PEPR2 Antibody

What is PEPR2 and why are antibodies against it important in plant immunology research?

PEPR2 (Perception of the Arabidopsis Danger Signal Peptide 2) is a leucine-rich repeat (LRR) receptor kinase located in the plasma membrane of Arabidopsis thaliana cells. It functions as a pattern recognition receptor (PRR) that specifically recognizes endogenous danger-associated molecular patterns (DAMPs), particularly Pep1 and Pep2 peptides . PEPR2 shares approximately 76% amino acid similarity with its homolog PEPR1 .

Antibodies against PEPR2 are essential research tools because:

• They enable the study of plant immunity mechanisms involving PEPR-mediated defense signaling

• They allow researchers to investigate the spatial and temporal expression patterns of PEPR2

• They facilitate the examination of PEPR2's interactions with other proteins in immune signaling cascades

• They help distinguish between PEPR1 and PEPR2 functions in plant defense responses

What validation methods should researchers use to ensure PEPR2 antibody specificity?

When validating PEPR2 antibodies, researchers should implement multiple complementary strategies:

Primary validation methods:

• Genetic knockout controls: Use pepr2 T-DNA insertion mutants (e.g., SALK_098161) alongside wild-type plants . This approach is considered the gold standard as it definitively demonstrates antibody specificity.

• Side-by-side comparison: Test multiple antibodies against the same target simultaneously using standardized protocols . This allows for direct performance comparison.

• Western blot validation: Confirm the antibody detects a protein of the expected molecular weight that is absent in pepr2 mutants .

Secondary validation methods:

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to block specific binding . Note that this method should never be used in isolation, as peptide antigen will block both specific and non-specific binding.

• Cross-reactivity assessment: Evaluate potential cross-reactivity with PEPR1 due to their high sequence similarity (76%) .

How can researchers use PEPR2 antibodies to differentiate between PEPR1 and PEPR2 functions despite their high sequence similarity?

Distinguishing between PEPR1 and PEPR2 functions requires careful experimental design:

Recommended approach:

• Differential tissue expression analysis: Perform immunohistochemistry with validated PEPR2 antibodies in different tissues, particularly focusing on roots where PEPR2 shows higher expression than PEPR1 .

• Mutant comparison studies: Design experiments using pepr1, pepr2, and pepr1/pepr2 double mutants to identify PEPR2-specific functions .

• Peptide specificity correlation: Compare antibody detection patterns with known binding specificities—PEPR1 recognizes Pep1-6, while PEPR2 only recognizes Pep1 and Pep2 .

• Epitope selection: Use antibodies raised against peptide sequences from non-conserved regions between PEPR1 and PEPR2 .

Analytical considerations:

• Root growth inhibition assays are particularly informative as pepr2 mutants show complete insensitivity to AtPep1 in roots, while pepr1 mutants retain sensitivity .

• Observe differential expression patterns: PEPR2 is particularly highly expressed in the radicle, though some overlap with PEPR1 expression exists in roots .

What methodological approaches should be used for studying PEPR2 phosphorylation events using antibodies?

PEPR2, like PEPR1, undergoes phosphorylation during immune signaling. To study these events:

Recommended protocol:

• Phospho-specific antibody selection: Use antibodies specifically raised against phosphorylated epitopes of PEPR2 .

• Temporal analysis: Perform time-course experiments after Pep1/Pep2 treatment (10 nM concentration) to capture rapid phosphorylation events .

• Phosphatase controls: Include samples treated with phosphatase inhibitors and comparison samples with phosphatase treatment to verify phospho-specificity .

• BIK1 interaction studies: Co-immunoprecipitate PEPR2 with BIK1 (botrytis-induced kinase 1), as BIK1 is directly phosphorylated by PEPR1/PEPR2 in response to Pep treatment .

Analysis tips:

• Use Pep peptide treatments at 10 nM concentration in liquid-grown seedlings to standardize responses .

• Monitor phosphorylation events within 15-30 minutes of Pep treatment, as peak PEPR2 activation occurs rapidly .

• Compare phosphorylation patterns between ethylene-treated and Pep-treated samples, as both treatments induce BIK1 phosphorylation in a PEPR-dependent manner .

How can PEPR2 antibodies be effectively used in co-immunoprecipitation studies to identify immune signaling partners?

Co-immunoprecipitation (Co-IP) is crucial for understanding PEPR2's protein interaction network:

Optimized Co-IP protocol:

• Tissue selection: Use tissues with known PEPR2 expression, particularly roots or seedlings treated with Pep1/Pep2 peptides to upregulate expression .

• Crosslinking consideration: Implement mild crosslinking (0.5% formaldehyde) to stabilize transient interactions between PEPR2 and signaling partners.

• Membrane protein extraction: Use specialized buffers containing mild detergents (0.5-1% NP-40 or Triton X-100) to effectively solubilize the plasma membrane-localized PEPR2 .

• Antibody coupling: Covalently couple purified PEPR2 antibodies to protein A/G beads to prevent antibody contamination in the eluted samples.

