CERK1 Antibody

What is CERK1 and what is its primary function in plants?

CERK1 (Chitin elicitor receptor kinase 1) is a receptor-like kinase that plays a crucial role in plant immunity by perceiving microbial signals. Its primary function is to recognize chitin oligosaccharides derived from fungal cell walls and bacterial peptidoglycans, initiating immune signaling cascades. In Arabidopsis thaliana, CERK1 is essential for chitin perception and subsequent immune responses, including the activation of mitogen-activated protein kinase (MAPK) cascades . CERK1 contains LysM domains in its extracellular portion that bind to chitin molecules, and an intracellular kinase domain that transmits the signal through phosphorylation events . This receptor not only recognizes fungal chitin but has also been shown to be involved in the perception of bacterial peptidoglycans, collaborating with other LysM domain-containing proteins like LYM1 and LYM3 .

How does CERK1 contribute to the plant immune system?

CERK1 contributes to plant immunity through several mechanisms:

• Recognition of MAMPs (Microbe-Associated Molecular Patterns): CERK1 directly binds to chitin oligosaccharides, particularly 1,4-β-d-(GlcNAc)6, as validated through in silico predictions and isothermal titration calorimetry binding assays .

• Signal transduction initiation: Upon chitin perception, CERK1 forms a complex with LYK5 and phosphorylates downstream targets such as the receptor-like cytoplasmic kinase PBL27 .

• MAPK cascade activation: CERK1-activated PBL27 subsequently phosphorylates MAPKKK5, which is the starting molecule of the MAPK cascade leading to the activation of MPK3, MPK4, and MPK6 .

• Transcriptional reprogramming: This signaling cascade ultimately leads to extensive changes in gene expression. RNA-seq analysis identified 339 genes whose expression was induced or suppressed by chitin treatment in a MAPKKK5-dependent manner, with many of these genes containing WRKY, NAC, and heat-shock transcription factor binding motifs in their promoters .

What is the molecular structure of CERK1?

CERK1 is a transmembrane receptor kinase with an expected molecular weight of approximately 67 kDa . The protein contains:

• An extracellular domain with LysM motifs that are responsible for binding to chitin oligosaccharides and other glycan structures .

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

• An intracellular kinase domain that possesses enzymatic activity for phosphorylating downstream signaling components such as PBL27 .

The molecular structure of CERK1 enables it to function at the cell surface, where it can perceive extracellular signals and transmit them to the intracellular environment through its kinase activity. While crystal structures of CERK1 in complex with chitin have been suggested through molecular docking calculations, complete structural validations through full molecular dynamics simulations remain limited .

How should researchers prepare samples for CERK1 detection using Western blot?

For optimal detection of CERK1 using Western blot, researchers should follow these methodological guidelines:

• Sample preparation: Extract microsomal membrane proteins from Arabidopsis thaliana tissues, as CERK1 is a membrane-localized receptor. The protocol described by LaMontagne et al. (2016) is recommended for this purpose .

• Protein denaturation: Use standard SDS-PAGE sample buffer with heating to denature the proteins properly.

• Gel electrophoresis: Separate proteins using SDS-PAGE, considering that CERK1 has an expected molecular weight of 67 kDa .

• Transfer: Transfer proteins to a nitrocellulose or PVDF membrane using standard protocols.

• Antibody dilution: Use the anti-CERK1 antibody at a dilution of 1:1000 to 1:3500 for Western blot applications .

• Controls: Include appropriate positive controls (wild-type Arabidopsis) and negative controls (cerk1 mutant) to validate specificity. The antibody has been confirmed to be reactive with Arabidopsis thaliana but not with Nicotiana benthamiana or Solanum lycopersicum .

• Detection: Use a secondary antibody compatible with the rabbit-derived primary antibody and an appropriate detection system.

What are the optimal conditions for storing and handling CERK1 antibodies?

For maximizing antibody shelf life and maintaining optimal performance, researchers should adhere to these guidelines when handling CERK1 antibodies:

• Storage format: The antibody is typically provided in lyophilized format and should be stored at -20°C in this state .

• Reconstitution: Add 50 μl of sterile water to reconstitute the lyophilized antibody (for a standard 50 μg quantity) .

• Post-reconstitution storage: After reconstitution, continue to store the antibody at -20°C and make small aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality .

