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) Reporting period: 2021-01-01 to 2022-12-31 Summary of the context and overall objectives of the project 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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) 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.