Figure 7.

OsMYBc binds to the OsHKT1;1 promoter in vivo. A, Structure of the OsHKT1;1 gene. The green bar represents sequence upstream of the start codon, and the gray bar stands for coding regions of OsHKT1;1. The blue boxes indicate the positions of the two OsMYBc-binding elements. The lines below the binding elements and coding region indicate the fragments for ChIP-PCR in B. B, ChIP assay indicates that OsMYBc binds the OsHKT1;1 promoter in vivo. Fragmented chromatin DNA of rice protoplasts expressing the OsMYBc-Flag fusion protein was immunoprecipitated using anti-Flag antibody. Fragment I (−422 to +13) containing two cis-elements was amplified by PCR. Fragment II (+226 to +665) was used as a negative control. Input, Total input chromatin DNA; Flag, DNA precipitated using Flag antibody; IgG, DNA precipitated using mouse IgG. Each assay was repeated more than three times with independent biological materials. C and D, Schematic diagram of the effector and reporter used for transactivation studies. The plasmid 35S:OsMYBc was used as the effector, the plasmid ProOsHKT1;1-629:GUS (P) and its mutant version mProOsHKT1;1-629:GUS (mP) were used as the reporter, and 35S:GFP was used as an internal control. The sequences containing mutated nucleotides are shown in D. E, Transactivation activity was detected by GUS staining after reporter and effector plasmids were coinfiltrated into N. benthamiana. F, Quantitative analysis of the GUS activity indicated in E. Asterisks indicate that the mean value is significantly different from that of the control: *, P < 0.05. Error bars represent se (n = 3). 4-MU, 4-Methylumbelliferone.