Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity.

Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal '*' module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.

Repurposing plant hormone receptors as chemically-inducible genetic switches for dynamic regulation in yeast.

Precise control of gene expression is critical for optimizing cellular metabolism and improving the production of valuable biochemicals. However, hard-wired approaches to pathway engineering, such as optimizing promoters, can take time and effort. Moreover, limited tools exist for controlling gene regulation in non-conventional hosts. Here, we develop a two-channel chemically-regulated gene expression system for the multi-stress tolerant yeast Kluyveromyces marxianus and use it to tune ethyl acetate production, a native metabolite produced at high titers in this yeast. To achieve this, we repurposed the plant hormone sensing modules (PYR1ABA/HAB1 and PYR1*MANDI/HAB1*) for high dynamic-range gene activation and repression controlled by either abscisic acid (ABA) or mandipropamid (mandi). To redirect metabolic flux towards ethyl acetate biosynthesis, we simultaneously repress pyruvate dehydrogenase (PDA1) and activate pyruvate decarboxylase (PDC1) to enhance ethyl acetate titers. Thus, we have developed new tools for chemically tuning gene expression in K. marxianus and S. cerevisiae that should be deployable across many non-conventional eukaryotic hosts.

Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions.

Plant hormones are a group of chemically diverse small molecules that direct processes ranging from growth and development to biotic and abiotic stress responses. Surprisingly, genome analyses suggest that classic animal nuclear hormone receptor homologs do not exist in plants. It now appears that plants have co-opted several protein families to perceive hormones within the nucleus. In one solution to the problem, the hormones auxin and jasmonate (JA) act as “molecular glue” that promotes protein-protein interactions between receptor F-boxes and downstream corepressor targets. In another solution, gibberellins (GAs) bind and elicit a conformational change in a novel soluble receptor family related to hormone-sensitive lipases. Abscisic acid (ABA), like GA, also acts through an allosteric mechanism involving a START-domain protein. The molecular identification of plant nuclear hormone receptors will allow comparisons with animal nuclear receptors and testing of fundamental questions about hormone function in plant development and evolution.

Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo.

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.

A Closed Form Model for Molecular Ratchet-Type Chemically Induced Dimerization Modules.

Chemical-induced dimerization (CID) modules enable users to implement ligand-controlled cellular and biochemical functions for a number of problems in basic and applied biology. A special class of CID modules occur naturally in plants and involve a hormone receptor that binds a hormone, triggering a conformational change in the receptor that enables recognition by a second binding protein. Two recent reports show that such hormone receptors can be engineered to sense dozens of structurally diverse compounds. As a closed form model for molecular ratchets would be of immense utility in forward engineering of biological systems, here we have developed a closed form model for these distinct CID modules. These modules, which we call molecular ratchets, are distinct from more common CID modules called molecular glues in that they engage in saturable binding kinetics and are characterized well by a Hill equation. A defining characteristic of molecular ratchets is that the sensitivity of the response can be tuned by increasing the molar ratio of the hormone receptor to the binding protein. Thus, the same molecular ratchet can have a pico- or micromolar EC50 depending on the concentration of the different receptor and binding proteins. Closed form models are derived for a base elementary reaction rate model, for ligand-independent complexation of the receptor and binding protein, and for homodimerization of the hormone receptor. Useful governing equations for a variety of in vitro and in vivo applications are derived, including enzyme-linked immunosorbent assay-like microplate assays, transcriptional activation in prokaryotes and eukaryotes, and ligand-induced split protein complementation.

The MATH-BTB BPM3 and BPM5 subunits of Cullin3-RING E3 ubiquitin ligases target PP2CA and other clade A PP2Cs for degradation.

Early abscisic acid signaling involves degradation of clade A protein phosphatases type 2C (PP2Cs) as a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. At later steps, ABA induces up-regulation of PP2C transcripts and protein levels as a negative feedback mechanism. Therefore, resetting of ABA signaling also requires PP2C degradation to avoid excessive ABA-induced accumulation of PP2Cs. It has been demonstrated that ABA induces the degradation of existing ABI1 and PP2CA through the PUB12/13 and RGLG1/5 E3 ligases, respectively. However, other unidentified E3 ligases are predicted to regulate protein stability of clade A PP2Cs as well. In this work, we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric cullin3 (CUL3)-RING-based E3 ligases (CRL3s), as PP2CA-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2, and HAB1. BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity, whereas bpm3 bpm5 plants show increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Using biochemical and genetic assays, we demonstrated that ubiquitination of PP2CA depends on BPM function. Given the formation of receptor-ABA-phosphatase ternary complexes is markedly affected by the abundance of protein components and ABA concentration, we reveal that BPMs and multimeric CRL3 E3 ligases are important modulators of PP2C coreceptor levels to regulate early ABA signaling as well as the later desensitizing-resetting steps.

