ERF114 Antibody

What is ERF114 and why is it important in plant immunity research?

ERF114 is a member of the APETALA2/ethylene-responsive factor (AP2/ERF) family of transcription factors that plays a crucial role in plant defense responses. It mediates resistance against various pathogens, particularly fungal pathogens like Verticillium dahliae and bacterial pathogens such as Pseudomonas syringae pv. tomato (Pst) DC3000 . ERF114 functions by directly binding to GCC-box elements in the promoters of defense-related genes, notably phenylalanine ammonia-lyase 1 (PAL1), to activate their transcription . This activation leads to increased lignin and salicylic acid (SA) accumulation, both of which are critical components of plant defense systems . The significance of ERF114 is highlighted by the fact that loss-of-function mutations in erf114 result in increased susceptibility to pathogens, while overexpression enhances resistance .

What experimental evidence confirms ERF114's role in plant immunity pathways?

Multiple experimental approaches have validated ERF114's role in plant immunity. CRISPR/Cas9-generated erf114 knockout mutants show significantly increased susceptibility to Pst DC3000 infection, as demonstrated by higher bacterial growth and more severe disease symptoms compared to wild-type plants . Conversely, overexpression lines (OE-ERF114) display enhanced resistance with reduced bacterial proliferation . Molecular analyses reveal that ERF114 positively regulates the expression of pathogenesis-related genes like PR1 . Additionally, reactive oxygen species (ROS) accumulation, an important defense response, is increased in OE-ERF114 plants but decreased in erf114 mutants following pathogen attack . Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assays (EMSA) have confirmed that ERF114 directly binds to the promoter of PAL1, a key enzyme in phenylpropanoid biosynthesis .

How should I design experiments to validate a new ERF114 antibody?

When validating a new ERF114 antibody, implement a comprehensive experimental design that includes:

• Specificity testing: Compare wild-type plants with erf114 knockout mutants in Western blot analyses. A specific antibody should show a band of appropriate molecular weight (~25-30 kDa) in wild-type samples that is absent or significantly reduced in mutant samples.

• Positive controls: Include samples from OE-ERF114 transgenic plants, which should show enhanced signal intensity compared to wild-type .

• Immunoprecipitation validation: Perform immunoprecipitation followed by mass spectrometry to confirm that the antibody pulls down ERF114 and its known interaction partners.

• Cross-reactivity assessment: Test the antibody against closely related ERF family members to ensure specificity, particularly within the AP2/ERF family.

• Functional validation: Use the antibody in ChIP experiments targeting known ERF114 binding sites, such as the GCC-box elements in the PAL1 promoter, to confirm functionality in chromatin immunoprecipitation applications .

How can I use ERF114 antibodies to investigate protein-DNA interactions during pathogen response?

ERF114 antibodies are valuable tools for examining protein-DNA interactions through chromatin immunoprecipitation approaches:

ChIP-qPCR Protocol for ERF114:

• Cross-link plant tissue (preferably leaves challenged with pathogens) using 1% formaldehyde for 10 minutes under vacuum.

• Extract and sonicate chromatin to achieve fragments of 200-500 bp.

• Immunoprecipitate using ERF114 antibody (5-10 μg per reaction) bound to protein A/G magnetic beads.

• Design primers targeting GCC-box regions in promoters of interest, particularly focusing on regions P1 and P2 in the PAL1 promoter as positive controls .

• Include IgG controls and input samples for normalization.

• Calculate enrichment as percent input or fold enrichment over IgG control.

Based on published research, ERF114 shows significant enrichment at GCC-box elements, particularly in the P1/2 region of the PAL1 promoter following pathogen challenge or elicitor treatment . Time-course experiments following PevD1 treatment or pathogen infection show dynamic binding patterns correlating with transcriptional activation of target genes .

What approaches can help resolve contradictory results between ERF114 transcript and protein levels?

Discrepancies between ERF114 transcript and protein levels may reflect complex post-transcriptional and post-translational regulatory mechanisms. To resolve such contradictions:

• Temporal analysis: Conduct detailed time-course experiments measuring both transcript levels (RT-qPCR) and protein levels (Western blot) following pathogen challenge. ERF114 transcripts are significantly elevated at specific time points post-infiltration with PevD1 or Pst DC3000 infection , but protein accumulation may lag.

• Protein stability assessment: Use cycloheximide chase assays to determine ERF114 protein half-life under different conditions. Transcription factors often exhibit rapid turnover rates.

• Proteasome inhibition: Treat samples with proteasome inhibitors (MG132) to determine if ERF114 is subject to proteasomal degradation.