• Handling: Before opening tubes, briefly spin them to ensure that all material is collected at the bottom and to avoid any losses from antibody adhering to the cap or sides of the tube .

• Working dilution preparation: When preparing working dilutions, use fresh buffer and maintain cold conditions to preserve antibody activity.

• Long-term storage: For long-term storage, keep the antibody in its original lyophilized state whenever possible, as this provides maximum stability.

What controls should be included when using CERK1 antibodies in immunological experiments?

Proper experimental controls are essential for validating results obtained with CERK1 antibodies:

• Positive control: Wild-type Arabidopsis thaliana samples should be included, as the antibody has confirmed reactivity with this species .

• Negative controls:

  • Samples from cerk1 knockout mutants to confirm antibody specificity

  • Samples from non-reactive species such as Nicotiana benthamiana and Solanum lycopersicum

  • Primary antibody omission control to assess non-specific binding of secondary antibodies

• Loading controls: Include detection of constitutively expressed proteins such as actin (similar to the Actin2 reference used in RT-PCR experiments mentioned in the research) .

• Treatment controls: When studying CERK1 responses to elicitors, include appropriate mock treatments alongside chitin treatments.

• Blocking controls: Perform peptide competition assays using the immunogen peptide to confirm antibody specificity, especially when using the antibody in new experimental contexts.

• Cross-reactivity assessment: Test potential cross-reactivity with closely related receptor kinases, particularly other LysM domain-containing proteins.

How does CERK1 interact with downstream signaling components?

CERK1 orchestrates downstream signaling through specific protein-protein interactions and phosphorylation events:

• Complex formation with co-receptors: CERK1 forms a chitin-induced complex with the related kinase LYK5, which enhances chitin perception .

• Phosphorylation of RLCKs: Upon activation, CERK1 phosphorylates the receptor-like cytoplasmic kinase PBL27, particularly at residues S138, T141, and S168. This phosphorylation is crucial for PBL27's kinase activity .

• MAPK cascade activation: Phosphorylated PBL27 interacts with and phosphorylates MAPKKK5, providing the missing link between chitin perception at the cell surface and MAPK cascade activation. The interaction between PBL27 and MAPKKK5 is chitin-dependent and occurs at the plasma membrane .

• Signal dissociation dynamics: After chitin perception, MAPKKK5 dissociates from PBL27, allowing for propagation of the signal. This dissociation is specific to chitin signaling and does not occur with flg22 treatment, indicating pathway specificity .

• Proteasomal regulation: Both PBL27 and MAPKKK5 protein levels appear to be regulated through proteasome-dependent degradation, as treatment with the proteasome inhibitor MG132 increases their protein levels .

What is the role of CERK1 in the MAPK cascade activation?

CERK1 plays a pivotal role in activating MAPK cascades in response to chitin perception:

• Initiation of signaling: CERK1 serves as the primary receptor for chitin, initiating the signaling cascade that leads to MAPK activation .

• PBL27 phosphorylation: CERK1 directly phosphorylates PBL27, enhancing its kinase activity. This phosphorylation is crucial for subsequent steps in the signaling pathway .

• MAPKKK5 activation: Phosphorylated PBL27 functions as a MAPKKK kinase, phosphorylating MAPKKK5. This phosphorylation is enhanced when PBL27 is first phosphorylated by CERK1, indicating a sequential activation mechanism .

• Downstream MAPK activation: MAPKKK5 subsequently leads to the activation of MPK3, MPK4, and MPK6, as chitin-induced activation of these MAPKs is reduced in mapkkk5 mutants .

• Transcriptional reprogramming: The activated MAPK cascade ultimately leads to significant changes in gene expression. RNA-seq analysis identified numerous genes whose expression is regulated through this pathway, many of which are involved in immune responses .

This signaling mechanism represents a crucial link between pattern recognition receptors at the plasma membrane and intracellular signaling cascades that orchestrate immune responses.

How do mutations in CERK1 affect plant immune responses?

Mutations in CERK1 significantly compromise plant immune responses to fungal and bacterial pathogens:

• Impaired chitin perception: In cerk1 knockout mutants, plants lose the ability to recognize chitin oligosaccharides, as demonstrated by the lack of immune responses to 1,4-β-d-(GlcNAc)6 (a chitin hexamer) .

