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.

An extracellular interactome map of plant receptor kinases Multicellular organisms, including plants, are exposed to ever-changing environments and they use cell-surface signaling systems to detect and respond to self and non-self-signals in the extracellular space. Receptor Kinases (RKs) have evolved as sophisticated environmental sensors to control key cellular processes, including growth and immunity. Plants, as sessile organisms constantly exposed to pathogen attacks and environmental fluctuation, are excellent models to discover paradigms of RK signaling. Despite two decades of work, only a handful of pathways that regulate plant growth and immunity have been elucidated. The main goal of this project was to understand how these sensing and signalling pathways are integrated within the plant. Based on a network map that detailed interactions between different RKs, I selected two candidates, named APEX1 and APEX2, as master regulators of diverse signalling pathways. To understand their function, I examined their exclusive and shared interacting partners. I found that APEX1 and 2 directly modulate growth-related processes, like root and hypocotyl growth. As plant growth is counterbalanced by immune responses, I also observed that APEX1 and 2 are negative regulators of plant defence. Collectively, the quantitative biophysical, biochemical, and genetic analyses I performed suggest that APEX1 and 2 operate as major regulators controlling the trade-off between growth and defence. The network map I used had to select APEX 1 and 2 already provided some evidence of how 225 RKs participate in a wide range of biological processes. However, the vast majority of the function of the remaining RKs and their system-level organization remain unknown. Using a high-throughput screen, I decided to extend this work and test for interactions between the remaining RKs as well as RK-like proteins (600 members). I completed the cloning of the remaining receptors and optimized the expression for each family of proteins. The material I generated will enable the screen I proposed, and the resulting data will be analyzed using advanced network studies to dissect the properties of RK subnetworks. These data will be made publicly available and will serve as a great resource for the international research community. Given the central role of RKs in multiple physiological processes and the large knowledge gap in sensing and signaling mechanisms, I anticipate that the results of this project will help to engineer strategies to boost plant disease immunity while improving growth.