• Translational regulation: Implement polysome profiling to assess translational efficiency of ERF114 mRNA under different conditions.

• Post-translational modifications: Investigate potential modifications using phosphorylation-specific antibodies or mass spectrometry approaches.

Creating a table comparing transcript and protein levels across different time points and conditions can help identify patterns and potential regulatory mechanisms:

How can I use ERF114 antibodies to study its role in chromatin remodeling during defense responses?

ERF114, as a transcription factor, likely functions within chromatin remodeling complexes to regulate defense gene expression. To investigate this aspect:

• Sequential ChIP (Re-ChIP): Perform initial ChIP with ERF114 antibody followed by a second round using antibodies against histone modifications (H3K27ac, H3K4me3) or chromatin remodelers to identify co-localization.

• ChIP-seq combined with ATAC-seq: Compare ERF114 binding sites (ChIP-seq) with changes in chromatin accessibility (ATAC-seq) following pathogen challenge to identify regions where ERF114 binding correlates with chromatin opening.

• Co-immunoprecipitation followed by mass spectrometry: Use ERF114 antibodies to pull down associated chromatin modifiers and remodelers from nuclear extracts of pathogen-challenged plants.

• Biochemical fractionation: Separate chromatin into loosely and tightly bound fractions to determine whether ERF114 associates preferentially with euchromatic regions.

• Immunofluorescence microscopy: Use fluorescently tagged ERF114 antibodies to visualize nuclear localization patterns and potential association with chromatin domains during pathogen response.

What are the optimal conditions for Western blot detection of ERF114?

Detecting transcription factors like ERF114 by Western blot requires optimized conditions:

Recommended Western Blot Protocol for ERF114 Detection:

• Sample preparation:

  • Extract nuclear proteins using a specialized nuclear extraction buffer (20 mM HEPES pH 7.5, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, protease inhibitor cocktail).

  • Include phosphatase inhibitors to preserve potential phosphorylation states.

  • Concentrate samples if necessary using TCA precipitation or similar methods.

• Gel electrophoresis:

  • Use 12% SDS-PAGE gels for optimal resolution of proteins in the 25-30 kDa range.

  • Load at least 50 μg of nuclear protein extract per lane.

  • Include positive controls from OE-ERF114 plants .

• Transfer conditions:

  • Use PVDF membrane (0.45 μm) with semi-dry transfer at 15V for 30 minutes.

  • Verify transfer efficiency with Ponceau S staining.

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

  • Incubate with ERF114 primary antibody (1:1000 dilution) overnight at 4°C.

  • Wash extensively (4 × 10 minutes) with TBST.

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature.

• Detection:

  • Use enhanced chemiluminescence with extended exposure times (up to 5 minutes) as needed .

  • For weak signals, consider using signal enhancement systems or more sensitive detection methods.

How should I optimize immunohistochemistry protocols for detecting ERF114 in plant tissues?

Immunohistochemical detection of transcription factors in plant tissues presents unique challenges due to low abundance and potential epitope masking. For optimal results:

• Tissue fixation and embedding:

  • Fix freshly harvested tissue in 4% paraformaldehyde for 2-4 hours under vacuum.

  • For better penetration, consider using a combination of paraformaldehyde and glutaraldehyde (4%/0.1%).

  • Dehydrate and embed in paraffin or resin, with paraffin preferred for better antigen preservation.

• Antigen retrieval:

  • Crucial step for transcription factor detection.

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) at 95°C for 20 minutes.

  • Allow slow cooling to room temperature (approximately 30 minutes).

• Blocking and antibody incubation:

  • Block with 2% BSA, 5% normal serum (matching secondary antibody host), and 0.3% Triton X-100 in PBS for 2 hours.

  • Incubate with ERF114 primary antibody (1:100-1:200) for 48 hours at 4°C.

  • Wash extensively (6 × 10 minutes) with PBS containing 0.1% Tween-20.

  • Incubate with fluorescently labeled secondary antibody (1:500) for 2 hours at room temperature.

• Controls and counterstaining:

  • Include erf114 mutant tissues as negative controls .

  • Counterstain nuclei with DAPI (1 μg/mL) for 10 minutes.

  • Consider dual staining with antibodies against nuclear markers for co-localization studies.

• Imaging and analysis:

  • Use confocal microscopy with appropriate filter sets.

  • Capture Z-stacks to ensure complete visualization of nuclear signals.

  • Perform quantitative analysis of nuclear signal intensity across different cell types and treatments.

