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

ended

Plasma membrane-localized receptor kinases (RKs) perceive diverse extracellular ligands, allowing plants to react to various stimuli. They function in signaling processes during all aspects of plants’ life, including development, biotic and abiotic stress responses as well as reproduction. In the last decade, a major paradigm has emerged that individual RKs have different biochemical functions. There are e.g. ligand-binding RKs, which require the formation of stable complexes with RK co-receptors in order to initiate signaling. In addition to this common mode of activation, it has been revealed that additional RKs can have regulatory functions within RK complexes, and that in planta several other proteins are part of these complexes to regulate – either positively or negatively – complex formation and initiation of downstream signaling. The proposed project aims to understand what the minimal components linking ligand binding to receptor activation and to the initiation of cellular outputs are. Furthermore, it has the goal to elucidate the structural mechanisms of receptor complex activation. To achieve this, two interconnected approaches will be pursued. The first will utilize protein expression in cell-culture to reconstitute the signaling pathway from ligand perception to activation of downstream targets. The second approach will use a cell-free in vitro expression system to reconstitute the complete RK complex in synthetic, membrane-mimicking nanodiscs. This will ultimately allow the analysis of ligand induced structural changes by cryo-electron microscopy within full length, membrane embedded RK complexes. The well-characterized RK FLAGELLIN SENSING 2 (FLS2) and its co-receptor RK BRI1-ASSOCIATED KINASE 1 (BAK1), which are core elements of the receptor complex mediating the perception of bacterial Flagellin and function in the induction of antibacterial immune responses, will be to this end employed as a model for the proposed project.