Fig. 7

RcMYB8 binds to RcPR5/1 and RcP5CS1 in vivo and in vitro. A Diagram showing the positions of four fragments containing putative MYB binding sites (P1, P2, P3 and P4) in the 2 kb promoter region of RcPR5/1. B Y1H analysis showing that RcMYB8 binds to the RcPR5/1 promoter fragment containing the MYB-binding sites (TAACCA). The promoter of RcPR5/1 was divided into four fragments. AbA (aureobasidin A), a yeast cell growth inhibitor, was used as a screening marker. The base concentration of AbA was 300 ng/mL. p53 was used as a positive control. C to F A luciferase complementation imaging assay showing that RcMYB8 accumulates RcPR5/1-P2 or RcPR5/1-P4 in tobacco leaves. The Agrobacterium strain GV3101 (pSoup-p19) harboring different constructs was infiltrated into different regions of tobacco leaves. Luciferase activities were recorded in these regions 3 days after infiltration. G EMSA analysis showing that RcMYB8 binds to the TAACCA motif of the RcPR5/1 promoter. The hot probe was a biotin-labeled fragment of the RcPR5/1 promoter containing the TAACAA sequence, and the cold probe was a nonlabeled competitive probe (10- and 50-fold larger amounts than that of the hot probe). GST-tagged RcMYB8 was purified. “-” represents the absence and “+” represents the presence of components in the reaction. H Binding of RcMYB8 to the RcP5CS1 promoter in the Y1H assay. The constructs pHis-RcP5CS1-P5 and pHis-RcP5CS1-P6. I Binding of RcMYB8 to the RcP5CS1 promoter region. A GST-RcMYB8 fusion protein expressed in Escherichia coli was purified. The bottom represents the schematic representation of the RcP5CS1 promoter, and it binding to the biotin-labeled probe contains TAACCA. J and K A luciferase complementation imaging assay showing that RcMYB8 accumulates RcP5CS1-P5 in tobacco leaves. The Agrobacterium strain GV3101 (pSoup-p19) harboring different constructs was infiltrated into different regions of tobacco leaves. Luciferase activities were recorded in these regions 3 days after infiltration