What are the best practices for quantifying ERF114 protein expression changes during pathogen infection?

Accurate quantification of ERF114 protein levels during pathogen infection requires careful experimental design and analysis:

• Experimental design considerations:

  • Establish a detailed time course following pathogen inoculation (0, 3, 6, 12, 24, 48, 72 hours).

  • Include both local (infected) and systemic (uninfected) tissues to assess systemic signaling.

  • Compare responses to different pathogens (e.g., Pst DC3000, Verticillium dahliae) and elicitors (e.g., PevD1) .

  • Always include appropriate controls: mock-treated plants, resistant and susceptible genotypes.

• Protein extraction optimization:

  • Use nuclear fractionation to enrich for transcription factors.

  • Standardize tissue collection, with equal leaf areas or weights across all samples.

  • Process all samples simultaneously to minimize technical variation.

• Quantification methods:

  • Use digital imaging systems with linear detection range .

  • Include standard curves with recombinant ERF114 protein if available.

  • Normalize to multiple reference proteins (nuclear markers like histone H3).

  • Implement technical replicates (at least 3) and biological replicates (at least 3).

• Data analysis approaches:

  • Apply appropriate statistical tests (ANOVA followed by post-hoc tests).

  • Use specialized software for densitometric analysis.

  • Present data as fold changes relative to time zero or mock-treated controls.

  • Create visualization that shows both the representative Western blot image and the quantification graph.

How can I design experiments to investigate ERF114's role in different plant species?

ERF114 orthologs exist across plant species with potentially conserved and divergent functions. For comparative functional studies:

• Ortholog identification:

  • Perform phylogenetic analysis of ERF family members across target species.

  • Confirm orthology through sequence alignment focusing on the conserved AP2/ERF domain.

  • Determine whether antibodies raised against Arabidopsis ERF114 cross-react with orthologs in other species.

• Expression analysis:

  • Compare expression patterns of ERF114 orthologs in response to pathogens across species.

  • ERF114 orthologs like NbERF114 in Nicotiana benthamiana show strong induction by PevD1 infiltration, similar to Arabidopsis ERF114 .

  • Determine if expression patterns correlate with species-specific disease resistance phenotypes.

• Functional complementation:

  • Express ERF114 orthologs from different species in Arabidopsis erf114 mutants.

  • Assess restoration of disease resistance to Pst DC3000 .

  • Quantify complementation by measuring bacterial growth, PR gene expression, and lignin/SA accumulation.

• Target gene conservation:

  • Perform ChIP-qPCR using ERF114 antibodies in different plant species.

  • Focus on promoters of conserved targets like PAL1 .

  • Compare binding affinities and transcriptional activation potentials across species.

What approaches can help distinguish ERF114's direct vs. indirect effects on defense pathways?

Distinguishing direct from indirect effects of ERF114 on defense pathways requires sophisticated experimental strategies:

• Inducible expression systems:

  • Utilize estradiol-inducible ERF114 expression systems, similar to the pER8-ERF114 constructs .

  • Perform time-course experiments following induction (0.5h, 1h, 3h) to identify immediate-early targets versus later responses.

  • Compare expression profiles with and without protein synthesis inhibitors (cycloheximide) to identify direct transcriptional targets.

• Genome-wide binding site identification:

  • Perform ChIP-seq using ERF114 antibodies following pathogen challenge.

  • Integrate with RNA-seq data to correlate binding events with transcriptional changes.

  • Focus analysis on genes with both ERF114 binding and rapid expression changes.

• Promoter analysis:

  • Conduct systematic mutagenesis of GCC-box elements in target promoters like PAL1 .

  • Use reporter gene assays to quantify the impact of mutations on ERF114-mediated activation.

  • Develop synthetic promoters with varying numbers and arrangements of GCC-boxes to define minimal requirements for ERF114 responsiveness.

• Protein-protein interaction mapping:

  • Identify ERF114 cofactors using proximity labeling approaches like BioID.

  • Determine if ERF114 functions within larger transcriptional complexes.

  • Assess whether ERF114 recruits chromatin modifiers to target promoters.

How can I investigate potential post-translational modifications of ERF114 during immune responses?

Post-translational modifications (PTMs) likely regulate ERF114 activity during immune responses. To investigate these modifications:

• Phosphorylation analysis:

  • Treat plant samples with phosphatase inhibitors during protein extraction.

  • Use Phos-tag gels to separate phosphorylated from non-phosphorylated forms.

  • Perform immunoprecipitation with ERF114 antibodies followed by phospho-specific antibody detection.

