EDS1 Antibody

What is EDS1 and why is it important in plant immunity research?

EDS1 (Enhanced Disease Susceptibility1) is a critical regulator of plant basal and receptor-triggered immunity. In Arabidopsis, EDS1 interacts with two related proteins, Phytoalexin Deficient4 (PAD4) and Senescence Associated Gene101 (SAG101), forming essential complexes for defense signaling. EDS1 is indispensable for resistance conditioned by the TIR-NB-LRR class of disease resistance proteins and functions as an essential component of plant resistance to biotrophic and hemibiotrophic pathogens . The protein has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases, suggesting that hydrolase activity on a lipid-based substrate may be central to its function .

What are the optimal fixation and permeabilization methods when using EDS1 antibodies for immunolocalization?

For effective immunolocalization studies of EDS1, researchers should use a 4% paraformaldehyde fixation (20 minutes at room temperature) followed by a mild detergent permeabilization (0.1% Triton X-100 for 10 minutes). This approach preserves both the nuclear and cytoplasmic pools of EDS1, which is crucial since EDS1 shuttles between these compartments. When studying EDS1 localization dynamics during immune responses, it's advisable to perform time-course fixations after pathogen treatment to capture the nuclear accumulation of EDS1 that occurs approximately 3 hours post-infection with avirulent pathogens like Pseudomonas syringae pv tomato DC3000 AvrRps4 .

How can I verify the specificity of my EDS1 antibody?

Specificity verification is essential when working with EDS1 antibodies. Use eds1 knockout mutants (such as eds1-2) as negative controls in Western blots and immunolocalization experiments. The complete absence of signal in these mutants confirms antibody specificity. Additionally, use complemented lines expressing tagged versions of EDS1 (such as EDS1-HA) as positive controls. For overexpression studies, compare antibody detection in wild-type plants versus plants expressing higher amounts of EDS1 to ensure proportionate signal intensity differences .

How does EDS1 subcellular distribution change during pathogen infection?

EDS1 exhibits dynamic subcellular redistribution during immune responses. Under normal conditions, EDS1 is present in both cytoplasmic and nuclear compartments. Upon infection with avirulent pathogens, particularly those recognized by TIR-NB-LRR receptors, there is a significant increase in nuclear accumulation of EDS1. This nuclear enrichment occurs approximately 3 hours post-infection with Pseudomonas syringae pv tomato DC3000 AvrRps4, preceding or coinciding with EDS1-dependent transcriptional reprogramming. Importantly, this early nuclear accumulation is not due to increased EDS1 gene expression but rather results from post-transcriptional mechanisms . The nuclear distribution of EDS1 is essential for resistance and transcriptional responses, while cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites .

What methods are most effective for tracking EDS1 nucleocytoplasmic shuttling in living cells?

For real-time analysis of EDS1 nucleocytoplasmic shuttling, fluorescence recovery after photobleaching (FRAP) combined with EDS1-GFP (or other fluorescent protein) fusions offers the most comprehensive data. When designing these experiments, consider:

• Using cell-specific promoters rather than constitutive promoters to maintain native-like expression levels

• Complementing eds1 null mutants with the fluorescent fusion to verify functionality

• Employing nuclear and cytoplasmic markers as references

For fixed-cell analysis, immunolocalization using EDS1-specific antibodies combined with cellular fractionation provides quantitative data on the relative distribution of EDS1. During pathogen challenge, perform analyses at multiple timepoints (0, 1, 3, 6, 9, and 24 hours post-infection) to capture the dynamic changes in EDS1 localization .

How do EDS1-interacting proteins affect its subcellular localization?

EDS1-interacting proteins significantly influence its subcellular distribution. For instance, EDS1-interacting J protein 1 (EIJ1) plays a crucial role in regulating EDS1 nuclear accumulation. Upon pathogen infection, EIJ1 relocates from the chloroplast to the cytoplasm, where it interacts with EDS1. This interaction restricts the pathogen-triggered trafficking of EDS1 to the nucleus, thereby compromising resistance at early infection stages. During disease development, EIJ1 is gradually degraded, allowing nuclear accumulation of EDS1 for transcriptional resistance reinforcement. Notably, avirulent pathogens like Pseudomonas syringae DC3000 (AvrRps4) can abolish this repressive action by rapidly inducing EIJ1 degradation during effector-triggered immunity responses .

What are the best conditions for co-immunoprecipitation of EDS1 with its interacting partners?

For optimal co-immunoprecipitation of EDS1 with its interaction partners (PAD4, SAG101, or EIJ1), use the following protocol:

• Extract proteins in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 2 mM EDTA, 5 mM DTT, with 0.1% Triton X-100 (for membrane-associated complex preservation)

• Include protease inhibitor cocktail and phosphatase inhibitors to prevent degradation and maintain post-translational modifications

• Perform extraction at 4°C with gentle agitation for 30 minutes

• Pre-clear lysates with protein A/G beads before immunoprecipitation

• Use mild wash conditions (at least 3 washes) to preserve weaker interactions

When investigating pathogen-induced changes in EDS1 complexes, collect samples at different timepoints after infection to capture the dynamic nature of these interactions, especially noting the 3-hour post-infection timepoint when nuclear EDS1 accumulation increases .

How do the molecular configurations of EDS1-PAD4 and EDS1-SAG101 complexes differ, and how can antibodies help distinguish them?

