Biology subjects

Stouthamer, J.

Publications and source records attributed to Stouthamer, J..

3 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into the role of Cysteine-Rich Receptor-Like Kinase 18 (CRK18) in Arabidopsis

Plants perceive and integrate diverse environmental signals through receptor kinase (RK) networks at the plasma membrane. Within this, Cysteine-Rich Receptor-Like Kinases (CRKs) constitute a large but not well-understood subfamily characterised by extracellular domains (ECDs) enriched in cysteine residues. CRKs have been implicated in plant responses to biotic and abiotic stress, as well as in developmental processes. Additionally, several CRKs have been proposed to act as redox sensors. Here, we investigate the homodimerization mechanism of Arabidopsis CRK18 and its regulation by redox conditions. By modulating pH and redox state, we assessed the stability and binding dynamics of the CRK18 ECD and its cysteine mutants. We also tested the plasma membrane localisation of all the cysteine mutants involved in the predicted disulfide bonds, and only CRK18C227A, C228A-ECD resembled the plasma membrane localization of wild-type CRK18. We combine co-immunoprecipitation, Forster resonance energy transfer-fluorescence lifetime imaging microscopy and microscale thermophoresis to quantify CRK18 self-association in planta and in vitro. Furthermore, we place CRK18 dimerization in a broader signalling context by identifying CRK18 interaction partners and CRK18-dependent signalling outputs. Using Arabidopsis CRK18 overexpression lines, we perform (phospho)proteomic and immunoprecipitation-mass spectrometry (IP-MS) analyses to map CRK18-centred signalling networks associated with stress-related responses. The constitutive activation of the CRK18 kinase domain and its interaction with many putative (cell wall) glycan-sensing RKs, including PERK15, suggest a regulatory role for CRK18. The absence of significant changes in the proteome and phosphoproteome of CRK18 overexpression lines in the absence of any trigger, and its restricted mobility after elicitation, suggest that CRK18 requires a stimulus for activation and possibly induces membrane microdomain reorganisation. This is in line with the infection assay with the nematode Heterodera schachtii, which causes modification and targeted damage to plant cell walls during infection, revealing that CRK18 acts as negative regulator of this process.

biochemistry↗

bioRxiv2026

Redox-dependent extracellular interaction networks of Cysteine-Rich Receptor-Like Kinases

Reactive oxygen species (ROS) regulate plant development and immunity, but how extracellular ROS signals are decoded and whether the Cysteine-rich Receptor-like Kinases (CRKs) truly serve as the long-suspected ROS sensors remains uncertain. Here, we combine high-throughput interactomics, redox proteomics, structural modelling, and genetics to map a ROS-dependent CRK interaction landscape in Arabidopsis thaliana. Using a redox-dependent interactome assay (RIACRK) on 40 CRK extracellular domains (ECDs), we identified ROS-modulated dimerisation networks with enhanced inter-community connectivity and hub redistribution in the presence of ROS. Integrating this with developmental and flg22-induced expression profiles reveals spatiotemporally limited subnetworks that likely function during activated immunity and leaf senescence, both of which are associated with extensive ROS production. Differential cysteine alkylation coupled with mass spectrometry shows that cysteines in a subset of CRK ectodomains undergo ROS-dependent oxidation. Notably, solvent-exposed, vicinal cysteines C228/C229 in CRK28 emerge as prime redox-sensitive candidates. CRK28 homodimerises and heterodimerises with CRK17 in vivo, and mutation of C228/C229 retains plasma membrane localisation but abolishes CRK28 homodimerisation, indicating a redox-controlled dimerisation switch. Loss of CRK28 delays senescence, while CRK28 overaccumulation from its native promoter causes dwarfism, premature senescence, autoimmune-like phenotypes, extensive phosphoproteome rewiring, and associations with Pathogenesis Related (PR) proteins, ROS-detoxifying enzymes, receptor(-like) kinases, and vesicle trafficking components. These results indicate that CRK28 is a potential ROS-regulated hub connecting extracellular redox signals to CRK network organisation, immune response, and age-related senescence.

plant biology↗

bioRxiv2025

Genetic diversity, predictive protein structures, and interaction networks of Cysteine-Rich Receptor-Like Kinases in Arabidopsis thaliana

Cysteine-rich receptor-like kinases (CRKs) are a large subfamily of plant receptor-like kinases (RLKs) implicated in immunity and development, yet their ligands, interaction partners, and mechanistic roles remain poorly defined. We combined population-genetic analyses and AlphaFold-based structural prediction to characterise the Arabidopsis thaliana CRK family. Phylogenetic reconstruction from 69 natural accessions resolved five well-supported CRK clades. Nucleotide diversity ({pi}) and neutrality tests revealed heterogeneous diversity across loci, with evidence of both positive and negative selection pressure acting on different CRKs. AlphaFold models of CRK extracellular domains (ECDs) recapitulate the DUF26 structure observed in Plasmodesmata Localizing Protein (PDLP)5/PDLP8 and ginkbilobin-2 but display distinct biochemical properties and disulfide-bond topologies. Pairwise AlphaFold dimer modelling of all 780 CRK-ECD combinations produced 145 high-confidence interaction models; [~]78% of these adopt a shared dimer conformation characterized by an extended intermolecular {beta}-sheet at the interface. Integrating evolutionary and structural approaches reveals clade-specific selective regimes and conserved structural features of CRK ECDs that likely underpin receptor-receptor interactions. Predicted high-confidence dimer interfaces suggest a general mode of CRK-ECD association that can guide targeted biochemical and genetic validation, accelerating functional dissection of this important receptor family.

bioinformatics↗