Fig. 9

HvANT1 and HvANT2 interact with HvWD40-140 proteins and form MBW complexes with three transcription factors to verify HvDFR promoter transcriptional activation A Validation of protein self-activation of HvANT1, HvANT2, and HvWD40-140. B Validation of interaction between HvANT1, HvANT2, and HvWD40-140 were analyzed via yeast two-hybrid assay. -LT denotes SD/-Leu-Trp, and-THA denotes SD/-Trp-His-Ade. The yeasts were grown in the absence of Leu and Trp (SD-LT) for selection of co-transformed cells. east cells were incubated to OD600 = 1 and diluted 1-, 10- or 100-fold for assay. The assay of the auto-activation of and interactions between the tested genes was conducted in the absence of Trp, His, and Ade (SM-THA). BD-53 + AD-T7 is a positive control, BD-53 + AD-T is a negative control. C Validation of interaction between HvANT1, HvANT2, and HvWD40-140 were analyzed via BiFC. HvANT1 was fused with the N-terminal and C-terminal fragments of yellow fluorescent protein (YFP), HvANT2 as fused with the N-terminal fragment, and HvWD40-140 as fused with the C-terminal fragment. It was found that HvnANT1 and HvnANT2 interacted with HvWD40-140 to form biomolecular fluorescent complexes. Images of YFP, chlorophyll autofluorescence, bright field, and YFP merged with bright field (Merge) are shown. The scale bar indicates 100 μm. D HvANT1-HvANT2-HvWD40-140 transcription factor complexes activate HvDFR promoter. Effector constructs and HvDFR promoter-driven reporter gene construct and promoter activity of HvDFR in tobacco leave protoplasts is activated by the co-expression of HvANT1 with HvANT2 and the HvWD40-140 genes. Promoter activities are expressed as the Firefly/Renilla luciferase activity ratio. The data are from three biological replicates and are expressed as the mean SD.