• Interaction validation: Confirm interactions using reverse Co-IP and include pepr2 mutant controls.

Key interaction targets to investigate:

• BIK1 and PBL1 (PBS1-like 1) receptor-like cytoplasmic kinases, which interact with both PEPR1 and PEPR2 .

• Components of the ethylene signaling pathway, given the connection between PEPR signaling and ethylene-mediated immunity .

• Proteins involved in the JA and SA signaling pathways, as PEPR activation leads to co-activation of these otherwise antagonistic pathways .

What approaches should be used when developing immunohistochemistry protocols with PEPR2 antibodies for spatiotemporal expression studies?

Optimized immunohistochemistry protocol:

• Fixation optimization: Use 4% paraformaldehyde with vacuum infiltration for proper fixation of plant tissues.

• Antigen retrieval: Implement mild heat-induced epitope retrieval to improve antibody accessibility to the PEPR2 antigen.

• Blocking considerations: Use 5% BSA with 0.3% Triton X-100 to reduce non-specific binding.

• Antibody dilution series: Test multiple antibody dilutions (1:100 to 1:1000) to determine optimal signal-to-noise ratio.

• Dual labeling: Combine PEPR2 antibodies with markers for cellular compartments to precisely locate PEPR2.

Experimental design for spatiotemporal studies:

• Wound-response time course: Examine PEPR2 expression at 15 min, 30 min, 1 hour, and 4 hours post-wounding to capture the rapid induction and subsequent decrease to basal levels .

• Tissue-specific expression: Compare expression in leaves versus roots, noting the higher expression of PEPR2 in root tissues .

• Treatment comparisons: Analyze expression patterns after treatment with methyl jasmonate, Pep peptides, and pathogen-associated molecular patterns, all of which induce PEPR2 transcription .

Include appropriate controls with pepr2 mutants and secondary antibody-only samples to confirm specificity.

How can researchers troubleshoot non-specific binding issues with PEPR2 antibodies?

Common causes and solutions for non-specific binding:

Validation strategy for suspected cross-reactivity:

• Compare staining patterns in wild-type, pepr1, pepr2, and pepr1/pepr2 double mutants .

• Perform peptide competition assays with peptides derived from both PEPR1 and PEPR2 .

• Use recombinant PEPR1 and PEPR2 proteins in dot blot assays to quantify cross-reactivity.

What considerations should be made when designing experiments to study PEPR2-mediated signaling using antibodies?

Experimental design considerations:

• Appropriate controls:

  • Include pepr2 single mutants and pepr1/pepr2 double mutants .

  • Use plants overexpressing PEPR2 as positive controls .

  • Include treatments with both Pep1 (recognized by PEPR1 and PEPR2) and Pep3 (recognized only by PEPR1) to distinguish receptor-specific responses .

• Treatment standardization:

  • Use standardized peptide concentrations (10 nM for seedling treatments) .

  • Apply peptides using consistent methods (liquid media for seedlings, petiole feeding for mature plants).

  • Conduct time-course experiments (0.5-4 hours) to capture dynamic responses .

• Readout selection:

  • Monitor defense marker genes like PR1 (SA pathway) and PDF1.2/PDF1.3 (JA/ET pathway) .

  • Assess root growth inhibition, which shows PEPR2-dominant effects .

  • Measure ROS production as a rapid response to PEPR activation .

• Pathway interactions:

  • Consider the role of PEPR2 in connecting local to systemic immunity .

  • Investigate interactions with ethylene signaling components, as ethylene-induced seedling growth inhibition is partially controlled by PEPR1/PEPR2 .

  • Examine cross-talk between SA and JA pathways, which are co-activated by PEPR signaling .

How can researchers use PEPR2 antibodies to investigate the role of PEPR2 in systemic acquired resistance?

Protocol for investigating PEPR2 in systemic immunity:

• Split-leaf/plant experimental design:

  • Challenge one half of leaves with pathogens or apply Pep peptides locally.

  • Collect both local (treated) and systemic (untreated) leaves at various timepoints.

  • Compare PEPR2 protein levels using quantitative immunoblotting.

• Protein localization changes:

  • Use subcellular fractionation followed by immunoblotting to track PEPR2 localization changes.

  • Perform immunohistochemistry on cross-sections from both local and systemic leaves.

• Signaling cascade analysis:

  • Use co-immunoprecipitation with PEPR2 antibodies to identify interacting partners in local versus systemic tissues.

  • Compare phosphorylation patterns of PEPR2 and downstream components like BIK1 in local and systemic leaves .

Key findings to validate:

• PEPR2-mediated signaling activates genetically separable JA and SA branches in systemic leaves .

• PROPEP2/PROPEP3 induction is restricted to pathogen challenge sites during systemic immunity, while PEPR activation occurs systemically .

• Local PEPR activation provides a critical step in connecting local to systemic immunity .

What techniques are available for quantifying PEPR2 protein levels in different experimental conditions?

