Recombinant Arabidopsis thaliana Probable inactive receptor kinase At5g67200 (At5g67200)

What is the predicted domain structure of At5g67200?

At5g67200 belongs to the family of leucine-rich repeat receptor-like kinases (LRR-RKs) in Arabidopsis thaliana. Its structure consists of:

• An N-terminal extracellular domain (ECD) containing leucine-rich repeats important for ligand recognition

• A transmembrane domain that anchors the protein to the plasma membrane

• A C-terminal cytoplasmic kinase domain that may be catalytically inactive

The protein shows structural similarities to other LRR-RKs involved in plant immunity and development. Structural analysis indicates At5g67200 is proximal to ARM1 in the ECD-based receptor similarity tree, suggesting potential functional relationships with ARM family receptors .

What experimental evidence supports At5g67200's classification as an inactive receptor kinase?

The classification of At5g67200 as a probable inactive receptor kinase is based on:

• Sequence analysis revealing mutations in catalytic residues essential for phosphorylation activity

• Similarity to other receptor kinases, such as the subgroup VIII receptor-like cytoplasmic kinases (RLCKs), where catalytically inactive members still perform critical roles in plant immunity

• Biochemical assays demonstrating absence of in vitro phosphorylation activity

Similar to MAZ and CARK7, which demonstrate no catalytic protein kinase activity in vitro but still function in plant immunity, At5g67200 likely exerts its biological effects through protein-protein interactions rather than enzymatic activity .

How is At5g67200 expressed across different developmental stages and tissues?

At5g67200 expression patterns vary across developmental stages and tissues:

Transcriptional profiling of the Arabidopsis root quiescent center did not identify At5g67200 among significantly expressed transcription factors, suggesting its expression may be regulated in a tissue-specific manner outside the root quiescent center .

How does At5g67200 contribute to pattern-triggered immunity (PTI)?

At5g67200 appears to function in plant immune signaling pathways, potentially through:

• Recognition of damage-associated molecular patterns (DAMPs) derived from plant cell walls

• Participation in receptor complexes that detect pathogen-associated molecular patterns (PAMPs)

• Signal transduction leading to mitogen-activated protein kinase (MAPK) activation

Though not directly confirmed for At5g67200, related LRR-RKs have been implicated in the recognition of cell wall-derived signals like rhamnogalacturonan I (RG-I) . At5g67200 was included in initial screenings for RG-I interaction, though reliable interaction values were not obtained .

What is the relationship between At5g67200 and MAPK signaling cascades?

The connection between At5g67200 and MAPK signaling likely involves:

• Potential participation in receptor complexes that activate MAPK cascades upon pathogen detection

• Possible role as a scaffold protein that facilitates interactions between signaling components

• Contribution to sustained immune signaling through protein-protein interactions

Research on related receptor kinases shows that RG-I treatment induces MAPK activation in Arabidopsis, similar to that observed with oligogalacturonides (OGDP10-15) treatment . This suggests At5g67200 might function in a similar signaling context, potentially in redundancy with other receptors.

What are the known or predicted protein interaction partners of At5g67200?

Based on studies of related receptor kinases, At5g67200 likely interacts with:

While direct interactions have not been extensively characterized for At5g67200 specifically, related receptor kinases like ARM1 form homo- and hetero-dimers with other family members . CPK28 has been shown to phosphorylate receptor-like cytoplasmic kinases such as MAZ and CARK7 on multiple residues critical for protein kinase activation , suggesting similar regulatory mechanisms might apply to At5g67200.

How can protein-protein interactions of At5g67200 be experimentally determined?

To identify protein interaction partners of At5g67200, researchers should employ multiple complementary approaches:

• In vitro techniques:

  • MicroScale Thermophoresis (MST) assay using His-tagged purified extracellular domain (ECD) labeled with fluorescent tags

  • Pull-down assays with recombinant At5g67200

  • Surface Plasmon Resonance (SPR) for kinetic analysis of interactions

• In vivo approaches:

  • Co-immunoprecipitation from plant tissues expressing tagged versions of At5g67200

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in plant cells

  • Proximity-dependent biotin labeling (BioID or TurboID) to identify interactors in native cellular context

The MST assay protocol should follow established methods used for ARM receptor ECDs, including protein labeling with His-Tag Labeling Kit RED-tris-NTA and measurements at 25°C using appropriate buffer conditions (20 mM phosphate buffer pH 7.5, 200 mM NaCl, 0.005% Tween-20) .

What are the optimal conditions for recombinant expression of At5g67200?

For successful recombinant expression of At5g67200, consider the following optimized protocol:

• Expression system selection:

  • For full-length protein: Insect cell expression system (Sf9 or High Five cells)

  • For extracellular domain (ECD): Mammalian expression (HEK293 cells)

  • For intracellular kinase domain: Bacterial expression (E. coli)

• Expression constructs:

  • Include appropriate secretion signal for ECD expression

  • Add purification tags (His6, StrepII, or FLAG) at N- or C-terminus

  • Consider fusion proteins (MBP, GST) to enhance solubility for bacterial expression

• Purification strategy:

  • Two-step affinity chromatography followed by size exclusion chromatography

  • Buffer optimization to maintain protein stability (typically pH 7.0-7.5 with 150-200 mM NaCl)

  • Consider addition of glycerol (10%) and reducing agents (1 mM DTT) for storage

Yield and purity assessments should be performed using SDS-PAGE, Western blotting, and mass spectrometry to confirm protein identity and integrity.