• Reduced MAPK activation: Chitin-induced activation of MPK3, MPK6, and MPK4 is compromised in cerk1 mutants, indicating that CERK1 is essential for MAPK cascade activation in response to chitin .

• Compromised transcriptional responses: The induction of defense-related genes following chitin treatment is significantly reduced in cerk1 mutants .

• Enhanced disease susceptibility: cerk1 mutants show increased susceptibility to fungal pathogens such as Alternaria brassicicola, highlighting CERK1's importance in fungal resistance .

• Altered response to bacterial peptidoglycans: Since CERK1 also contributes to peptidoglycan perception in collaboration with LYM1 and LYM3, cerk1 mutants also show compromised responses to bacterial PAMPs .

• Specificity in immune pathway defects: Interestingly, while cerk1 mutants are defective in chitin-induced immunity, they maintain normal responses to other PAMPs such as flg22, indicating pathway specificity .

How can CERK1 antibodies be used to study receptor complex formation?

CERK1 antibodies can be powerful tools for investigating receptor complex dynamics using several methodological approaches:

• Co-immunoprecipitation (Co-IP) assays: CERK1 antibodies can be used to pull down CERK1 and analyze its interacting partners under different conditions (e.g., before and after chitin treatment). This approach was used to demonstrate the chitin-induced disassociation between PBL27 and MAPKKK5 .

• In vivo proximity labeling: CERK1 antibodies can validate results from proximity labeling approaches (such as BioID or APEX) that aim to identify proteins in close proximity to CERK1 in living cells.

• Bimolecular Fluorescence Complementation (BiFC) validation: CERK1 antibodies can confirm expression levels of CERK1 in BiFC experiments examining interactions with potential partners, as was done for studying PBL27-MAPKKK5 interactions at the plasma membrane .

• Super-resolution microscopy: CERK1 antibodies can be used in immunofluorescence microscopy to study receptor clustering and complex formation at nanoscale resolution following chitin treatment.

• Protein-protein interaction dynamics: Using CERK1 antibodies in time-course immunoprecipitation experiments can reveal the temporal dynamics of complex formation and dissociation following chitin perception.

• Cross-linking studies: Chemical cross-linking followed by immunoprecipitation with CERK1 antibodies can capture transient interactions that might be missed with standard Co-IP approaches.

How can phosphorylation states of CERK1 be analyzed in experimental settings?

Analyzing CERK1 phosphorylation states is critical for understanding its activation and regulation:

• Phospho-specific antibodies: While not mentioned in the search results, phospho-specific antibodies targeting known phosphorylation sites on CERK1 would enable direct detection of activated CERK1.

• Mobility shift assays: Western blot analysis using standard CERK1 antibodies can detect phosphorylation-induced mobility shifts, as phosphorylated proteins often migrate differently in SDS-PAGE.

• Phosphatase treatments: Treating samples with phosphatases before Western blot analysis with CERK1 antibodies can confirm that observed mobility shifts are due to phosphorylation.

• Mass spectrometry: Immunoprecipitation with CERK1 antibodies followed by mass spectrometry analysis can identify specific phosphorylation sites and quantify phosphorylation levels, similar to the phosphoproteome analysis mentioned for studying in vivo phosphorylation .

• In vitro kinase assays: CERK1 antibodies can be used to immunoprecipitate CERK1 for subsequent in vitro kinase assays to study its activity under different conditions.

• Mutagenesis validation: CERK1 antibodies can be used to confirm the expression of phosphosite mutants (e.g., alanine or phosphomimetic substitutions) in complementation studies.

How can researchers analyze CERK1-dependent transcriptional changes?

Several methodological approaches can be used to analyze CERK1-dependent transcriptional changes:

• RNA sequencing (RNA-seq): This powerful approach was used to identify CERK1-dependent genes by comparing transcriptional changes in wild-type and mapkkk5 mutant plants upon chitin treatment. A similar approach could be applied directly to cerk1 mutants .

• Data analysis pipeline: For RNA-seq data, researchers should follow a rigorous bioinformatics pipeline that includes:

  • Mapping reads to the reference genome (e.g., using TopHat for Arabidopsis thaliana)

  • Counting reads per gene (e.g., using HTSeq)

  • Identifying differentially regulated genes (DRGs) with appropriate statistical cutoffs (q-value < 0.05)

  • Clustering analysis to identify genes with similar expression patterns

• Motif enrichment analysis: To identify transcription factors potentially involved in CERK1-mediated responses, researchers can analyze promoters of CERK1-dependent genes for enriched cis-elements, as was done to identify WRKY, NAC, and heat-shock transcription factor binding sites .

