Fig. 4
Confirmation of PbrWRKY62 as the upstream regulator of PbrADC1 gene. a Dual-luciferase assay. Transformants containing empty pSAK277 and each report vector were used as controls. b Y1H assay. Yeast cell co-transformed with pGAD7-p53 & p53-AbAi was used as a positive control, while yeast cell with pGADT7-AD & PbrADC1proS1-pAbAi and pGADT7-AD & PbrADC1proS2-pAbAi, respectively, were used as negative controls. c ChIP-qPCR analysis. Pear calli overexpressing the empty pCAMBIA1300-GFP plasmid was used as a negative control. d In vitro EMSA assay. FAM luciferase-labelled PbrADC1 promoter fragments, containing the W-box elements (TTGACC) and its mutant (TTGACC → TTTAGC), were named as PbrADC1proS1 probe, PbrADC1proS2 probe, PbrADC1proS1mut probe, and PbrADC1proS2mut probe, respectively, and the unlabeled PbrADC1 promoter fragments containing the W-box elements were used as competitor probes. The presence and absence of His protein, His-PbrWRKY62 protein, labeled probe, or competitor probe were indicated by “ + ” and “ − ”, respectively. Competitor probe concentrations were 50-fold (50 ×) and 100-fold (100 ×) those of the labeled probe. e BiFC assay for the self-interaction of PbrWRKY62. Transformants containing YFPN & YFPC, YFPN & PbrWRKY62-YFPC, and YFPC & PbrWRKY62-YFP.N were used as controls. Data represented the mean value of three biological replicates, and different lowercase letters meant significance between samples (p < 0.05)