Elwira Smakowska-Luzan

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 stress responses, developmental processes, and some have been proposed to act as redox sensors. Here, we investigated the structural and functional properties of Arabidopsis thaliana CRK18, focusing on cysteines in the ECD, homodimerization, and signalling context. Biophysical analyses showed that disulphide bonds are essential for CRK18 ECD stability, with conserved DUF26 cysteines particularly important for proper folding. Consistently, mutation of most predicted disulphide pairs caused partial endoplasmic reticulum retention of full-length CRK18, whereas mutation of the non-conserved vicinal C227–C228 pair preserved plasma membrane localization. Mass photometry, microscale thermophoresis, co-immunoprecipitation, and FRET–FLIM demonstrated that CRK18 forms homodimers both in vitro and in planta. Dimerization was largely unaffected by pH, moderate redox perturbations, or mutation of C227–C228. Multiscale molecular dynamics simulations identified a stable closed ECD dimer in which the vicinal disulphides become solvent-exposed at the interface and display unusually low predicted pKa values, suggesting a potential regulatory role in redox-dependent signalling. IP-MS identified CRK18-associated receptor kinases, including PERK15, WAK1, MIK2, and SOBIR1, along with proton pumps and transporters, indicating that CRK18 might play a role in cell-wall integrity, apoplastic homeostasis, and immunity. In particular, the decrease in CRK18 mobility after flg22 elicitation supports the observation that CRK18 undergoes stimulus-dependent reorganization at the plasma membrane. Finally, CRK18 overexpression reduced, whereas crk16/crk17/crk18 mutations increased, plant susceptibility to the cyst nematode Heterodera schachtii, which damages plant cell walls. Collectively, these findings establish CRK18 as a disulphide-stabilized plasma membrane homodimer embedded within cell-surface signalling networks, with its solvent-exposed vicinal cysteines as predicted candidates for stress-responsive regulation.

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.

Cysteine-rich receptor-like kinases (CRKs) are a large subfamily of plant receptor-like kinases 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 characterize the Arabidopsis thaliana CRK family. Phylogenetic reconstruction from 69 natural accessions resolved 5 well-supported CRK clades. Nucleotide diversity (π) 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 modeling 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 β-sheet at the interface. Integrating evolutionary and structural approaches reveal 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.

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 (π) 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 β-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.

In plants, peptides play an irreplaceable role as intercellular communication molecules, triggering signal transduction by activating plasma membrane-localized receptors. Of specific interest here are the cysteine-rich peptides (CRPs), which are well-characterized for their disulfide bonds that enhance structural stability and functional specificity. Although the first CRP, systemin, was identified over three decades ago, our understanding of the evolutionary trajectories, functional diversity, and underlying mechanisms of CRPs remains limited. This review focuses on two main families of CRPs: the Rapid Alkalinization Factor (RALF) and the Epidermal Patterning Factor (EPF)/EPF-Like peptides. We thus explore the diverse and, so far, identified signalling pathways in which the peptides have a pivotal function. We organize our tour by providing a comprehensive overview of the discovery of peptides, structural diversity, and biological functions. Particularly, emphasis is placed on their roles in plant growth, development, reproduction, defence against biotic and abiotic stresses, and plant–bacteria symbiosis.

SUMMARY: Over the past few decades, significant advancements have been made in understanding how plasma‐membrane localised receptor kinases (RKs) detect signals and activate responses to various stimuli. Numerous examples of ligand‐induced receptor activation mechanisms and their downstream consequences have been characterised in detail. The crucial role of post‐translational modifications (PTMs), such as the phosphorylation of receptor kinases, has been demonstrated concerning different cellular responses. Given the diverse structures and architectures of the extracellular domains (ECDs) of RKs, it is probable that various forms of PTMs also play an essential role in receptor activation, including cysteine oxidative modifications triggered by reactive oxygen species (ROS). The function of cysteine oxidative modifications as functional redox switches that modulate protein structure and function has been extensively studied across various multicellular organisms. Based on biochemical and structural characteristics, the family of cysteine‐rich receptor‐like kinases (CRK) emerges as excellent candidates for proteins regulated in a redox‐dependent manner. This review provides a concise overview of cysteine's biochemical and structural properties in its role as a molecular redox switch. Drawing on the currently available literature, we describe how cysteine‐redox signalling is maintained, particularly in plant cells. We further focus on extracellular ROS perception and the role of CRKs as promising candidates for ROS sensors in Arabidopsis thaliana . We discuss the structural and biochemical properties of CRKs, their involvement in plant growth and defence processes, and our perspective on why CRKs could be key components of the ROS sensing machinery or ROS sensors, especially regarding the dimerization abilities of CRKs. Finally, we highlight the current challenges in identifying and quantifying cysteine oxidative modifications and propose methods for detecting ROS‐modified cysteines that may be promising for investigating the role of CRKs in extracellular ROS perception and signalling.

The plant cell wall not only serves as a physical barrier against pathogens but, when damaged, also functions as a source of cell wall-derived molecules that play crucial roles in plant immunity as damage-associated molecular patterns (DAMPs). While oligogalacturonides from homogalacturonan are well-studied DAMPs, the immune-signaling potential of other cell wall components remains largely unexplored. Conventional genetic and biochemical approaches aimed at identifying ligand-receptor pairs in plant immunity have been limited by the vast diversity of potential ligand molecules and functional redundancy of putative receptors. Here, we developed a high-throughput screening pipeline that simultaneously examines multiple interactions between plant cell wall-derived glycans and >350 extracellular domains (ECDs) of receptor kinases and receptor like proteins in Arabidopsis, resulting in the screening of >40,000 interactions. We discovered a group of leucine-rich repeat receptor kinases named ARMs (AWARENESS of RG-I MAINTENANCES) that interact with rhamnogalacturonan-I (RG-I), a major component of pectin. RG-I treatment induced pattern-triggered immunity responses, with distinct kinetics compared to oligogalacturonide responses. We identified RG-I oligosaccharide structures required for interaction with ARM receptors and immune activation, and found that ARM receptors are redundantly involved in plant immunity. Collectively, our approach provides a powerful platform for discovering glycan-receptor pairs in plants, facilitating a more comprehensive understanding of cell wall surveillance mechanisms in plant immunity.

The abscission of floral organs and emergence of lateral roots in Arabidopsis is regulated by the peptide ligand INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and the receptor protein kinases HAESA (HAE) and HAESA-LIKE 2 (HSL2). During these cell separation processes, the plant induces defense-associated genes to protect against pathogen invasion. However, the molecular coordination between abscission and immunity has not been thoroughly explored. Here we show that IDA induces a release of cytosolic calcium ions (Ca2+) and apoplastic production of reactive oxygen species, which are signatures of early defense responses. In addition, we find that IDA promotes late defense responses by the transcriptional upregulation of genes known to be involved in immunity. When comparing the IDA induced early immune responses to known immune responses, such as those elicited by flagellin22 treatment, we observe both similarities and differences. We propose a molecular mechanism by which IDA promotes signatures of an immune response in cells destined for separation to guard them from pathogen attack.