Fig. 3: OsGF14h interacts with OsHOX3 and OsVP1 to modulate the ABA-responsive pathway.

a Y2H assay. The interaction between (OsGF14h^WR04-6 and OsGF14h^SN9816) and (OsHOX3 and OsVP1). Strains carrying two indicated constructs were grown on a synthetic medium. b Luciferase complements imaging assays. Agrobacterium carrying different plasmids, as indicated, were co-expressed in Nicotiana benthamiana, the colors representing interaction strength. c Pull-down assay. GST-OsHOX3 and GST-OsVP1 were used as baits and the pull down of His-OsGF14h^WR04-6 and His-OsGF14h^SN9816 were detected by the anti-His antibody. d Co-IP assay. Input means protein number before the experiment. GFP-IP means the protein number detected after co-IP buffer washing. e, f Performance (e) and coleoptile length (f) of WT^WR04-6 and two overexpression lines, and WT^kiaake and mutant under anaerobic stress. Scale bar = 1 cm. n = 5 biologically independent experiments. g Relative expression of OsVP1 in WT^WR04-6 and knockout line at different hours under anaerobic stress. n = 3 biologically independent samples. h Y1H assay showing the interaction between OsHOX3 and the promoter of OsPYL5. -AbA, 0 ng/mL; +AbA, 200 ng/mL. i ChIP-qPCR assays. Enrichment of OsHOX3 in OsPYL5 promoter. The upper schematic indicates the locations of the DNA fragments used for ChIP-qPCR. The red square indicates the element. A/T rich indicates a high A/T base region. Error bars indicate the means ± SD for independent experiments. j Dual-LUC assays. OsHOX3 binding OsPYL5 promoter. The schematic indicates effector and reporter constructs. k Expression of OsPYL5 at the 12th hour of WT^kiaake and hox3 mutant, and at the 36th hour of WT^WR04-6, knockout line and expression lines under anaerobic (blue background) and aerobic conditions. n = 3 biologically independent samples. l EMSA assay. The competitor was unlabeled and the concentrations were 10× and 20× of the biotin-labeled probe. 0.5× and 1× OsGF14h^WR04-6 protein amount of OsHOX3 was added. In a, b, c, d, h, i, j, l, representative experiments from three independent experiments are shown. In f, g, j, k data were presented as means ± SD, P values are indicated by unpaired two-tailed Student’s t-test. Source data are provided as a Source Data file.