• Quantitative RT-PCR validation: Selected genes identified through RNA-seq should be validated using qRT-PCR, using appropriate reference genes such as Actin2 (At2g18780) .

• ChIP-seq analysis: Chromatin immunoprecipitation followed by sequencing can identify direct binding targets of transcription factors activated downstream of CERK1 signaling.

• Reporter gene assays: Promoters of CERK1-regulated genes can be fused to reporter genes to study their activation dynamics in wild-type versus cerk1 mutant backgrounds.

What are common issues when detecting CERK1 in plant samples and how can they be resolved?

Researchers may encounter several challenges when working with CERK1 antibodies:

• Low signal intensity: CERK1 may be expressed at relatively low levels, as indicated by the low expression level of MAPKKK5 in the signaling pathway . To improve detection:

  • Optimize protein extraction protocols specifically for membrane proteins

  • Increase protein loading amount

  • Use more sensitive detection systems

  • Optimize antibody concentration (1:1000 to 1:3500 range recommended)

• Non-specific bands: To improve specificity:

  • Include appropriate controls (cerk1 mutant samples)

  • Optimize blocking conditions

  • Adjust antibody dilution

  • Perform peptide competition assays

• Protein degradation: CERK1 levels may be regulated through proteasomal degradation , which can complicate detection. Researchers should:

  • Include protease inhibitors in extraction buffers

  • Consider adding proteasome inhibitors like MG132 during sample preparation

  • Maintain samples at cold temperatures throughout processing

  • Process samples quickly to minimize degradation

• Cross-reactivity issues: While the antibody is specific to Arabidopsis thaliana CERK1, it does not react with Nicotiana benthamiana or Solanum lycopersicum . When working with other species:

  • Test antibody reactivity before proceeding with experiments

  • Consider developing species-specific antibodies if needed

  • Use genetic approaches (e.g., epitope tagging) as alternatives

• Difficulty detecting phosphorylated forms: Phosphorylation-specific mobility shifts may be subtle. Researchers can:

  • Use Phos-tag SDS-PAGE to enhance separation of phosphorylated proteins

  • Develop phospho-specific antibodies for key phosphorylation sites

  • Use mass spectrometry-based approaches for phosphorylation analysis

How can researchers validate antibody specificity for CERK1 in different experimental systems?

Validating antibody specificity is crucial for reliable results:

• Genetic validation: The most rigorous approach is to compare antibody reactivity in wild-type plants versus cerk1 knockout mutants, where the specific band should be absent in the mutant.

• Recombinant protein controls: Express and purify recombinant CERK1 (or its immunogenic region) to use as a positive control in Western blots.

• Peptide competition assay: Pre-incubate the antibody with excess immunogenic peptide before using it in experiments; this should eliminate specific binding.

• Species specificity testing: As the antibody is known to react with Arabidopsis thaliana but not with Nicotiana benthamiana or Solanum lycopersicum , researchers can use these species as positive and negative controls, respectively.

• RNA interference: Compare antibody reactivity in plants where CERK1 expression has been reduced through RNAi approaches.

• Epitope-tagged CERK1: Express CERK1 with an epitope tag and perform parallel detection with both the CERK1 antibody and an antibody against the tag to confirm specific recognition.

• Mass spectrometry validation: Perform immunoprecipitation with the CERK1 antibody followed by mass spectrometry analysis to confirm that the pulled-down protein is indeed CERK1.

What strategies can improve the detection of CERK1-associated protein complexes?

Detecting CERK1-associated protein complexes can be challenging but several strategies can improve results:

• Crosslinking approaches: Use membrane-permeable crosslinkers to stabilize transient interactions before cell lysis and immunoprecipitation with CERK1 antibodies.

• Optimized lysis conditions: Use mild detergents that preserve protein-protein interactions while still solubilizing membrane proteins effectively.

• Timing considerations: Since CERK1 interactions may be dynamic and change rapidly after chitin perception, perform careful time-course experiments to capture relevant complexes at the appropriate time points .

• Subcellular fractionation: Enrich for plasma membrane fractions where CERK1 is localized before immunoprecipitation to reduce background.