Agrochemical control of plant water use using engineered abscisic acid receptors.

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 responses. 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.

Optimized small-molecule pull-downs define MLBP1 as an acyl-lipid-binding protein.

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand-binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC-MS to identify candidate binding ligands. We optimized this method using ABA-PYL interactions and show that ABA co-purifies with wild-type PYL5 but not a binding site mutant. The Kd of PYL5 for ABA is 1.1 µm, which suggests that the method has sufficient sensitivity for many ligand-protein interactions. Using this method, we surveyed a set of 37 START domain-related proteins, which resulted in the identification of ligands that co-purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co-purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein-metabolite interaction and better understand protein-ligand interactions in plants.

Toward Development of Fluorescence-Quenching-Based Biosensors for Drought Stress in Plants.

Abscisic acid (ABA) is a drought stress signaling molecule, and simple methods for detecting its levels could benefit agriculture. Here, we present proof-of-concept detection for ABA in aqueous solutions by the use of a mixture of Cyanine 5.5 (Cy5.5) fluorophore- and BHQ3 quencher-conjugated endogenous ABA receptor pyrabactin resistance 1 like proteins (PYL3). These dye-conjugated PYL3 protein form dimers in solutions without ABA and monomerize upon ABA binding. When they are in dimers, fluorescence of Cy5.5 is either nearly completely quenched by the BHQ3 or 20% quenched by another Cy5.5. Consequently, mixtures of equal amounts of the two protein conjugates were used to detect ABA in aqueous solution. As the ABA concentration increased from <1 µM to 1 mM, the intensity of fluorescence detected at around 680 nm from the mixture was more than doubled as a result of ABA-induced monomerization, which leads to halt of quenching and recovery of fluorescence of Cy5.5 in monomers. Kinetic modeling was used to simulate the fluorescence response from the mixture and the results generally agree with the experimentally observed trend. This work demonstrates that fluorescence measurements of a single dissociation reaction in one spectral region are adequate to assess the ABA concentration of a solution.

Small Molecule Probes of ABA Biosynthesis and Signaling.

The phytohormone ABA mediates many physiological and developmental responses, and its key role in plant water relations has fueled efforts to improve crop water productivity by manipulating ABA responses. ABA's core signaling components are encoded by large gene families, which has hampered functional studies using classical genetic approaches due to redundancy. Chemical approaches can complement genetic approaches and have the advantage of delivering both biological probes and potential agrochemical leads; these benefits have spawned the discovery and design of new chemical modulators of ABA signaling and biosynthesis, which have contributed to the identification of ABA receptors and helped to define PYR1 and related subfamily III receptors as key cellular targets for chemically manipulating water productivity. In this review, we provide an overview of small molecules that have helped dissect both ABA signaling and metabolic pathways. We further discuss how the insights gleaned using ABA probe molecules might be translated to improvements in crop water productivity and future opportunities for development of small molecules that affect ABA metabolism and signaling.

Chemical-Induced Inhibition of Blue Light-Mediated Seedling Development Caused by Disruption of Upstream Signal Transduction Involving Cryptochromes in Arabidopsis thaliana.

Plants have a remarkable ability to perceive and respond to various wavelengths of light and initiate regulation of different cascades of light signaling and molecular components. While the perception of red light and the mechanisms of its signaling involving phytochromes are largely known, knowledge of the mechanisms of blue light signaling is still limited. Chemical genetics involves the use of diverse small active or synthetic molecules to evaluate biological processes. By combining chemicals and analyzing the effects they have on plant morphology, we identified a chemical, 3-bromo-7-nitroindazole (3B7N), that promotes hypocotyl elongation of wild-type Arabidopsis only under continuous blue light. Further evaluation with loss-of-function mutants confirmed that 3B7N inhibits photomorphogenesis through cryptochrome-mediated light signaling. Microarray analysis demonstrated that the effect of 3B7N treatment on gene expression in cry1cry2 is considerably smaller than that in the wild type, indicating that 3B7N specifically interrupts cryptochrome function in the control of seedling development in a light-dependent manner. We demonstrated that 3B7N directly binds to CRY1 protein using an in vitro binding assay. These results suggest that 3B7N is a novel chemical that directly inhibits plant cryptochrome function by physical binding. The application of 3B7N can be used on other plants to study further the blue light mechanism and the genetic control of cryptochromes in the growth and development of plant species.

