Extended Data Fig. 5: Characterization of PYR1*MANDI and HAB1* overexpression effects.
From: An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity
(a) Col-0 wild type and two 35S::GFP-PYR1*MANDI strains were treated with 1 µM mandipropamid, thermographed after 24 hours, treated seven days later with 10 µM mandi, and again imaged 24 hours later. A previously constructed 35 S::PYR1MANDI transgenic line was included as a positive control (1 µM treatment only). These images show that mandipropamid treatments do not cross active endogenous ABA signaling in the 35S::GFP-PYR1*MANDI transgenic lines; the positive control 35S::PYR1MANDI strain shows a strong thermal response to ABA, as previously described; the experiment was conducted one time. (b) Seed germination responses of wild type, 35S::PYR1MANDI, 35S::GFP-PYR1*MANDI, and 35S::GFP-HAB1* transgenic strains to ABA (0.5 µM), mandipropamid (0.5 µM), or mock treatments. Mandi inhibits germination of 35S::PYR1MANDI seeds due to activation of ABA signaling but does not in 35S::GFP-HAB1* strains. (c) Overexpression of wild-type HAB1 causes ABA insensitivity, which can be observed indirectly as reduced leaf temperature caused by elevated transpiration. Two 35S::GFP-HAB1* transgenic lines were grown alongside wild-type Columbia and abi1 (Col-0) as a positive control (it possesses growth defects and reduced leaf temperature due to aba insensitivity). The GFP-HAB1* plants show leaf temperatures comparable to wild-type plants, indicating that the GFP-HAB1* protein does not strongly impair plant guard cell ABA responses. (d) GFP-PYR1*MANDI and GFP-HAB1* protein levels were established by western blotting using an anti-GFP antibody; as described above, the three lines had similar protein expression levels. The experiment was conducted once, and the entire scanned gel is presented in the supplemental information.
