Fig. 5: BoERF1L regulates male fertility by the direct repression of Ms-cd1 expression.

a Yeast one-hybrid assay. WTpro represents a 66-bp probe (–624 to –559 upstream of ATG) from the WT promoter. MTpro represents a 65-bp probe (–624 to –559 upstream of ATG) from the DGMS01-20 promoter. Yeast strains were cultured on SD/-Leu medium with 200 ng/mL AbA. b EMSA analysis. The biotin-labeled probes are indicated. Competition for BoERF1L binding was performed with excess unlabeled WTpro probe at 20×, 30× and 40× the amount of labeled probe. c The dual-luciferase transient expression assay shows that BoERF1L represses transcription of the Ms-cd1_PWT gene promoter. Luciferase intensity was imaged and quantified using a Tanon 5200 system. d qRT‒PCR analysis of BoERF1L expression in various tissues of 01-20. e Flowers, anthers and pollen grains stained with Alexander solution among WT and boerf1l mutants. Scale bars, 3 mm for flowers, 2 mm for anthers, 50 μm for pollen grains. f, g Transverse section, TEM and SEM analysis of anthers in WT and the boerf1l mutant. E epidermis, En endothecium, ML middle layer, T tapetum, Tes tetrads, Msp microspore, Te tectum, Ba bacula, In intine, Ne nexine. Scale bars, 50 μm in (f1–f6); 4 μm in (g1), (g2), (g4) and (g6); 2 μm in (g2) and (g5). Data are presented as the means ± SD (n = 10 for (c), n = 3 for (d)). A two-tailed unpaired t-test was used for statistical analysis. Experiments were repeated three times independently with similar results. Source data are provided as a Source Data file.