Rising temperatures and lessening fresh water supplies are threatening agricultural productivity and have motivated efforts to improve plant water use and drought tolerance. During water deficit, plants produce elevated levels of abscisic acid (ABA), which improves water consumption and stress tolerance by controlling guard cell aperture and other protective responses1,2. One attractive strategy for controlling water use is to develop compounds that activate ABA receptors, but agonists approved for use have yet to be developed. In principle, an engineered ABA receptor that can be activated by an existing agrochemical could achieve this goal. Here we describe a variant of the ABA receptor PYRABACTIN RESISTANCE 1 (PYR1) that possesses nanomolar sensitivity to the agrochemical mandipropamid and demonstrate its efficacy for controlling ABA responses and drought tolerance in transgenic plants. Furthermore, crystallographic studies provide a mechanistic basis for its activity and demonstrate the relative ease with which the PYR1 ligand-binding pocket can be altered to accommodate new ligands. Thus, we have successfully repurposed an agrochemical for a new application using receptor engineering. We anticipate that this strategy will be applied to other plant receptors and represents a new avenue for crop improvement.
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We thank N. Chen for technical assistance constructing the K59R pocket library, J. Mandal for RNA-seq library preparation, D. Jensen for protein production, J. Bailey-Serres for comments on the manuscript and M. Nuccio, M. Nina and F. Early for suggestions regarding candidate agrochemicals. This work was supported in part by the National Science Foundation (IOS 1258175, MCB 1022378 to S.R.C.), Syngenta Corporation (S.R.C. and F.P.), and a United States–Israel Binational Agricultural Research and Development Postdoctoral Fellowship F1-440-2010 (to A.M.).
S.R.C. receives research funding from Syngenta Corporation and is an inventor on a patent application related to the work described.
Extended data figures and tables
To create a screening platform for identifying orthogonal ligand–PYR1 receptor pairs, we inactivated the intrinsic ABA responsiveness of PYR1 by introducing the K59R mutation and constructed all possible substitution mutations in 25 ligand-proximal residues. Each mutant receptor in this ‘pocket library’ was tested for responsiveness to 15 agrochemicals (at 100 μM) using a yeast two-hybrid assay that reports agonist-induced binding of receptor to HAB1 (ref. 6). The 15 compounds screened are listed in the Methods. The table on the left-hand side summarizes the results of the 7,125 ligand–receptor interactions tested. Orthogonal receptors were identified for 4 of the 15 compounds tested, shown on the right. Coloured boxes in the table indicate the specific mutations that confer responsiveness to one or more of the compounds screened. An asterisk indicates a specific mutation that was used in subsequent combinatorial mutagenesis experiments. Eight of the 25 residues mutagenized did not confer ligand responsiveness and are not shown in the table.
A, Flow chart for the receptor optimization process. The mutations identified at each step are shown as insets in each box and the minimum concentration of mandipropamid required to elicit a detectable response in yeast two-hybrid assays for the mutants is shown on the left. B, Summary of the mutations tested using the yeast two-hybrid assay at different stages of the optimization process.
A, PYR1(K59R/V81I/F108A/F159L) is a sensitive mandipropamid receptor. Crystallization experiments with PYR1MANDI were unsuccessful; however, reverting S122G and Y58H present in PYR1MANDI to wild-type residues yielded the quadruple mutant receptor, which crystallized successfully. Recombinant PYR1MANDI and PYR1(K59R/V81I/F108A/F159L) proteins were tested side by side for activity in HAB1 PP2C activity assays. The recombinant PYR1(K59R/V81I/F108A/F159L) mutant protein was the same material used in crystallization experiments (which had its 6×His tag proteolytically removed) whereas the PYR1MANDI protein used did not have its tag cleaved. B, The Fo − Fc density present in the binding pocket of the PYR1(K59R,V81I,F108A,F159L)–mandipropamid–HAB1 structural model after several rounds of refinement in the absence of ligand; Fo − Fc is shown using a sigma level of 3. C, The profile of the unbiased electron density shown in B allowed us to unambiguously place mandipropamid into the structural model. D, PYR1(K59R/V81I/F108A/F159L) adopts a closed-gate conformation in the presence of mandipropamid and HAB1.
A, Ligplot of mandipropamid interactions with PYR1(K59R/V81I/F108A/F159L) and HAB1 in the ternary complex. B, Ligplot of ABA interactions with PYR1 and HAB1 in the ternary complex of previously published coordinates (Protein Data Bank accession 3QN1), shown for comparison. Ligplots were made using LigPlot+31. In both plots the residues from PYR1 are denoted by chain A and HAB1 by chain B.
Extended Data Figure 5 PYR1MANDI is functional in Arabidopsis using seed germination and root growth inhibition assays.