Novel Vein Patterns in Arabidopsis Induced by Small Molecules.

The critical role of veins in transporting water, nutrients, and signals suggests that some key regulators of vein formation may be genetically redundant and, thus, undetectable by forward genetic screens. To identify such regulators, we screened more than 5000 structurally diverse small molecules for compounds that alter Arabidopsis (Arabidopsis thaliana) leaf vein patterns. Many compound-induced phenotypes were observed, including vein networks with an open reticulum; decreased or increased vein number and thickness; and misaligned, misshapen, or nonpolar vascular cells. Further characterization of several individual active compounds suggests that their targets include hormone cross talk, hormone-dependent transcription, and PIN-FORMED trafficking.

ACCERBATIN, a small molecule at the intersection of auxin and reactive oxygen species homeostasis with herbicidal properties.

The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides.

50 years of Arabidopsis research: highlights and future directions.

922 I. 922 II. 922 III. 925 IV. 925 V. 926 VI. 927 VII. 928 VIII. 929 IX. 930 X. 931 XI. 932 XII. 933 XIII. Natural variation and genome-wide association studies 934 XIV. 934 XV. 935 XVI. 936 XVII. 937 937 References 937 SUMMARY: The year 2014 marked the 25(th) International Conference on Arabidopsis Research. In the 50 yr since the first International Conference on Arabidopsis Research, held in 1965 in Göttingen, Germany, > 54 000 papers that mention Arabidopsis thaliana in the title, abstract or keywords have been published. We present herein a citational network analysis of these papers, and touch on some of the important discoveries in plant biology that have been made in this powerful model system, and highlight how these discoveries have then had an impact in crop species. We also look to the future, highlighting some outstanding questions that can be readily addressed in Arabidopsis. Topics that are discussed include Arabidopsis reverse genetic resources, stock centers, databases and online tools, cell biology, development, hormones, plant immunity, signaling in response to abiotic stress, transporters, biosynthesis of cells walls and macromolecules such as starch and lipids, epigenetics and epigenomics, genome-wide association studies and natural variation, gene regulatory networks, modeling and systems biology, and synthetic biology.

Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.

The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.

Chemical manipulation of plant water use.

Agricultural productivity is dictated by water availability and consequently drought is the major source of crop losses worldwide. The phytohormone abscisic acid (ABA) is elevated in response to water deficit and modulates drought tolerance by reducing water consumption and inducing other drought-protective responses. The recent identification of ABA receptors, elucidation of their structures and understanding of the core ABA signaling network has created new opportunities for agrochemical development. An unusually large gene family encodes ABA receptors and, until recently, it was unclear if selective or pan-agonists would be necessary for modulating water use. The recent identification of the selective agonist quinabactin has resolved this issue and defined Pyrabactin Resistance 1 (PYR1) and its close relatives as key targets for water use control. This review provides an overview of the structure and function of ABA receptors, progress in the development of synthetic agonists, and the use of orthogonal receptors to enable agrochemical control in transgenic plants.

Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance.

Abscisic acid (ABA) is an essential molecule in plant abiotic stress responses. It binds to soluble pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor receptors and stabilizes them in a conformation that inhibits clade A type II C protein phosphatases; this leads to downstream SnRK2 kinase activation and numerous cellular outputs. We previously described the synthetic naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in Arabidopsis thaliana. Here we describe quinabactin, a sulfonamide ABA agonist that preferentially activates dimeric ABA receptors and possesses ABA-like potency in vivo. In Arabidopsis, the transcriptional responses induced by quinabactin are highly correlated with those induced by ABA treatments. Quinabactin treatments elicit guard cell closure, suppress water loss, and promote drought tolerance in adult Arabidopsis and soybean plants. The effects of quinabactin are sufficiently similar to those of ABA that it is able to rescue multiple phenotypes observed in the ABA-deficient mutant aba2. Genetic analyses show that quinabactin's effects in vegetative tissues are primarily mediated by dimeric ABA receptors. A PYL2-quinabactin-HAB1 X-ray crystal structure solved at 1.98-Å resolution shows that quinabactin forms a hydrogen bond with the receptor/PP2C "lock" hydrogen bond network, a structural feature absent in pyrabactin-receptor/PP2C complexes. Our results demonstrate that ABA receptors can be chemically controlled to enable plant protection against water stress and define the dimeric receptors as key targets for chemical modulation of vegetative ABA responses.