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. 2023 Aug 5;35(11):4133–4154. doi: 10.1093/plcell/koad214

Figure 4.

Figure 4.

GhERF108 acts as a coactivator of GhARF7-1 and GhARF7-2 to coregulate GhMYBL1 expression. A) Diagram of vectors and AuxREs in GhMYBL1 promoter for Dual-LUC assay, ChIP-qPCR analysis, and EMSA. (Bottom) Horizontal line represents the GhMYBL1 promoter, the ovals represent AuxREs in the promoter, the chip1 to chip4 indicate the fragments detected by ChIP-qPCR, and the probe2 and probe3 indicate the fragments used in EMSA. The numbers along the gene model are relative to the ATG. B) Dual-LUC assay of GhERF108 and GhARF7-1/7-2 on GhMYBL1 promoter activity. The relative LUC activities were normalized to the reference REN LUC. The corresponding effector (+) and empty vector (−) were cofiltrated. The mean value and Sd are calculated from 3 biological replicates. The different letters indicate significant difference (P < 0.05) between the 2 groups according to the Tukey HSD test. C, D) EMSA of GhARF7-1 C) and GhARF7-2 D) binding to the AuxREs in the GhMYBL1 promoter. Biotin-labeled probes were incubated with GST-GhARF7-1 or GST-GhARF7-2 in vitro. Unlabeled probes were used for competition, and biotin-labeled mutated AuxREs were used as negative controls. Normal (up) or mutated AuxREs (down) are shown in C and D. Values marked with different letters indicate statistically significant differences (P < 0.05) between each group according to the Tukey HSD multiple range test. E, F) ChIP-qPCR analysis of GhARF7-1 E) and GhARF7-2 F) bind to the GhMYBL1 promoter. Equal amounts of anti-GhARF7-1/7-2 antibodies were used in 21 DPA fibers of both WT and GhERF108 RiL6 transgenic cotton. The ChIP signal is expressed as the percentage of immunoprecipitated DNA in the total input DNA. mock, ChIP without anti-GhARF7-1 and anti-GhARF7-2 but added lgG antibody as negative control; RiL6, the GhERF108 RNAi line. The mean value and Sd are calculated from 3 biological replicates. Tukey HSD test demonstrated that there were significant differences (P < 0.05) between the mock and ChIP groups. G, H) DNA–protein pull-down assay of GhERF108 acting as a coactivator of GhARF7-1 G) and GhARF7-2 H) to bind to the GhMYBL1 promoter. GhERF108-His protein was incubated with GST-GhARF7-1 protein and labeled probe2 or GST-GhARF7-2 protein and labeled probe3 in vitro, using empty GST protein and biotin beads as a negative control. The precipitated proteins were analyzed by immunoblotting with anti-His or anti-GST antibody.