EDS1 forms molecularly distinct complexes with either PAD4 or SAG101 without additional plant factors. These complexes differ in size and intracellular distribution patterns:

To distinguish these complexes using antibodies:

• Use size exclusion chromatography followed by immunoblotting with EDS1-specific antibodies

• Perform sequential immunoprecipitations with PAD4 and SAG101 antibodies

• Use co-immunofluorescence with differentially labeled antibodies for colocalization studies

Importantly, the EDS1-PAD4 complex is necessary for basal resistance involving transcriptional upregulation of PAD4 itself and mobilization of salicylic acid defenses, while the dissociated forms of EDS1 and PAD4 are fully competent in signaling receptor-triggered localized cell death at infection foci .

How can chromatin immunoprecipitation with EDS1 antibodies help identify its nuclear targets?

While EDS1 is not known to directly bind DNA, it influences transcriptional reprogramming during immune responses. Chromatin immunoprecipitation (ChIP) using EDS1 antibodies can identify genomic regions where EDS1 associates with chromatin-bound transcription factors or regulatory complexes. When designing EDS1 ChIP experiments:

• Use crosslinking conditions optimized for protein-protein (not just protein-DNA) interactions (1-2% formaldehyde for 10-15 minutes)

• Include appropriate controls (eds1 mutants, IgG controls, and input samples)

• Perform ChIP at different timepoints after pathogen infection to capture dynamic interactions

• Consider sequential ChIP (re-ChIP) approaches to identify genomic regions where EDS1 co-localizes with known interacting transcription factors

Analysis should focus on defense-related genes, particularly those involved in salicylic acid signaling, as EDS1 functions upstream of salicylic acid-dependent PR1 mRNA accumulation .

How do post-translational modifications affect EDS1 function and antibody recognition?

Post-translational modifications (PTMs) of EDS1 likely regulate its activity, subcellular localization, and protein interactions. Research suggests that the rapid nuclear accumulation of EDS1 after pathogen challenge (occurring before transcriptional upregulation) is regulated by post-transcriptional mechanisms, possibly including PTMs . When using antibodies to study EDS1:

• Consider generating or using modification-specific antibodies (phospho-EDS1, acetyl-EDS1) for detecting specific modified forms

• Compare antibody recognition patterns in samples treated with phosphatase or deacetylase inhibitors versus untreated controls

• Use 2D gel electrophoresis followed by Western blotting to separate differentially modified EDS1 forms

• For mass spectrometry studies, immunoprecipitate EDS1 at different timepoints during immune responses to identify infection-induced PTMs

Different epitopes may be masked or exposed depending on EDS1's modification state or interaction with partner proteins, potentially affecting antibody recognition .

What are the common issues when detecting EDS1 by immunoblotting and how can they be resolved?

When detecting EDS1 by immunoblotting, researchers commonly encounter several issues:

For optimal results, extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 2 mM EDTA, with complete protease inhibitor cocktail. Remember that EDS1 levels increase approximately 2-3 fold upon pathogen infection or salicylic acid treatment .

How can I resolve inconsistent data between immunolocalization and biochemical fractionation of EDS1?

Discrepancies between immunolocalization and biochemical fractionation results for EDS1 subcellular distribution are not uncommon. To resolve these inconsistencies:

• Validate fractionation purity: Use established markers for each cellular compartment (e.g., histone H3 for nuclei, GAPDH for cytoplasm)

• Optimize fixation conditions: Overfixation can mask epitopes while underfixation can allow protein redistribution

• Consider dynamic redistribution: EDS1 shuttles between compartments, so rapid sample processing is crucial

• Account for extraction bias: Some extraction methods may preferentially release EDS1 from certain compartments

• Check antibody accessibility: Nuclear EDS1 may be associated with chromatin complexes that hinder antibody recognition

If discrepancies persist, quantitative approaches like ratiometric imaging of fluorescently-tagged EDS1 in live cells can provide additional verification. For nuclear EDS1 detection specifically, consider using nuclear export inhibitors (leptomycin B) as positive controls to increase nuclear accumulation .

How might proximity labeling combined with EDS1 antibodies reveal novel interaction partners?

Proximity labeling approaches such as BioID or TurboID fused to EDS1 offer powerful methods to identify transient or context-specific interaction partners. These methods can reveal the dynamic EDS1 interactome during immune responses:

• Generate transgenic plants expressing EDS1-BioID/TurboID in eds1 mutant backgrounds

• Activate immune responses using avirulent pathogens or immune elicitors

• Allow biotin labeling of proximal proteins (1-24 hours depending on the system)

• Purify biotinylated proteins using streptavidin beads

• Identify labeled proteins by mass spectrometry

• Validate candidates using co-immunoprecipitation with EDS1 antibodies

This approach is particularly valuable for identifying components of transcriptional complexes that form with nuclear EDS1 during immunity, potentially revealing how EDS1 influences gene expression without directly binding DNA. Compare labeled proteins from nuclear versus cytoplasmic fractions to understand compartment-specific interactions .

How can quantitative proteomics with EDS1 antibodies help understand temporal dynamics of immunity?

Quantitative proteomics combined with EDS1 immunoprecipitation can reveal the dynamic changes in EDS1 protein complexes during immune responses:

• Perform time-course experiments after pathogen infection (0, 1, 3, 6, 12, 24 hours)

• Immunoprecipitate EDS1 complexes using specific antibodies

• Analyze samples using label-free quantitative proteomics or isobaric tagging (TMT/iTRAQ)

• Generate temporal interaction networks showing protein association changes

• Correlate changes with defense gene expression profiles and resistance phenotypes

This approach can identify when EDS1 associates with different partner proteins and how these associations correlate with the transition from early to late immune responses. For example, tracking the dynamics of EIJ1-EDS1 interaction could reveal how pathogen-triggered EIJ1 degradation allows nuclear accumulation of EDS1 for transcriptional resistance reinforcement .