• Bimolecular Fluorescence Complementation (BiFC): This technique was successfully used to visualize interactions between PBL27 and MAPKKK5 at the plasma membrane and could be adapted for studying CERK1 interactions.

• Advanced proteomics approaches: Techniques such as proximity-dependent biotin identification (BioID) or APEX-based proximity labeling coupled with CERK1 antibody validation can identify proteins in close proximity to CERK1 in living cells.

• Quantitative interaction proteomics: Stable isotope labeling with amino acids in cell culture (SILAC) or tandem mass tag (TMT) labeling combined with immunoprecipitation using CERK1 antibodies can identify interaction partners that change in abundance following chitin treatment.

How does CERK1 function in different plant species beyond Arabidopsis thaliana?

Understanding CERK1 function across different plant species represents an important research frontier:

• Evolutionary conservation: While the search results focus on Arabidopsis thaliana, researchers should investigate whether CERK1 orthologs in other plant species perform similar functions in chitin perception and immune signaling.

• Crop plant studies: Since the current antibody does not react with Nicotiana benthamiana or Solanum lycopersicum , developing antibodies against CERK1 orthologs in crop plants would facilitate research on chitin-triggered immunity in agriculturally important species.

• Functional conservation testing: Complementation studies with CERK1 orthologs from different species in the Arabidopsis cerk1 mutant background could reveal the degree of functional conservation.

• Receptor complex variation: Investigating whether CERK1 orthologs in different plant species form complexes with different co-receptors could reveal evolutionary adaptations in chitin perception mechanisms.

• Downstream signaling comparison: Comparative studies of CERK1-dependent signal transduction pathways across plant species could identify conserved and divergent aspects of chitin-triggered immunity.

• Host-pathogen co-evolution: Examining how CERK1 variants in different plant species recognize chitin from their specific pathogens could provide insights into host-pathogen co-evolution.

What is the relationship between CERK1-mediated immunity and other plant defense pathways?

Understanding how CERK1 signaling integrates with other immune pathways is crucial for a comprehensive view of plant immunity:

• Cross-talk with other PRR pathways: The search results indicate that PBL27-MAPKKK5 disassociation is specific to chitin signaling and does not occur with flg22 treatment , suggesting pathway specificity. Further research should investigate potential points of convergence and divergence between CERK1 and other PRR signaling pathways.

• MAPK cascade integration: Since MAPKKK5 activates MPK3, MPK4, and MPK6 , which are also activated by other immune receptors, research should focus on how specificity in downstream responses is achieved.

• Hormonal crosstalk: Investigating how CERK1 signaling influences and is influenced by defense hormones such as salicylic acid, jasmonic acid, and ethylene would provide insights into defense network architecture.

• Effector-triggered immunity interaction: Research on how CERK1-mediated PTI interacts with effector-triggered immunity (ETI) could reveal important aspects of layered plant immune responses.

• Priming mechanisms: Studies on whether prior activation of CERK1 signaling primes plants for enhanced responses to subsequent pathogen encounters would illuminate important aspects of immune memory.

• DAMPs sensing integration: CERK1 does not bind the damage-associated molecular pattern (DAMP) cellohexaose (1,4-β-d-(Glc)6), and immune responses to this molecule are not impaired in cerk1 mutants . Research into how DAMP and MAMP sensing pathways interact would be valuable.

What role does CERK1 play in non-immune plant processes?

Expanding research beyond immunity to investigate potential roles of CERK1 in other plant processes:

• Development regulation: Many immune receptors also play roles in plant development. Research should investigate whether CERK1 contributes to developmental processes in addition to immunity.

• Beneficial microbe interactions: Since CERK1 recognizes chitin, which is also present in beneficial fungi like mycorrhizae, studies on how plants differentiate between pathogenic and beneficial chitin sources would be valuable.

• Abiotic stress responses: Investigating potential roles of CERK1 in responses to abiotic stresses such as drought, salt, or temperature extremes could reveal unexpected functions.

• Cell wall sensing: Given CERK1's ability to bind various glycan structures , research on whether it contributes to cell wall integrity sensing would be informative.

• Nutrient signaling: Studies on whether CERK1 plays a role in nutrient sensing or response to nutrient availability could uncover new functions.

• Root-microbe interactions: Since roots are constantly exposed to microbial signals, research on CERK1's specific roles in root immunity and microbiome establishment would be valuable.