Fig. 6: PbHILT1 interacts with PbHSFA1b and enhances its transcriptional activity.
A PbMBF1c expression in the control, PbHILT1-overexpressing (OE), PbHILT1mut OE, and PbHILT1-silenced (RNAi) lines under 25 °C or 38 °C, detected using RT-qPCR. PbHILT1mut was generated using an adenine insertion after the initiation codon of PbHILT1. B Protein accumulation in nucleus of PbHILT1 after heat stress by western blot. Cytoplasm and nuclear protein was extracted from leaves of ‘Hongbaoshi’ exposed to 25 °C or 38 °C for 6 h. β-actin and Histone 3 (H3) are the representatives in cytosol and nucleus respectively. C PbMBF1c expression in the control, PbHSFA1b OE, and PbHSFA1b RNAi lines subjected to a 6 h treatment at 38 °C, as determined using RT-qPCR. D ChIP assays showing the direct binding of PbHSFA1b to the PbMBF1c promoter at 38 °C but not 25 °C. E Bimolecular fluorescence complementation assays in tobacco leaves with DAPI. Confocal images were captured 48 h after Agrobacterium infiltration. Bars = 50 μm. F Interaction between PbHILT1 and PbHSFA1b, as determined by Co-IP. G Split-luciferase (LUC) assays showing the enhancement of PbHSFA1b transcriptional activity by PbHILT1. pPbMBF1c::LUC was co-expressed with p35S::PbHILT1 and/or p35S::PbHSFA1b in tobacco leaves. Bar = 1 cm. The experiments were performed independently three times, and error bars represent the standard deviation. Significant differences were determined using a two-tailed Student’s t test (*P < 0.05, **P < 0.01).
