeated three times independently with similar results. C Subcellular localization of the Ym1 fused with nucleus exporting (Ym1-GFP-NES) and localization (Ym1-GFP-NLS) sequences. GFP and the fusion proteins, Ym1-GFP-NLS, and Ym1-GFP-NES, were expressed in N. benthamiana leaves by Agro-infiltration. The signals were observed under a confocal microscope at 2 dpi. Histone 2B (H2B-RFP) was used as nucleus-localized marker protein. The arrows point the merged signals within nucleus. Bars = 25 μm. The experiment was repeated three times independently with similar results. D Analysis of cell death-inducing activity of the Ym1-GFP-NES and NLS fusion proteins. The cell death triggered by each fusion protein was scored at 4 dpi (leaf panel). A thumbnail diagram indicates the positioning of each expressed fusion protein.