Figure 6.

Translocation of OsNAC4 into the nucleus and phosphorylation of OsNAC4 during HR cell death. (A) Time-dependent accumulation of OsNAC4 in cultured rice cells after inoculation with the avirulent N1141 strain. The upper panels represent the amount of OsNAC4 protein in the nuclear fraction. Nuclear fractions (10 μg protein) were separated by SDS–PAGE and transferred to a nitrocellulose membrane. Then OsNAC4 were detected by immunoblotting with an anti-OsNAC4 antibody (top part). The same amount of each nuclear fraction was separated by SDS–PAGE and proteins were detected by silver staining (middle part). The lower panel represents the proportion of OsNAC4 in the cytosolic fraction. Total protein (10 μg) from each fraction was separated by SDS–PAGE and transferred to a nitrocellulose membrane. Then OsNAC4 were detected by immunoblotting with an anti-OsNAC4 antibody (upper part). The same amount of each cytosolic fraction was separated by SDS–PAGE and proteins were detected by silver staining (lower part). The purity of each fraction was analysed by immunoblotting using an antihistone H3 antibody (nucleus-specific antibody) and an anti-OsUSP (cytosol-specific antibody). (B) Immunogold labelling of OsNAC4 using anti-OsNAC4 antibody. A control cell line (upper panels) and the NR2-4 OsNAC4 knock-down line (middle panels) were visualized 0 h (left panels) and 12 h (right panels) after inoculation with the avirulent N1141 strain. A control cells treated with preimmune serum were visualized 0 h (left panels) and 12 h (right panels) after inoculation with the N1141 strain (lower panels). Bar represents 1 μm. (C) The number of gold particles in the nucleus and cytosol when the OsNAC4 protein was immunogold labelled. The number of gold particles in nucleus and cytosol were visually counted. Pre-IS, preimmune serum; N, nucleus; C, cytosol. Each data represent the average of the particle number in five counted area (1 μm²). Bars indicate the standard errors. (D) The localization of NES-fused OsNAC4-DsRed and OsNAC4-DsRed after 6 h transformation in rice protoplasts. NES fused to the N-terminal of OsNAC4-DsRed (left panel) and OsNAC4-DsRed (right panel). Bar represents 10 μm. (E) Cell death in protoplasts transfected with the NES-OsNAC4-DsRed and OsNAC4-DsRed (control) vectors. Six hours after transfection, dead protoplasts were scored using a light microscope. Y-axis values represent the percentage of transfected protoplasts that were determined to be dead. Each determination was done with at least 200 cells in each of three independent experiments. Bars indicate the standard errors. (F) The time-dependent accumulation of OsNAC4 in nuclei isolated from cultured rice cells after inoculation with the avirulent N1141 strain. The left three lanes represent nuclear OsNAC4 in water-treated cells, whereas the right three lanes represent OsNAC4 in cells treated with 2 μM staurosporine. Total protein (10 μg) from each fraction was separated by SDS–PAGE and transferred to a nitrocellulose membrane. The same amount of each fraction was separated by SDS–PAGE, and proteins were detected by silver staining (lower part). (G) Phosphorylation of the OsNAC4 protein accumulated in nuclei. Rice protoplasts were expressed with OsNAC4 and cytosol and nuclei fractions were isolated. Both protein extracts were subjected to immunoprecipitation (IP) with anti α-OsNAC4, and the same amounts of immunoprecipitated proteins were immunoblotted (IB) with the indicated antibodies. A full colour version of this figure is available at The EMBO Journal online.