Reconstitution of a Minimal Signaling-Active Plant Receptor Kinase Complex - Towards the Structural Determination of Ligand-Induced Activation Mechanisms

Informations projet

MINIREX

N° de convention de subvention: 892398

DOI

10.3030/892398

Projet clôturé

Date de signature de la CE 20 Juillet 2020

Date de début 1 Janvier 2021

Date de fin 31 Decembre 2022

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 203 149,44

Contribution de l’UE

€ 203 149,44

Coordonné par

UNIVERSITAT ZURICH
Switzerland

Periodic Reporting for period 1 - MINIREX (Reconstitution of a Minimal Signaling-Active Plant Receptor Kinase Complex - Towards the Structural Determination of Ligand-Induced Activation Mechanisms)

Période du rapport: 2021-01-01 au 2022-12-31

Résumé du contexte et des objectifs généraux du projet

Plants are the foundation of all human life. Their photosynthetic activity ultimately provides the nutrition and the oxygen-rich atmosphere we need to survive, yet this capacity is challenged by ever-changing environmental conditions. Growing populations require increasing performance from crop species, while accelerating anthropogenic climate change limits yields.
For plants, just as for all organisms, the first step of physiological response lies in the perception of environmental changes and adequate translation into biochemical events. To monitor and respond to alterations in extracellular conditions, plants deploy cell surface-localized receptors, such as receptor kinases (RKs).
The RK family represents one of the largest plant protein families and thereby poses a challenge to characterization via solely genetic approaches, even using state-of-the-art tools. Thus, despite the critical importance of understanding the fundamental basis of RK signalling, the characterization of a minimal functional RK complex remains elusive. I therefore combined both cell-based and cell-free approaches to address this critical knowledge gap.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

During the project MINIREX, the aim to reconstitute a functioning plant receptor kinase complex in a synthetic, membrane mimicking lipid bilayer was pursued. To this end, the suitability of different protein expression systems was evaluated. Therefore, both expression of plant proteins in a cell-free wheat-germ extract based expression system and expression of those proteins in mammalian HEK293T cells were established in the host laboratory. For both systems, modular cloning systems were developed and successfully established and will be available for further projects utilizing those techniques. Remarkably, at the end of the funding period a protocol allowing for scalable coexpression of a plant receptor like kinase with its co-receptor kinase has been established, which will allow to generate the material required for structural dissemination of the arising bi-kinase complex. With regards to the proposed functional reconstitution of plant signalling pathways in human cell lines, the NADPH-oxidase activation assays were successfully established in the host laboratory. Extensive optimization efforts have been pursued throughout the project time to circumvent technical challenges.

During this project, two peer-reviewed open access publication have been published which were directly supported by the action.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

Results and insights generated through this project will allow findings clearly extending the state of the art in the field of plant immunity research. While the expected scientific outputs of the project have no direct socio-economic impact with regards to endeavored patents, they could be of immense value in the future nonetheless. Understanding plant immune responses is of great necessity to address agronomic problems of the presence and future, and getting mechanistic insights into pattern perception – that will likely arise from this project – might pave the way for future biostimulant development that may help agricultural production.

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