Fig. 5.

RhNAP binding to cis-elements in the promoter of RhCKX6. A Wild-type and mutant probes derived from the RhCKX6 promoter. The wild-type cis-element and its nucleotide substitutions in the mutants are underlined. Interaction between GST-RhNAP and the biotin-labeled probe on a native PAGE gel. Purified protein (3 μg) was incubated with 25 pM of the biotin-labeled wild-type probe. Non-labeled probe with different concentrations (from 10 to 100 x) was added for the competition test. B Transactivation activity of RhNAP with the RhCKX6 promoter in yeast. GAD-RhNAP, but not GAD itself, activates expression of the LacZ reporter gene driven by the wild-type 31-bp fragment of the RhCKX6 promoter. The mutated fragment abolishes activation of the LacZ reporter gene expression. C Regulation of the RhCKX6 promoter activity by RhNAP in A. thaliana mesophyll protoplasts. The effector constructs contained GFP-RhNAP or GFP alone, driven by the super1300 promoter. The reporter constructs contained the RhCKX6 promoter (− 1308 bp to − 1 bp upstream of ATG). Protoplasts were co-transformed with different combinations of effector and reporter constructs and the relative GUS activity indicated the promoter activity. Normalized GUS activities are presented as the means ± standard deviation (n = 6). The difference was statistically significant (Student’s t-test, P < 0.01) as denoted by asterisks