Figure 3.

Improved chloroacetamide response and cross-reactivity of four variants isolated in consecutive rounds of mutagenesis and selection. (A) Two snapshots of a homologous ABA-bound PYL extracted from a crystal struture (PDB code 3KB3).¹⁵ ABA is highlighted in pink. Other colored stick models indicate residues that were mutated in one or more of the said variants. The table below details the substitutions present in each BdPYL3 variant. Color scheme for mutated positions is consistent with the structural snapshots above. (B, C) Plasmid-encoded Y2H assay with GAL4-regulated mScarlet-I as a reporter gene in a 96-well plate format. (B) Dose response of each variant to ABA and five chloroacetamides. (C) Nonspecific activities were tested by treating each variant with a panel of herbicides at 100 μM (DMSO served as mock). All 12 herbicides activated at least one BdPYL3 mutant in the initial screen. The elevated basal activity of variants 1d12 and 2f is evident by their fluorescence in the mock treatment. Asterisks indicate a statistically significant difference from the mock treatment (p-value < 0.05, Dunnett’s test). In vivo binding curves of other isolated variants and of an isogenic line bearing an empty pBD-GAL4 vector are provided in Figure S4. The Y axes are uniform and shared across all plots in panels B and C.