EDS1 Antibody

What is EDS1 and why are antibodies against it important for plant immunity research?

EDS1 is a key immune regulator mediating basal resistance to virulent pathogens and receptor-triggered immunity in plants, particularly Arabidopsis. It functions through interactions with related proteins PAD4 (Phytoalexin Deficient4) and SAG101 (Senescence Associated Gene101) to form distinct protein complexes essential for defense signaling . Antibodies against EDS1 are critical research tools that enable detection and tracking of EDS1 protein during immune responses, investigation of its subcellular localization, and analysis of protein-protein interactions that mediate its function.

EDS1 antibodies help researchers investigate how EDS1 activities are distributed between the cytoplasm and nucleus, which is crucial as EDS1 forms different-sized complexes with distinct intracellular distributions that likely perform non-redundant functions in immune response pathways .

How do I determine the optimal fixation method when using EDS1 antibodies for immunolocalization?

For effective immunolocalization of EDS1 using antibodies, consider these methodological approaches:

• Paraformaldehyde fixation: Use 4% paraformaldehyde for 20-30 minutes at room temperature, which preserves protein structure while maintaining antigenicity.

• Nucleus vs. cytoplasm detection: Since EDS1 shuttles between nucleus and cytoplasm, combining fixation with gentle detergent permeabilization (0.1% Triton X-100) improves antibody access to both compartments.

• Validation controls: Always include eds1 mutant plants as negative controls to verify antibody specificity . When studying nuclear-cytoplasmic distribution, include controls with known nuclear or cytoplasmic markers.

• Co-localization studies: For studying EDS1-PAD4 or EDS1-SAG101 interactions, perform double immunolabeling using antibodies against both proteins with appropriate controls for cross-reactivity .

The importance of proper fixation cannot be overstated, as studies have demonstrated that EDS1 undergoes nucleocytoplasmic shuttling during immune responses, with receptor-stimulated increases in nuclear EDS1 preceding or coinciding with defense gene expression changes .

What are the best extraction methods for preserving EDS1 protein complexes for antibody detection?

To effectively preserve EDS1 protein complexes for antibody detection, consider the following methodological approach:

This methodology is critical because studies show EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without requiring additional plant factors . Loss of interaction with EDS1 reduces PAD4 post-transcriptional accumulation, suggesting EDS1 physical association stabilizes PAD4. Improper extraction could disrupt these interactions, leading to misleading results .

For nuclear fraction analysis, use differential centrifugation with nuclear lysis buffer containing higher detergent concentrations (0.5-1% NP-40) after separating nuclear and cytoplasmic fractions, as demonstrated in studies examining nuclear EDS1 accumulation during plant immunity responses .

How can I distinguish between free EDS1 and EDS1 in complex with PAD4 or SAG101 using antibody-based techniques?

Distinguishing between free EDS1 and its complexes requires sophisticated immunological approaches:

• Sequential immunoprecipitation: First immunoprecipitate with anti-EDS1 antibodies, then probe the precipitate with anti-PAD4 or anti-SAG101 antibodies. This allows detection of specific complexes.

• Size exclusion chromatography followed by immunoblotting: Separate protein complexes based on size, then use EDS1 antibodies to identify fractions containing EDS1. Different molecular weights correspond to free EDS1 (~72 kDa) versus EDS1-PAD4 (~120 kDa) or EDS1-SAG101 complexes .

• Native gel electrophoresis: Preserves protein-protein interactions during electrophoresis, allowing antibody detection of intact complexes with different mobility compared to free EDS1.

• Proximity ligation assays: Use paired antibodies against EDS1 and PAD4 or SAG101 to generate fluorescent signals only when proteins are in close proximity, enabling visualization of specific complexes within cellular compartments.

Research has shown that the EDS1-PAD4 complex is essential for basal resistance involving transcriptional upregulation of PAD4 and mobilization of salicylic acid defenses, while dissociated forms of EDS1 and PAD4 are still competent in signaling receptor-triggered localized cell death . Therefore, distinguishing between free and complexed forms is crucial for understanding their differential roles in immune signaling.