A, Germination of wild-type and three independent PYR1MANDI transgenic lines (lines 1, 2 and 3) on Petri plates containing 250 nM mandipropamid. Images taken 3 days after stratification. B, Dose–response curves for germination sensitivity to mandipropamid in the two independent PYR1MANDI transgenic lines and wild-type lines. The PYR1MANDI transgenic line 3 shown in A was excluded from this experiment because its germination is not inhibited by mandipropamid at the concentrations tested. C, PYR1 protein levels in the wild-type and three PYR1MANDI transgenic lines (detected using an anti-PYR1 antibody). The bottom panel is a loading control showing the rubisco large subunit. D, Selective inhibition of root primary growth by mandipropamid in PYR1MANDI genotypes. E, Fresh weights of 7-week-old wild-type and transgenic plants (n = 20). F, Flowering times of the wild-type and transgenic genotypes used. Neither of the two PYR1MANDI lines characterized displayed statistically significant differences in their fresh weight or flowering time in comparison to wild-type controls; however, PYR1 overexpression decreased fresh weight in comparison to the wild type (P < 0.05, two-sided t-test). Error bars show standard deviation (s.d.).
A, B, To compare the ABA and mandipropamid responses of wild-type and PYR1MANDI Arabidopsis genotypes respectively, we treated 3-week-old plants with mock solutions (0.02% Silwet) or either 50 µM ABA (wild type Columbia) or 1 µM mandipropamid (PYR1MANDI). Leaf surface temperatures were monitored immediately before treatment and at 24 h intervals for 7 days after treatment. A, B, Representative images from the experiment (A) and quantification of mean leaf temperatures (B). These data show that the response induced by mandipropamid in Arabidopsis is more persistent than that induced by ABA, which we speculate may be because mandipropamid evades metabolism by the CYP707A enzymes that mediate ABA catabolism. Asterisks indicate a significantly warmer mean leaf temperature (at a P < 0.01 cutoff) relative to the mock-treated control analysed at the same time point, as determined using a two-sided t-test (n = 8 or n = 7 for wild-type and PYR1MANDI samples, respectively); error bars show s.d.
A–D, Transgenic tomato plants expressing the 35S::PYR1MANDI transgene were made as described in the Methods. A single-insert line was obtained that segregated plants with high (H) and low (L) PYR1MANDI protein levels as well as non-transgenic (N, Null) segregants. A, Western blot analyses of SDS–PAGE separated proteins from T2 segregants using an anti-PYR1 antibody. The bottom panel shows the large subunit of Rubisco as a loading control. B, Thermography of representative T2 transgenic plants and wild-type control before and 24 h after treatment with a solution containing 25 μM mandipropamid. The wild-type and H plant images shown in B are the same images shown in Fig. 4. C, Quantification of mean leaf temperatures (measured from the three oldest leaves) before and after mandipropamid treatment. The asterisk indicates a statistically significant (P < 0.05) difference between the high expressing PYR1MANDI line in comparison to the null segregant control, as determined using a two-sided t-test; n = 2 for each of the four sample groups. Error bars show s.d. D, Analysis of an independent primary transgenic tomato PYR1MANDI line. The line was grown alongside a wild-type control and treated with a mock solution and then thermographed 24 h later. Three days after this, the plants were treated with a solution containing 10 μM mandipropamid and thermographed 24 h after application.
Extended Data Figure 8 Mandipropamid confers drought tolerance to the PYR1MANDI genotype in Arabidopsis.
A, B, The genotypes shown were treated with 1 μM mandipropamid or mock solution two times over the course of a water deprivation experiment. Photographs were taken 24 h after re-watering. Drought experiments were conducted on three separate occasions over an 8-month period; each experiment was conducted using a minimum of three replicate pots each containing four plants of either the wild type, a 35S::GFP–PYR1 wild-type overexpressor line (PYR1OX), or one of two independent 35S::PYR1MANDI lines. A, Shown are representative images 24 h after recovery for one set of replicates. B, Summary of survival data for the three independent experiments conducted. The inset survival values have been separated for each experiment. The images shown in A are from the second experiment conducted and those in B are taken from the third experiment conducted. The numerator of each value is the number of plants that survived 24 h after re-watering and the denominator is the total number of plants tested. Experiment 1 initiated the water deprivation at 2 weeks after germination while experiments 2 and 3 initiated at 3 weeks; further experimental details are described in Methods. The mock-treated wild-type control and mandipropamid-treated PYR1MANDI (line 1) pots were photographed separated for Fig. 4.
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Park, SY., Peterson, F., Mosquna, A. et al. Agrochemical control of plant water use using engineered abscisic acid receptors. Nature 520, 545–548 (2015). https://doi.org/10.1038/nature14123
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