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Drought discovery to improve drought tolerance in crops

Periodic Reporting for period 1 - DrugCrops (Drought discovery to improve drought tolerance in crops)

Reporting period: 2017-03-03 to 2019-03-02

In order to produce food in a sustainable way for an additional 2 billion people by 2050, crop yield needs to increase significantly. This becomes even more important in the face of climate change, where predictions indicate an increase in extreme droughts in important agricultural areas worldwide. The plant hormone abscisic acid, protect plants against these stresses by coordinating adaptative responses to reinforce plant stress tolerance. In the last years, the major class of ABA receptors has been described and extensive structural data is available for the receptors of the dicot model plant Arabidopsis thaliana. However, in crops, that provide more than 50% of the diet worldwide, this knowledge is very limited. The goal of DrugCrops is to generate and use data on the ABA receptors in the model crop plant Setaria viridis (Setaria) and in maize to discover novel ABA receptor agonists to improve drought resistance using small molecules. The Experienced Researcher (ER) has been working in collaboration with different partner research organizations to fulfill the following objectives:
-Objective 1: Identification and characterization of the ABA signaling core module in Maize and Setaria. The different components of the ABA signaling core module have not been characterized in detail in maize or Setaria. Here, we will identify and characterize i) the ABA receptors, ii) the PP2Cs and iii) the SnRK2s involved in the ABA signaling core for both Maize and Setaria.

-Objective 2: Development of new ABA agonsits. We will use high-throughput screening and virtual docking methods to obtain the first set of small molecules able to activate stress tolerance in crops. The best agonists will be characterized in greenhouse experiments to assess their activity on stress tolerance.
-Objective 3: Structural analysis of PYR/PYL/RCAR proteins in complex with synthetic agonists. The crystal structure ligand-receptor will be generated for the best 2 compounds. This data has the potential to be used by the ER in the future to design novel ABA agonists and will help him to establish his own laboratory.
1. Explanation of the work carried per WP.

WP 1. Identification and characterization of the ABA signaling core in Maize and Setaria.
In this WP1 we have:
- Identified the different ABA receptors of maize along with 4 different PP2Cs.
- The amino acid sequence of these proteins has been used to find the Setaria counterparts by bioinformatics surveys in the Phytozome ( and Setariabase ( databases. These are in total 8 ABA receptors, 5 PP2C phosphatases and 2 SnRK2 kinases.
- Produced the different receptors and PP2Cs as active recombinant proteins.
- Performed the biochemical characterization of these proteins.
- Assessed the in planta functionality of Setaria ABA receptors.

The ER has performed all this work in the receiving institution (CSIC). For some of the tasks, the ER had the help of MSc students.

A paper describing these findings is under preparation.

WP 2. Development of new ABA agonists to control stress tolerance in crops.
In this WP2 we have:
- Developed a method for medium (96-well plates) and high (384-well plates) throughput screening of chemical libraries to identify ABA receptor agonists, in collaboration with the BioFarma lab, one of the nodes of the EU-OPENSCREEN network. The method is based on in vitro PP2C-phosphatase assays. Two manuscripts have been accepted for publication in the Methods in Molecular Biology series (Lozano-Juste et al., 2019 and García-Maquilón et al., 2019). The manuscripts will be deposited in open access repositories (bioRxiv) once we get the permit from the editor (tentative date September 2019).
- By using this method we have screened 5,000 molecules and found IRE1, a small molecule able to activate SvPYL1 and ZmPYL1 ABA receptors. We have also identified a new molecule, IRE2, able to inhibit the HAB1 PP2C phosphatase. Two papers are in preparation to publish this work.
- Identified a novel ABA receptor agonist by in silico screening of small molecules in collaboration with Dr. Armando Albert (IQFR-CSIC).
- Evaluated the in vitro and in vivo potency of the different compounds identified.

The ER has performed this WP2 in the receiving institution (IBMCP-CSIC) and visiting the BioFarma lab (USC) and Dr. Armando Albert lab (IQFR-CSIC). For some of the tasks, the ER had the help of MSc students.

WP 3. X-ray studies of PYR/PYL/RCAR proteins with synthetic agonists.
In this WP3 we have:
- Elucidated the crystal structure of one of the new small molecules identified in the previous WP2 in complex with an ABA receptor and a PP2C phosphatase.
- Generated crystals of Setaria and Maize ABA receptors. We will perform the X-ray study of these crystals in the upcoming months.

The ER has performed this WP3 in Dr. Armando Albert lab (IQFR-CSIC). For some of the tasks, the ER had the help of MSc students.

2. Exploitation and dissemination of result.

The support of the MSC action has been referenced and promoted in all publications,
presentations, workshops, public events, outreach activities and any actions related to DrugCrops.

These included:

Invited speaker:
-Biotechnology Havana 2017 “Agricultural Biotechnology in the XXI

Oral presentations:
-12TH Congress of the International Plant Molecular Biology, Montpellier, France, 2018.
Poster presentations:
XIV RBMP, Salamanca, Spain, 2018.
At The Forefront Of Plant Research, Ghent, Belgium, 2017.

Invited speaker:
Institute of Plant Sciences IPS2, Paris-Saclay, France, 2018.
Universidad de Valencia, 2018.
Universidad de Valencia, 2017.


6 publications described in the publications section.

-Exploitation of results and intellectual property.

We have identified small molecules able to activate ABA signaling in crops. The ER is now considering filling a patent for each of those compounds. We are gathering data to submit an application to the CSIC Technology Transfer Office that will assist with the patent applications.
In this project we have expanded the previous knowledge about the ABA signaling core components, describing and characterizing the PYR/PYL ABA receptors and the PP2C phosphatases of the C4 monocot species Setaria viridis and Zea mays. We have also discovered 2 novel ABA agonist with moderate activity over specific ABA receptors. We have identified a specific compound with activity towards PYL1-like ABA receptors but not towards PYR1-like receptors. Additionally, the structure of one of these compounds in complex with the PYL1 receptor and the HAB1 phosphatase has been elucidated. During the 2 years of the MSC the ER has published 6 papers in international journals that are all freely available through open repositories.
The MSC grant has helped the ER to gain experience as independent scientist and it has been instrumental to establish productive collaborations. For instance, the ER has developed part of the project in collaboration with the BioFarma group, leaded by Mabel Loza. Through this collaboration the ER was introduced to a biotech company and together with the ER they have obtained funding to continue this project. The small molecules identified in DrugCrops will contribute to produce resource-efficient terrestrial food technologies for a sustainable food chain, which is a key priority that impacts all EU citizens.