Extended Data Fig. 11: SCREW1 and ABI suppress ABA-induced OST1 phosphorylation.
From: Phytocytokine signalling reopens stomata in plant immunity and water loss
a, ABI1 suppresses ABA-induced OST1 phosphorylation. Protoplasts were co-transfected with OST1-Flag and ABI1-HA with the indicated ratio of DNAs, followed by treatment with 1 µM SCREW1 for 5 min before adding 1 µM ABA for an additional 5 min. Proteins were separated with Mn2+-Phos-tag (top three) or regular SDS-PAGE (middle two) and detected with anti-HA or anti-Flag antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of bands corresponding to phosphorylated ABI1 (pABI1) and OST1 (pOST1) from four independent immunoblots were quantified by ImageJ (bottom two). The relative phosphorylation of OST1 represents the ratio of phosphorylated to unphosphorylated OST1. The phosphorylation of ABI1 without treatment was set as 1. Data are shown as mean ± s.d. (n = 4, biologically independent repeats). b, ABI2 suppresses ABA-induced OST1 phosphorylation. Protoplasts were co-transfected with ABI2-HA and OST1-Flag followed by treatment with 1 µM SCREW1 before adding 1 µM ABA. Proteins were separated with Mn2+-Phos-tag (top two) or regular SDS-PAGE (bottom three) and detected with anti-HA or anti-Flag antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of bands from four independent immunoblots were analysed by ImageJ (bottom). The relative phosphorylation of OST1 represents the ratio of phosphorylated to unphosphorylated OST1. Data are shown as mean ± s.d. (n = 4, biologically independent repeats). c, SCREW1 suppresses ABA-induced OST1 phosphorylation in a dosage-dependent manner. Protoplasts were transfected with OST1-Flag followed by treatments with 0, 10, 100 or 1,000 nM SCREW1 for 5 min before adding 1 µM ABA for another 5 min. Proteins were separated with Mn2+-Phos-tag (top two) and regular SDS-PAGE (middle two) and detected with anti-Flag antibodies. The protein loading is shown by CBB staining for RBC. Signal intensities of bands from four independent immunoblots were analysed by ImageJ. The relative phosphorylation of OST1 represents the ratio of phosphorylated to unphosphorylated OST1. Data are shown as mean ± s.d. (n = 4, biologically independent repeats). d, NUT interacts with ABI1 in protoplasts. Protoplasts were co-transfected with ABI1-HA and NUT-Flag or a control vector. Proteins were immunoprecipitated with anti-Flag agarose beads and detected with anti-HA or anti-Flag antibodies (top two). Proteins before IP are shown as input controls (bottom two). e, GST-NUTCD interacts with ABI1 in vitro. GST or GST-NUTCD proteins were immobilized on glutathione sepharose beads and incubated with His-ABI1-HA followed by immunoblotting with anti-HA antibodies (top). The protein loading is shown by CBB (bottom). Experiments were repeated three times with similar results. Data were analysed by one-way (b, c) or two-way (a) ANOVA followed by Tukey’s test. Exact P values are provided in the graphs and Supplementary Table 3.
