An extracellular interactome map of plant receptor kinases

Multicellular organisms use complex cell surface signaling systems to detect and appropriately respond to self and non-self-signals in the extracellular space. Receptor Protein Kinases (RKs) are found in membranes of animals and plants, and have been used throughout evolution to control key cellular processes such as growth and immunity. Combined, the genomes of the model animal species human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), frog (Xenopus laevis), rat (Rattus norvegicus), fruit fly (Drosophila melanogaster) and roundworm (Caenorhabditis elegans) code for approximately 160 RKs. In stark contrast, the small genome of the plant model organism Arabidopsis thaliana alone codes for 400 RKs. Plants, as sessile organisms constantly exposed to pathogen attacks along with environmental fluctuations, are an excellent model to discover paradigms of RKs signaling. However, only a small number of these receptors have been characterized in Arabidopsis. RKs display a typical three-part structure with an extracellular domain (ECD), a single transmembrane domain and, in most cases, a functional kinase domain inside the cell. ECDs of cell surface receptors are both interaction sites and regulatory modules for receptor activation. ECD interactions determine response specificity and dictate the downstream signaling cascades that modulate fundamental signaling pathways. How ECD interactions occur in combinatorial modules to produce signal-competent receptor complexes is a very difficult question to address. Although large-scale protein interaction data have been generated in the last decade, extracellular proteins are greatly underrepresented in these data sets due to technical challenges. Systems biology and proteomics approaches have so far not properly accounted for the transient nature and low biochemical tractability of RK interactions. The main goal of this project is to understand how immune and developmental signals are generated by RKs at the cell surface. For this, it is essential to discover the composition and dynamics of cell surface complexes. Therefore, the proposed project involves testing more than 150.000 putative ECD interactions between members of different RK families using a sensitive high-throughput binding technology (CSIRK). This innovative approach allows screening for interactions between ECDs with very low affinity, a great improvement over other methods. The resulting resource will be leveraged using advanced network studies to implement algorithms that allow detection of network communities and elucidation of the self-assembling properties of RK subnetworks. These data will be implemented to the Arabidopsis BAR platform and, therefore, will serve as great resource for the international research community. Finally, the RK networks will be assigned specific biological functions using molecular genetics together with a broad spectrum of biochemical approaches. By determining new hubs of growth and defence signalling pathways, this work represents an important step towards a systematic and comprehensive understanding of cell surface signalling.

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