Figure 7.

Direct binding of TgNAP to promoters of TgPOD12 and TgPOD17. A, Schematic diagram of the TgPOD12 and TgPOD17 promoters. The black circles indicate the position of the partial promoter fragments within CACG motifs. P1: promoter of TgPOD12; P2: promoter of TgPOD17. B, The prey and bait constructs used for Y1H assay. mP1 and mP2 are mutated versions of P1 and P2, in which the CACG was replaced with AAAG. PADH1, promoter of alcohol dehydrogenase1. GAL4AD, galactose-specific transcription enhancing factor 4 activation domain. C, Growth of yeast cells co-transformed with various prey+bait combinations on selective medium (SD/-Ura/-Leu) with (right) or without (left) AbA. AbA, Aureobasidin A. Positive control: p53-AbAi+pGAD-p53; Negative control: bait+pGADT7. D, EMSA analysis of specific binding of TgNAP to the promoters of TgPOD12 and TgPOD17. The purified His-TgNAP protein and biotin-labeled probe of designed fragments containing CACG motif or mutated AAAG motif were used. Competitor was unlabeled probe at 100- and 200-fold. +: presence; −: absence. E, The schematic of the TgNAP effector and pTgPOD12::LUC, pTgPOD17::LUC reporters. T35S, the terminator of CaMV 35S, respectively. MCS, multiple cloning sites. LUC, firefly luciferase. REN, Renilla luciferase. F, Live image of transcriptional activation of the TgPOD12 and TgPOD17 promoters by TgNAP in N. benthamiana leaves using Dual-LUC system. G, Quantitative analysis of dual-LUC transient expression assays of the promoter activity in N. benthamiana protoplasts. LUC/REN ratio of SK-TgNAP + pGreen0800 empty vector was used as the negative control and was considered as 1 for data normalization. Error bars indicate SE (n = 3). Different letters denote significant differences at P < 0.05 analyzed by Tukey’s test.