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. Author manuscript; available in PMC: 2020 Nov 13.
Published in final edited form as: Nat Plants. 2018 May 28;4(6):365–375. doi: 10.1038/s41477-018-0160-7

Figure 6. ORE1 and KIR1 homo- and heterodimers directly control expression of dPCD-associated genes.

Figure 6

a) ChIP-qPCR showing the enrichment of dPCD-associated promoter fragments 24 h after estradiol-induced overexpression of pRPS5A::XVE≫KIR1-GFP. b) ChIP-qPCR showing the percentage of enrichment of dPCD- associated genes 48 h after estradiol-induced overexpression of pRPS5A::XVE≫ORE1-GFP. In both A and B, gray bars indicate the estradiol-treated Col-0 control, red bars show estradiol-treated pRPS5A::XVE≫KIR1-GFP or pRPS5A::XVE≫ORE1-GFP, respectively. Promoter fragments from ACTIN2 and EEF1A were used as negative controls. A mean of two biological replicates per experiment is shown (each biological replicate includes two technical repeats). c) Electrophoretic mobility shift assay. Purified His6-MBP-KIR1 and His6-MBP-ORE1 bind in vitro to a 40-bp sequence of the EXI1, RNS3, and BFN1 promoters, a clear band shift can be seen for the tested promoter fragments. Generally, weaker band shifts were observed, when a non-labeled competitor probe (400-fold excess) was added. In contrast, strong band shifts were detected again when a mutated competitor was added. d) Left panel: Y1H auto-activation assay showing transcriptional activation of full-length ORE1 and KIR1 proteins fused to the DBD domain in absence of a DAD-fused protein partner. C-terminally truncated versions do not auto-activate transcription in yeast any more. Right panel: Y2H assay showing interactions of ORE1 and KIR1 as homodimers, and of ORE1 and KIR1 as heterodimers. Note that DBD-KIR1 interacts with DAD- ORE1, but not vice versa. e) Transient expression assay (TEA) in tobacco BY-2 protoplasts quantifying the activation of dPCD promoters by p35S::KIR1 and p35S::ORE1 constructs. Promoter activation was measured based on firefly luciferase activity controlled by pBFN1, pEXI1, and pRNS3, and normalized to Renilla luciferase controlled by the constitutive p35S promoter. Note that co-transfection with both KIR1 and ORE1 did not lead to an additive, nor to a synergistic effect on promoter activation of all tested dPCD-associated genes. To account for the doubled amount of TF added in the case of dimerization assays, the respective control assays contained doubled amounts of KIR1 and ORE1 (4µg total TF plasmid).