Quantitative approaches for PEPR2 measurement:

• Quantitative Western blotting:

  • Use internal loading controls (actin, tubulin) for normalization.

  • Implement standard curves using recombinant PEPR2 protein or peptide standards.

  • Use fluorescent secondary antibodies for wider linear range of detection.

• ELISA-based quantification:

  • Develop sandwich ELISA using capture and detection antibodies against different PEPR2 epitopes.

  • Include serial dilutions of samples to ensure measurements within the linear range.

  • Normalize to total protein concentration.

• Mass spectrometry-based approaches:

  • Use immunoprecipitation with PEPR2 antibodies followed by targeted mass spectrometry.

  • Employ Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) for absolute quantification.

  • Include isotopically labeled peptide standards for accurate quantification.

Experimental applications:

• Compare PEPR2 protein levels in various tissues, with special attention to roots where PEPR2 shows dominant effects .

• Track changes in PEPR2 levels following treatment with Pep peptides, methyl jasmonate, or pathogen challenge .

• Investigate PEPR2 protein stability and turnover rates in different signaling conditions.

How might antibodies against phosphorylated forms of PEPR2 advance our understanding of plant immune signaling?

Potential applications of phospho-specific PEPR2 antibodies:

• Kinetics of PEPR2 activation:

  • Map the temporal sequence of phosphorylation events following ligand binding.

  • Identify differences in phosphorylation patterns between various elicitor treatments (Pep1 vs. Pep2).

  • Compare phosphorylation dynamics between PEPR1 and PEPR2 to understand their functional divergence.

• Spatial distribution of active PEPR2:

  • Use immunohistochemistry with phospho-specific antibodies to visualize where PEPR2 activation occurs.

  • Determine if PEPR2 phosphorylation patterns differ between local and systemic tissues during immune responses.

• Signaling network mapping:

  • Identify phosphorylation-dependent protein interactions using phospho-PEPR2 antibodies in Co-IP studies.

  • Use proximity labeling techniques coupled with phospho-PEPR2 antibodies to identify transient interaction partners.

Technical development needs:

• Generation of site-specific phospho-antibodies targeting key phosphorylation sites in PEPR2.

• Validation of phospho-antibodies using phosphatase treatments and phospho-mimetic/phospho-dead PEPR2 variants.

• Development of high-throughput assays to monitor PEPR2 phosphorylation states in various conditions.

What novel approaches could be developed using PEPR2 antibodies to study root hair development regulation?

Recent research has shown that Plant Elicitor Peptides (Peps) regulate root hair development in Arabidopsis . Novel approaches with PEPR2 antibodies could include:

Innovative methodological approaches:

• Single-cell resolution studies:

  • Use highly specific PEPR2 antibodies for immunohistochemistry to visualize PEPR2 distribution at the single-cell level in developing root hairs.

  • Combine with markers for cell differentiation stages to correlate PEPR2 expression with developmental transitions.

• Protein-DNA interaction studies:

  • Develop ChIP protocols using PEPR2 antibodies to identify if PEPR2 signaling directly regulates transcription factors.

  • Focus on known root hair regulators like CPC and GL2, which are affected by Pep2 treatment .

• Micropipette-based approaches:

  • Apply Pep peptides locally to specific root zones while monitoring PEPR2 localization and phosphorylation status.

  • Combine with live-cell imaging using fluorescent-labeled PEPR2 antibody fragments.

Research questions to address:

• How does PEPR2 signaling alter the expression pattern of GL2 and CPC to regulate root hair development?

• Does PEPR2 localization change during root hair initiation and elongation?

• Are there tissue-specific phosphorylation patterns of PEPR2 in root epidermal cells?

• How does PEPR2 signaling integrate with other known root hair development pathways?

How can multiplexed antibody approaches advance our understanding of PEPR1/PEPR2 co-regulation and differential functions?

Advanced multiplexed approaches:

• Multi-color immunofluorescence:

  • Develop protocols using spectrally distinct fluorophores conjugated to PEPR1 and PEPR2 antibodies.

  • Apply in tissue sections to visualize differential expression patterns.

  • Include co-staining for downstream signaling components (e.g., BIK1, defense markers).

• Sequential immunoprecipitation strategies:

  • Use PEPR2 antibodies for primary IP followed by PEPR1 antibodies (or vice versa).

  • Identify protein complexes containing both receptors versus receptor-specific complexes.

  • Apply to tissues at different stages of immune response to track dynamic changes.

• Proximity ligation assays:

  • Implement in situ proximity ligation assay using antibodies against PEPR1 and PEPR2.

  • Quantify interaction frequency in different cell types and conditions.

  • Combine with treatment by different Pep peptides to analyze receptor complex formation.

Applications to biological questions:

• How do PEPR1 and PEPR2 differentially contribute to local versus systemic immunity?

• Do these receptors form heterocomplexes during signaling, and does this affect ligand specificity?

• How does receptor expression change in response to different pathogens and does this correlate with pathogen lifestyle?

• Can patterns of receptor co-expression predict specific immune outcomes?