The nuclear localization signal of a pollen-specific, desiccation-associated protein of lily is necessary and sufficient for nuclear targeting

The lily LLA23 protein represents a novel member of water-deficit/ripening-induced protein family (Plant Cell Physiol. 41: 477-485, 2000). Examination of the C-terminal half of LLA23 reveals the presence of basic regions that are reminiscent of a nuclear localization signal (NLS). To investigate the nuclear targeting property of NLS in LLA23, a green fluorescent protein (GFP) gene fused with the C-terminal half of LLA23 (GFP-LLA23) was constructed. In addition, the GFP alone and a mutGFP-DeltaLLA23 that had the sequence for the putative NLS deleted were also constructed. All these three constructs were separately inserted into a bamboo mosaic potexvirus (BaMV) vector. Infection of BaMV in Chenopodium quinoa caused local lesions in leaves where the green fluorescence of fusion proteins could be visualized by fluorescence microscopy. The RNA blot and immunoblot analyses of BaMV-infected leaves indicated that the recombinant subgenomic RNA and the resulting protein were strongly detected. Fluorescence microscopic studies revealed that the NLS in LLA23 exhibited a property of nuclear targeting, showing highly condensed spots of green fluorescence in leaf cells, whereas GFP alone was apparently distributed throughout the cytoplasm. In contrast, a deletion of the NLS sequence resulted in exclusively cytoplasmic localization of the fusion protein. The nuclear location of the GFP-LLA23 protein in leaf cells was further confirmed by staining with 4',6-diamidino-2-phenylindole. These results clearly demonstrate that the putative NLS in LLA23 is necessary and sufficient for import of the LLA23 protein into the nucleus.