Extended Data Fig. 1: Characterization of interactions between module components identified along the directed evolutionary trajectory to the PYR1*MANDI/HAB1* CID module.
From: An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity
(a, b) Y2H interaction assays using the mutant CID components shown. These assays use derivatives of pBD-PYR1 and pACT-HAB1 and are assayed in the S. cerevisiae Y2H reporter strain Y190. Colony overlay assays with an X-gal substrate were used to visualize β-galactosidase activity. The yeast strains were tested on different concentrations of the compounds listed (DMSO carrier solvent in the mock 0 µM control wells). (c) Recombinant GST-HAB1 and the mutants shown were produced in E. coli, and phosphatase activity was measured using a spectrophotometric assay with a 4-MUP substrate, as described in the methods; activity is reported as %GST-HAB1 control. Bars show the mean of triplicate assays, and error bars are the standard deviation. (d) Recombinant GST-HAB1, 6x-His-PYR1, and 6X-His-PYR1*MANDI were produced in E. coli, and phosphatase activity was measured using a phosphopeptide substrate and malachite green; these data confirm that PYR1*MANDI does not inhibit HAB1 PP2C activity. The x-axis shows log10 of the ABA or mandi concentrations tested (µM). Activity is plotted relative to the mock control, which lacks test ligands but contains a receptor and phosphatase; 95% confidence intervals are shown.
