Fig. 3: PYR1* has a malleable ligand-binding pocket.

a, Structures of ligands screened. To establish if the new ligand-responsive receptors could be isolated using the orthogonal module, PYR1*^MANDI was subjected to saturating mutagenesis using NNK oligonucleotides. The resultant library was screened using a reverse two-hybrid strain against a panel of 10 organophosphate ligands at 100 μM, and hits were retested and sequenced. b, The table shows the changes to PYR1*^MANDI’s ligand-binding pocket (and neighboring residues) for the organophosphate receptors identified (Y2H and sequence data are provided in Extended Data Fig. 3); the right-hand side of the table shows the minimal concentration that yielded a positive signal in β-gal-based Y2H assays. c, An azinphos-ethyl-responsive mutant was subjected to two rounds of affinity optimization using Y2H-based selections. The mutations identified at each round of mutagenesis and selection are shown; the minimal ligand concentration yielding a positive signal in β-gal-based Y2H assays is shown at the right. The bottom panel shows β-gal-based Y2H assays conducted on PYR1*^AZIN. d, The PYR1*^AZIN/HAB1* module functions orthogonally to the WT ABA response module. Shown are pair-wise tests of the WT or * components with one another tested for activation by ABA or azinphos-ethyl (10 µM). Gen., generation.