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Data repository of raw and processes data of the redox-dependent interactome assay (RIA CRK-CRK) described in chapter 5. The repository includes a description of CRK constructs used, experimental design of the in vitro assay, raw spectrophotometry data of each plate measured, processed data after statistical analysis as described in Materials and Methods of Chapter 5, CRK ECD charge distribution quantification and centrality measurements after network analysis as described in Materials and Methods of Chapter 5.

Data repository of raw and processes data of the redox-dependent interactome assay (RIA CRK-LRR) described in chapter 4. The repository includes a description of CRK and LRR constructs used, experimental design of the in vitro assay, raw spectrophotometry data of each plate measured, processed data after statistical analysis as described in Materials and Methods of Chapter 4, detailed description of node number as plotted in the RIA networks of Chapter 4, and centrality measurements after network analysis as described in Materials and Methods of Chapter 4.

Data repository of Immunoprecipitation followed by mass spectrometry (IP-MS) data performed at different stages of Arabidopsis thaliana development. Data repository of proteomic and phosphoproteomic mass spectrometry (MS) data performed on the whole tissue (TF) or membrane-enriched (MF) fraction of 5-week-old rosettes of Arabidopsis thaliana. Description of Arabidopsis lines used in the study Data from Perseus software with statistical analysis (Student's test) for comparison between genotypes of interest and the Col-0 control. Analysis of protein list organized by Fold change (log2) and FDR of CRKs overexpressing lines compared to Col-0. Developmental stage code; 2W (2-week-old), 4W (4-week-old) and 5W (5-week-old) plants used in the study.

Plant cells are contained inside a rigid network of cell walls. Cell walls are highly dynamic structures that act both as a structural material and as a hub for a wide range of signaling processes. Despite its crucial role in all aspects of the plant life cycle, live dynamical imaging of the cell wall and its functional properties has remained challenging. Here, we introduce CarboTag, a modular toolbox for live functional imaging of plant walls. CarboTag relies on a small molecular motif, a pyridine boronic acid, that targets its cargo to the cell wall, is non-toxic and ensures rapid tissue permeation. We designed a suite of cell wall imaging probes based on CarboTag in any desired color for multiplexing. Moreover, we created new functional reporters for live quantitative imaging of key cell wall features: network porosity, cell wall pH and the presence of reactive oxygen species. CarboTag opens the way to dynamical and quantitative mapping of cell wall responses at subcellular resolution.

Plants have evolved mechanisms to abscise organs as they develop or when exposed to unfavorable conditions.1 Uncontrolled abscission of petals, fruits, or leaves can impair agricultural productivity.2,3,4,5 Despite its importance for abscission progression, our understanding of the IDA signaling pathway and its regulation remains incomplete. IDA is secreted to the apoplast, where it is perceived by the receptors HAESA (HAE) and HAESA-LIKE2 (HSL2) and somatic embryogenesis receptor kinase (SERK) co-receptors.6,7,8,9 These plasma membrane receptors activate an intracellular cascade of mitogen-activated protein kinases (MAPKs) by an unknown mechanism.10,11,12 Here, we characterize brassinosteroid signaling kinases (BSKs) as regulators of floral organ abscission in Arabidopsis. BSK1 localizes to the plasma membrane of abscission zone cells, where it interacts with HAESA receptors to regulate abscission. Furthermore, we demonstrate that YODA (YDA) has a leading role among other MAPKKKs in controlling abscission downstream of the HAESA/BSK complex. This kinase axis, comprising a leucine-rich repeat receptor kinase, a BSK, and an MAPKKK, is known to regulate stomatal patterning, early embryo development, and immunity.10,13,14,15,16 How specific cellular responses are obtained despite signaling through common effectors is not well understood. We show that the identified abscission-promoting allele of BSK1 also enhances receptor signaling in other BSK-mediated pathways, suggesting conservation of signaling mechanisms. Furthermore, we provide genetic evidence supporting independence of BSK1 function from its kinase activity in several developmental processes. Together, our findings suggest that BSK1 facilitates signaling between plasma membrane receptor kinases and MAPKKKs via conserved mechanisms across multiple facets of plant development.

The field of plant receptor biology has rapidly expanded in the past three decades. However, the demonstration of direct interaction between receptor-ligand pairs remains a challenge. Identifying and quantifying protein-ligand interactions is crucial for understanding how they regulate certain physiological processes. An important aspect is the quantification of different parameters of the interaction, like binding affinity, kinetics, and ligand specificity that drive the formation of signaling complexes. In this chapter, we discuss Isothermal Titration Calorimetry (ITC) as a label-free technique to measure thermodynamic parameters of ligand binding with high accuracy and reproducibility. We provide a detailed guideline how to design, perform, analyze, and interpret ITC measurements using as an example the interaction between the SCHENGEN3/GASSHO1 (SGN3/GSO1) leucine-rich repeat receptor-like kinase and its sulfated peptide ligand CASPARIAN STRIP INTEGRITY FACTOR 2 (CIF2).

Interactions between extracellular domains (ECDs) are crucial for many physiological processes in the cell, most importantly perception of its environment. However, studying these often-transient interactions can be challenging. Here we describe a method that allows for in vitro detection of extracellular domain interactions through an oligomerization-based cell surface interaction (CSI) assay. In a CSI, bait- and prey-tagged proteins are produced and secreted by Drosophila S2 cells to ensure proper folding and post-translational modifications. Subsequently, Bait (FC fragment) and Prey (pentamer domain and alkaline phosphatase) tags allow the detection of interactions in protein A-coated 96 wells plates through a colorimetric readout. Due to the easy detection of interactions this approach can be used for high-throughput screening and mapping of extracellular interaction networks.