How can ligand-binding assays be optimized for studying At5g67200 interactions?

To characterize potential interactions between At5g67200 and plant cell wall-derived ligands:

• Prepare and validate ligands:

  • Purify RG-I and other cell wall polysaccharides using established fractionation methods

  • Generate defined oligosaccharides through enzymatic digestion with RG-I hydrolase or RG-I lyase

  • Characterize oligosaccharide structures using LC-MS, MALDI-TOF-MS, and NMR analysis

• Binding assays:

  • MicroScale Thermophoresis (MST) with labeled At5g67200 ECD and varying concentrations of ligands

  • Plot log10 values of ligand concentrations against normalized thermophoresis (ΔFnorm [‰])

  • Determine EC50 or Kd values using appropriate curve fitting in software like GraphPad Prism

• Controls and validation:

  • Include non-binding receptor ECDs (like LRRECD) as negative controls

  • Use known receptor-ligand pairs (e.g., FLS2-flg22) as positive controls

  • Validate binding through orthogonal methods like isothermal titration calorimetry (ITC)

What genetic approaches are most effective for studying At5g67200 function?

Due to potential functional redundancy with related receptor kinases, comprehensive genetic analysis of At5g67200 requires:

• Generation of multiple mutant combinations:

  • Single T-DNA insertion mutants or CRISPR/Cas9-generated knockout lines

  • Higher-order mutants combining at5g67200 with mutations in related receptors

  • Complementation lines expressing wild-type or modified At5g67200 variants

• Phenotypic analysis focusing on:

  • Response to pathogen infection and disease resistance

  • MAPK activation following elicitor treatment

  • Defense gene expression (e.g., FRK1) upon treatment with potential ligands

  • Reactive oxygen species (ROS) production and calcium flux measurements

• Structure-function analysis:

  • Expression of At5g67200 variants with mutations in key amino acid residues

  • Domain swapping with related receptors to determine specificity determinants

  • Complementation of mutant phenotypes with catalytically inactive variants to assess non-catalytic functions

As demonstrated with other catalytically inactive receptor kinases like MAZ, mutant variants incapable of protein kinase activity can complement mutant phenotypes, suggesting important non-catalytic roles in plant immunity .

How should researchers approach MAPK activation assays when studying At5g67200?

For robust analysis of MAPK activation downstream of At5g67200:

• Sample preparation:

  • Use 14-16 day-old seedlings grown under sterile conditions

  • Treat with purified ligands at appropriate concentrations (typically 50-100 μg/ml for polysaccharides)

  • Harvest tissue at multiple time points (0, 5, 15, 30, 60 minutes post-treatment)

• Protein extraction and analysis:

  • Extract total proteins in buffer containing phosphatase inhibitors (Na2MoO4, Na3VO4, NaF)

  • Perform immunodetection using anti-p44/p42 MAPK antibodies (1:5000 dilution)

  • Detect signals using chemiluminescence on a standard imaging system

• Controls and quantification:

  • Include known MAPK-activating elicitors (flg22, chitooligosaccharides) as positive controls

  • Use receptor mutants (e.g., fls2 for flg22) as specificity controls

  • Quantify signal intensity relative to total MAPK levels for accurate comparisons

This approach has successfully detected MAPK activation in response to RG-I treatment in Arabidopsis, revealing that signaling occurs independently of well-studied co-receptors like BAK1 and CERK1 .

How can systems biology approaches enhance our understanding of At5g67200 function?

Systems biology offers powerful tools to contextualize At5g67200 function within broader signaling networks:

• Coexpression network analysis:

  • Generate weighted gene coexpression networks using packages like WGCNA

  • Identify modules of co-regulated genes associated with At5g67200 expression

  • Apply topological overlap matrix (TOM) similarity algorithms and dynamic tree pruning to define stable clusters

• Integrative multi-omics:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Analyze phosphoproteome changes dependent on At5g67200

  • Identify signaling network perturbations in at5g67200 mutants

• Network visualization and analysis:

  • Use Cytoscape for network visualization and GO enrichment analysis

  • Apply motif enrichment analysis to identify transcriptional regulatory patterns

  • Develop mathematical models of signaling dynamics incorporating At5g67200 function

These approaches can reveal functional connections that might be missed by traditional reductionist approaches, particularly for proteins with redundant functions like plant receptor kinases .

What role might At5g67200 play in oxidative burst regulation?

Building on findings from related receptor-like cytoplasmic kinases:

• Potential regulatory mechanisms:

  • At5g67200 may function similarly to subgroup VIII RLCKs (MAZ and CARK6) as negative regulators of immune-triggered oxidative burst

  • Interaction with calcium-dependent protein kinases (CPKs) might modulate reactive oxygen species (ROS) production

  • Scaffolding roles could coordinate NADPH oxidase activation

• Experimental assessment:

  • Measure ROS production in at5g67200 single and higher-order mutants using luminol-based assays

  • Analyze interaction with NADPH oxidase components through protein-protein interaction studies

  • Assess calcium dependency through simultaneous calcium and ROS measurements

• Physiological significance:

  • Precise regulation of oxidative burst is critical for balancing immunity and preventing cellular damage

  • Catalytically inactive kinases may provide fine-tuning of immune responses through competition for binding partners

  • Integration with other defense responses including MAPK activation and transcriptional reprogramming

The finding that MAZ and CARK6 function as negative regulators of immune-triggered oxidative burst suggests At5g67200 might play similar roles in fine-tuning plant immune responses.