| NPR1 (NON-EXPRESSOR OF PR GENES 1) is a critical regulator of systemic acquired resistance (SAR), an inducible defense response in plants. NPR1 transduces salicylic acid (SA), an important signal molecule for plant defense response. NPR1 also regulates biosynthesis of SA, and tolerance to SA stress. In this study, several approaches were taken in attempt to dissect the structure-function relationships of NPR1. First, NPR1 was shown to localize in the nucleus using an NPR1-Green Fluorescent Protein (GFP) fusion protein, and NPR1 nuclear localization correlated with onset of SAR. When NPR1 nuclear localization was controlled by fusing it with the hormone-binding domain of mammalian Glucocorticoid Receptor (GR-HBD), NPR1 nuclear localization was shown to be essential for its functioning.; Second, using a yeast two-hybrid screen, NPR1 was found to interact with members of the TGA family of the bZIP transcription factors. These factors, TGA2 (AHBP-1b), OBF5 and TGA6 were able to bind to the SA-responsive element of PR-1 promoter, therefore could be a link between NPR1 and SA-responsive promoters. When a truncated form of TGA2 was expressed in Arabidopsis, the resulted transgenic lines displayed dominant-negative phenotypes similar to that of npr1 mutants, indicating that TGA2 and NPR1 interact in planta. Biochemical evidence indicated that this interaction was specific and enhanced by SA treatment. Using a chimera-reporter system, a chimeric TGA2GAL4 transcription factor was found to activate a UASGAL::GUS reporter gene in response to SA, and this activation was abolished in the npr1 mutant. These genetic data clearly demonstrate that TGA2 is an SA-responsive and NPR1-dependent transcription activator.; Finally, SA-induced expression of PR genes was found to require the activity of protein phosphatases, but not protein kinases. Site-directed mutagenesis of two N-terminal serine residues was found to affect a specific, but not all, NPR1 function. Serine 11 and 15-to-alanine mutations failed to complement npr1 mutants for tolerance to exogenous SA, while complementing SA-induced expression of PR genes. This indicated that the SA-detoxification and activation of PR gene expression are two separate functions of NPR1. |