What controls should I implement when studying EDS1 nuclear translocation using antibodies?

When studying EDS1 nuclear translocation, implement these essential controls:

• Genetic controls: Include eds1 mutant plants (e.g., eds1-2) to validate antibody specificity and exclude false positive signals .

• Subcellular fractionation validation:

  • Use nuclear markers (e.g., histone H3) and cytoplasmic markers (e.g., GAPDH) to confirm fractionation purity

  • Perform reciprocal detection to ensure no cross-contamination between fractions

• Time course controls: Monitor EDS1 localization at multiple timepoints after stimulus, as studies show receptor-stimulated increases in nuclear EDS1 precede or coincide with defense gene induction .

• Stimulus-specific responses:

  • Include appropriate treatments (e.g., pathogen, SA)

  • Use plants with constitutive immunity (e.g., snc1 mutants) as positive controls for enhanced nuclear accumulation

  • Compare with overexpressor lines that have elevated EDS1 levels but no auto-immune phenotype

• Quantification method: Employ nuclear/cytoplasmic ratio measurements with standardized thresholding to objectively assess translocation .

Research has demonstrated that simple overexpression of EDS1 does not produce auto-immune phenotypes, despite increased nuclear and cytoplasmic EDS1 levels. This suggests that nuclear translocation alone is insufficient and additional signals generated by activated immune receptors are necessary for full immune activation .

How can I use antibodies to investigate the EDS1-NPR1 interaction in transcriptional regulation during immune responses?

To investigate the EDS1-NPR1 interaction in transcriptional regulation during immune responses:

• Co-immunoprecipitation (Co-IP): Use anti-EDS1 antibodies to precipitate protein complexes, then probe with anti-NPR1 antibodies to detect interaction. Include controls with NPR1 mutant variants (npr1-2, nim1-2, npr1-1, npr1-5) known to lose interaction with EDS1 .

• Chromatin immunoprecipitation (ChIP):

  • Perform ChIP with anti-EDS1 antibodies to identify EDS1 recruitment to promoters of defense genes like PR1

  • Compare wild-type with npr1 mutant plants to determine NPR1-dependency of EDS1 recruitment

  • Target the F4 (as-1) region of the PR1 promoter, where EDS1 binding is NPR1-dependent

• Sequential ChIP: First immunoprecipitate with anti-NPR1 antibodies, then with anti-EDS1 antibodies to identify genomic regions where both proteins co-localize.

• Proximity-dependent labeling: Use antibodies to validate BioID or APEX2-based proximity labeling results that identify proteins near EDS1 and NPR1 in living cells.

Research has shown that EDS1 functions as an autonomous transcriptional coactivator with intrinsic transactivation domains and physically interacts with the CDK8 subunit of Mediator. Upon salicylic acid induction, EDS1 is directly recruited by NPR1 onto the PR1 promoter via physical NPR1-EDS1 interactions, thereby potentiating PR1 activation . Additionally, EDS1 stabilizes NPR1 protein, while NPR1 transcriptionally up-regulates EDS1, creating a positive feedback loop critical for robust immune activation .

What methodologies can address the challenge of detecting low-abundance EDS1 complexes in specific cellular compartments?

Detecting low-abundance EDS1 complexes in specific cellular compartments requires specialized techniques:

• Targeted subcellular enrichment:

  • Nuclei isolation using sucrose gradient ultracentrifugation

  • Membrane fractionation to isolate microdomains where signaling complexes might localize

  • Cytoskeletal purification to identify transport-associated EDS1 complexes

• Signal amplification methods:

  • Tyramide signal amplification (TSA) that can increase sensitivity by 10-100 fold

  • Quantum dot-conjugated secondary antibodies with higher signal-to-noise ratio

  • Antibody-based proximity ligation assays that generate fluorescent signals only when target proteins interact

