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. 2017 Dec 28;69(5):1109–1123. doi: 10.1093/jxb/erx444

Fig. 6.

Fig. 6.

Substitution of Ser37 by Ala reduces the phosphorylation and the transactivation activity of AabZIP1. (A) Diagram of the AabZIP1 mutant. The polypeptides indicate the conserved protein kinase target sequences in the C1 domain of AREBs. The triangle indicates the location of the amino acid substitution. (B) A yeast two-hybrid assay was used to investigate the interaction of AabZIP1S37A with AaAPK1. BD, DNA-binding domain; AD, activating domain; –TL: synthetic medium without threonine and leucine; –TLH: synthetic medium without threonine, leucine, and histidine; –TLHA: synthetic medium without threonine, leucine, histidine, and adenosine. (C) Bimolecular fluorescence complementation (BiFC) assay to detect the interactions of AaAPK1 (fused with N-terminal fragment of yellow fluorescent protein, YFP, in pEarleyagte201-YFPN) with AabZIP1S37A (fused with C-terminal fragment of YFP in vector pEarleyagte202-YFPC). Construct pairs were co-infiltrated into leaves of N. benthamiana. The empty pEarleyagte202-YFPC construct was used as a negative control. DAPI-stained nuclei were used as control. (D) In vitro kinase assay to compare the phosphorylation of AabZIP1 and AabZIP1S37A by AaAPK1. The GST-AabZIP1or GST-AabZIP1S37A protein was incubated with AaAPK1 in kinase assay buffer, either in the presence (+) or absence (–) of alkaline phosphatase and ATP. After incubation, the proteins were separated using 8% Phos-tag SDS-PAGE gel with Phos-tag and detected by Coomassie Blue staining. Asterisks indicate the phosphorylated GST-AabZIP1 or GST-AabZIP1S37A. (E–H) Transient transactivation analysis of AabZIP1S37A. Transient transactivation of the ADS, CYP71AV1, DBR2, and ALDH1 promoter-LUC fusion gene by AabZIP1 and the mutated AabZIP1S37A by transiently expressing in tobacco leaves. The reporter is indicated at the top of each graph, and the presence (+) or absence (–) of the effectors is indicated at the bottom. The values were determined by calculating the ratio of LUC activity to REN activity (LUC/REN) and then compared with the 35S::YFP control. *, significant difference at P<0.05 as determined by t-tests. Data are means ±SD (n=3). (This figure is available in colour at JXB online.)