Protoplast Transformation and Dual-Luciferase Reporter Assay

Protoplasts were isolated from the rosette leaves of 4-week-old Col-0 as previously (Sun et al., 2013). The dual-luciferase assay was performed according to (Hellens et al., 2005). To characterize the core GEG promoter sequences, four fragments of the GEG promoter, named P1365, P880, P580, P260 (with the numbers representing the base pairs to the start of each fragment upstream from the ATG translational start site) were fused into pGREEN0800-LUC to generate reporter vectors. The modified pBluescript vector (pBS) (Paul et al., 2016) was used as an effector. For the interaction study of GhMIF and the GEG promoter, the pGREEN0800-LUC plasmid incorporated with pGEG₃₂₀ (-580∼-261) was used as the reporter, and the pBS-GhMIF was used as an effector. Meanwhile, the pGREEN0800-LUC plasmid incorporated with pGEG₁₇₀ (-430∼-261) was used as the unspecific binding control. The effectors were co-introduced with the reporters into the protoplasts as previously described (Yoo et al., 2007) and the transformed protoplasts were incubated at room temperature for 20–22 h. The dual-luciferase assay was performed as described by the manufacturer (Dual-Luciferase^® Reporter Assay, Promega, United States), and the Firefly and Renilla luciferase activities were detected using an Enspire multi-mode microplate reader (PerkinElmer Inc., United States). Three biological replicates were performed for all experiments.