• Advanced microscopy:

  • Super-resolution techniques (STED, PALM, STORM) to visualize complexes below diffraction limit

  • FRET-FLIM microscopy to detect protein-protein interactions directly in cellular compartments

  • Correlative light-electron microscopy to combine immunofluorescence with ultrastructural localization

• Mass spectrometry validation:

  • Immunoprecipitation followed by targeted mass spectrometry

  • Comparison of stoichiometry between different complexes (EDS1-PAD4 vs. EDS1-SAG101)

  • Mapping post-translational modifications that might regulate complex formation

Research has shown that changes in nuclear EDS1 levels become equilibrated with the cytoplasmic EDS1 pool, and cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites . The difficulty in detecting these dynamics necessitates combining multiple approaches to overcome sensitivity limitations.

How can I resolve antibody cross-reactivity issues when studying EDS1 in species other than Arabidopsis?

When studying EDS1 in non-Arabidopsis species, address antibody cross-reactivity challenges through:

• Epitope mapping and selection:

  • Perform sequence alignment of EDS1 orthologs to identify conserved and variable regions

  • Select conserved epitopes for cross-species reactivity or species-specific regions for selective detection

  • Target regions in the EP domain, which forms a positive surface lining a cavity created by the heterodimer that is essential for pathogen resistance

• Validation strategy:

  • Express the target species' EDS1 protein recombinantly as a positive control

  • Include genetic knockouts/knockdowns of EDS1 in the target species as negative controls

  • Perform peptide competition assays to confirm specificity

• Multi-antibody approach:

  • Use multiple antibodies targeting different EDS1 epitopes

  • Confirm results with both monoclonal (high specificity) and polyclonal (broader epitope recognition) antibodies

  • Validate findings with tagged EDS1 constructs expressed in the target species

• Pre-absorption technique:

  • Incubate antibodies with recombinant proteins or peptides from closely related species

  • Remove cross-reactive antibodies before using for detection in target species

  • Quantify specificity improvement through western blot signal ratios

EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without additional plant factors, but the precise interactions may vary between species . Research has shown that EDS1 heterodimers are recruited by Toll-interleukin1-receptor domain NLRs (TNLs) to transcriptionally mobilize resistance , making accurate antibody detection across species critical for comparative studies of plant immunity mechanisms.

What troubleshooting approaches can resolve contradictory results between immunolocalization and biochemical fractionation of EDS1?

When facing contradictory results between immunolocalization and biochemical fractionation of EDS1, implement these troubleshooting approaches:

• Sample preparation reconciliation:

  • Use identical tissue harvesting conditions, plant age, and treatment timing

  • Process samples for both techniques in parallel from the same biological material

  • Control for fixation artifacts by comparing different fixatives (paraformaldehyde vs. glutaraldehyde)

• Technical validation:

  • Perform reciprocal validation using GFP-tagged EDS1 for direct visualization

  • Complement with cellular markers to verify compartment identity

  • Use multiple antibodies targeting different EDS1 epitopes to rule out epitope masking

• Quantitative assessment:

  • Implement quantitative image analysis for immunolocalization data

  • Perform quantitative western blotting on fractionated samples

  • Compare relative distributions rather than absolute values

• Temporal dynamics consideration:

  • Assess EDS1 localization at multiple timepoints after stimulus

  • Consider that contradictions may reflect dynamic shuttling between compartments

  • Account for potential redistribution during sample processing

Research shows EDS1 undergoes nucleocytoplasmic shuttling, with receptor-stimulated increases in nuclear EDS1 preceding or coinciding with defense gene induction . Studies also indicate that while nuclear EDS1 is essential for resistance to biotrophic and hemi-biotrophic pathogens and for transcriptional reprogramming, cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites . This dynamic equilibrium between compartments may naturally lead to apparent contradictions if methods capture different temporal states.

How can I optimize antibody-based methods to distinguish between different functional states of EDS1?

To optimize antibody-based methods for distinguishing between different functional states of EDS1:

• Phosphorylation-state specific antibodies:

  • Develop antibodies that specifically recognize phosphorylated or non-phosphorylated EDS1

  • Validate with phosphatase treatments and phospho-mimetic mutants

  • Apply in both immunoblotting and immunolocalization studies

• Conformation-sensitive approaches:

  • Use limited proteolysis followed by epitope-specific antibody detection to reveal structural changes

  • Apply proximity-based labeling (BioID, APEX) to identify partners specific to different functional states

  • Develop antibodies against regions that undergo conformational changes upon complex formation

• Combined IP-activity assays:

  • Immunoprecipitate EDS1 from different cellular contexts

  • Assess associated proteins, post-translational modifications, and activities

  • Compare wild-type EDS1 with functional variants like eds1 L262P that cannot bind PAD4

• Dynamic tracking methodology:

  • Perform time-course analyses following immune activation

  • Track correlations between EDS1 state changes and downstream signaling events

  • Combine with genetic backgrounds that freeze EDS1 in specific functional states

Research has demonstrated that the EDS1-PAD4 complex is necessary for basal resistance involving transcriptional upregulation of PAD4 itself and mobilization of salicylic acid defenses, while dissociated forms of EDS1 and PAD4 are fully competent in signaling receptor-triggered localized cell death at infection sites . Additionally, studies show that the same EDS1 EP-domain surface is recruited by both TNL and CNL receptors for resistance against bacterial pathogens, signaling via three genetically separable resistance sectors .

How can EDS1 antibodies be used to investigate the coordination between EDS1-PAD4 and EDS1-NPR1 signaling modules in plant immunity?

EDS1 antibodies provide powerful tools to investigate coordination between EDS1-PAD4 and EDS1-NPR1 signaling modules:

• Sequential immune event mapping:

  • Use time-course immunoprecipitation to track temporal shifts between EDS1-PAD4 and EDS1-NPR1 complex formation

  • Correlate with transcriptional outputs specific to each module

  • Apply in both basal immunity and effector-triggered immunity contexts

• Spatial distribution analysis:

  • Perform subcellular fractionation followed by co-immunoprecipitation to isolate compartment-specific complexes

  • Use super-resolution immunofluorescence to visualize colocalization patterns

  • Track redistribution of complexes during immune activation

• Functional separation techniques:

  • Use chromatin immunoprecipitation to distinguish direct transcriptional roles of EDS1-NPR1

  • Apply metabolite analysis combined with EDS1-PAD4 immunoprecipitation to connect with upstream SA biosynthesis

  • Develop antibodies specific to different EDS1 complexes based on conformational differences

• Interaction network mapping:

  • Use antibody-based proximity-dependent labeling to identify proteins near each complex

  • Apply sequential immunoprecipitation to isolate proteins that may bridge between pathways

  • Validate with mutants defective in specific interactions

Research indicates that EDS1-PAD4 acts upstream of salicylic acid by antagonizing the coronatine/jasmonic acid-MYC2 branch at early timepoints in immunity, whereas chromatin-binding EDS1-NPR1 directly activates SA signaling and plays a major function at later stages of local and systemic immunity . These temporally and physically different EDS1-PAD4 and EDS1-NPR1 modules are crucial for robust and sustained immune responses, with EDS1-NPR1 synergistically accelerating SA signaling during transcriptional reprogramming and pathogen resistance .

What methodological approaches can resolve contradictory findings regarding EDS1 nuclear vs. cytoplasmic functions?

To resolve contradictory findings regarding EDS1 nuclear versus cytoplasmic functions:

• Compartment-restricted EDS1 expression systems:

  • Create synthetic EDS1 variants with strong nuclear localization signals (NLS) or nuclear export signals (NES)

  • Develop inducible systems to control nuclear import/export timing

  • Validate compartment restriction using antibody-based immunolocalization

• Spatiotemporal dynamics analysis:

  • Implement high-resolution time-course sampling following immune activation

  • Track EDS1 movement between compartments using antibody detection in fractionated samples

  • Correlate with defense response progression metrics

• Interaction-specific functional assessment:

  • Use antibodies to immunoprecipitate compartment-specific EDS1 complexes

  • Identify unique interaction partners through mass spectrometry

  • Validate functions through genetic analysis of partner proteins

• Integrated multi-method approach:

  • Combine genetic (eds1 mutants), biochemical (fractionation), and cytological (immunofluorescence) evidence

  • Develop mathematical models of EDS1 shuttling to predict experimental outcomes

  • Test model predictions with targeted perturbations of nuclear transport

Research has shown that balanced nuclear and cytoplasmic activities of EDS1 are required for robust plant immunity, with nuclear EDS1 directing transcriptional changes while cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites . Evidence points to post-transcriptional processes regulating receptor-triggered accumulation of EDS1 in nuclei, which become equilibrated with the cytoplasmic EDS1 pool . The identification of an essential EP-domain surface in EDS1 heterodimers, which lines a cavity created by the heterodimer, highlights structural features that may function differently in nuclear versus cytoplasmic contexts .

How can antibodies help identify novel EDS1 interaction partners in different plant tissues and stress conditions?

Antibodies are powerful tools for identifying novel EDS1 interaction partners across diverse tissues and stress conditions:

• Tissue-specific immunoprecipitation (IP):

  • Perform IP with anti-EDS1 antibodies from different plant tissues (leaves, roots, flowers)

  • Compare interaction profiles using mass spectrometry

  • Validate tissue-specific interactions with reciprocal co-IP and immunoblotting

• Stress-induced interactome changes:

  • Subject plants to various stresses (pathogens, abiotic stressors, hormones)

  • Perform time-course IP to capture dynamic interaction changes

  • Develop targeted antibody panels for candidate interactors identified through proteomics

• In situ proximity labeling:

  • Combine EDS1 antibodies with biotin-conjugated secondary antibodies and peroxidase

  • Generate reactive biotin species that label proteins in proximity to EDS1 in fixed tissues

  • Identify labeled proteins through streptavidin pulldown and mass spectrometry

• Comparative analysis methodology:

  • Create a reference table of interaction partners across conditions

  • Develop quantitative metrics for interaction strength based on co-IP efficiency

  • Perform network analysis to identify condition-specific interaction modules

Research has demonstrated that EDS1 interacts with NPR1 and functions as an autonomous transcriptional coactivator with intrinsic transactivation domains, physically interacting with the CDK8 subunit of Mediator . Additional studies show EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without requiring additional plant factors . These findings suggest EDS1 likely engages in different protein complexes depending on cellular context, with both constitutive and induced interactions contributing to immune function.

What are the most critical methodological considerations when using EDS1 antibodies for interdisciplinary plant immunity research?

When using EDS1 antibodies for interdisciplinary plant immunity research, prioritize these critical methodological considerations:

• Antibody validation strategy:

  • Include genetic controls (eds1 mutants) in all experiments

  • Perform epitope mapping to understand what specific region is recognized

  • Validate cross-reactivity with related proteins (PAD4, SAG101) and orthologs from different species

• Contextual interpretation framework:

  • Recognize that EDS1 functions in distinct molecular configurations with separable roles

  • Account for dynamic redistribution between compartments when interpreting results

  • Consider how experimental conditions might alter EDS1 complex stability

• Multi-method integration approach:

  • Complement antibody-based detection with independent methods (tagged proteins, mass spectrometry)

  • Correlate biochemical findings with genetic phenotypes

  • Develop coherent models that reconcile results from different methodologies

• Standardized reporting guidelines:

  • Document detailed antibody characteristics (epitope, validation, conditions)

  • Report quantitative metrics rather than only representative images

  • Include all necessary controls in publications to enable reproduction