  • For comprehensive analysis, use mass spectrometry to identify specific phosphorylation sites.

• Ubiquitination and SUMOylation studies:

  • Co-immunoprecipitate ERF114 from plants treated with proteasome inhibitors.

  • Probe Western blots with anti-ubiquitin or anti-SUMO antibodies.

  • Perform in vitro ubiquitination/SUMOylation assays with purified components.

  • Create lysine-to-arginine mutants at predicted modification sites and test functional consequences.

• Stability and turnover analysis:

  • Conduct cycloheximide chase experiments to determine ERF114 half-life.

  • Compare protein stability under normal conditions versus pathogen challenge.

  • Assess whether PTMs alter protein stability or subcellular localization.

• Functional consequences of modifications:

  • Generate phosphomimetic (S/T to D/E) and phospho-null (S/T to A) mutants of predicted sites.

  • Test these variants for altered DNA binding ability using EMSA .

  • Assess transcriptional activation potential using reporter gene assays.

  • Determine if modifications affect protein-protein interactions or subcellular localization.

How does ERF114 coordinate with other transcription factors during plant immune responses?

ERF114 functions within a complex transcriptional network regulating plant immunity. To understand these interactions:

• Co-expression network analysis:

  • Perform time-course RNA-seq following pathogen challenge in wild-type and erf114 mutants .

  • Identify transcription factors with similar or antagonistic expression patterns.

  • Construct co-expression networks highlighting potential regulatory relationships.

• Hierarchical relationships:

  • Use inducible expression systems to determine if ERF114 regulates other transcription factors.

  • Create double mutants between erf114 and other immunity-related transcription factor mutants.

  • Assess epistatic relationships by analyzing disease phenotypes and defense marker expression.

• Promoter occupation studies:

  • Conduct sequential ChIP experiments to identify co-occupation of promoters by multiple transcription factors.

  • Compare ERF114 binding sites with those of other ERF family members to identify unique and shared targets.

  • Investigate potential antagonistic or synergistic relationships at co-occupied promoters.

• Protein-protein interactions:

  • Perform yeast two-hybrid or split-luciferase complementation assays to identify direct interactions with other transcription factors.

  • Use co-immunoprecipitation with ERF114 antibodies followed by mass spectrometry to identify interacting partners in planta.

  • Investigate whether these interactions are constitutive or induced by pathogen challenge.

What methods can help decipher ERF114's role in systemic acquired resistance?

Systemic acquired resistance (SAR) involves long-distance signaling from infected to uninfected tissues. To investigate ERF114's role in SAR:

• Split-leaf experiments:

  • Inoculate one half of a leaf with Pst DC3000 and collect the other half at various time points.

  • Compare ERF114 protein levels and binding activity in local versus distal tissues.

  • Assess if ERF114-dependent genes like PAL1 are activated in systemic tissues .

• Grafting approaches:

  • Generate grafted plants with different combinations of wild-type, erf114 mutant, and OE-ERF114 rootstocks and scions .

  • Challenge rootstocks with pathogens and assess SAR responses in scions.

  • Determine if ERF114 is required in local tissues, systemic tissues, or both for effective SAR.

• Mobile signal identification:

  • Analyze phloem sap composition from wild-type and erf114 plants following pathogen challenge.

  • Focus on known SAR signals (methyl salicylate, pipecolic acid, glycerol-3-phosphate) and their precursors.

  • Determine if ERF114 regulates genes involved in synthesis or perception of these mobile signals.

• Systemic chromatin dynamics:

  • Perform ChIP-seq for ERF114 in both local and systemic tissues following pathogen challenge.

  • Analyze whether ERF114 binding patterns differ between these tissues.

  • Investigate if ERF114-mediated chromatin changes in local tissues can be transmitted to or recapitulated in systemic tissues.

How can I design experiments to assess ERF114's contribution to broad-spectrum disease resistance?

ERF114 appears to confer resistance against diverse pathogens, suggesting a role in broad-spectrum immunity. To evaluate this:

• Pathogen panel testing:

  • Challenge wild-type, erf114 mutant, and OE-ERF114 plants with diverse pathogens beyond those already tested :

    • Additional bacterial pathogens (different Pseudomonas strains, Xanthomonas)

    • Fungal pathogens (Botrytis cinerea, Alternaria species)

    • Oomycetes (Phytophthora, Pythium)

    • Viral pathogens (mosaic viruses)

  • Quantify disease progression and severity across pathogen types.

• Mechanistic investigations:

  • Determine if ERF114-regulated defenses (lignin, SA) are effective against diverse pathogens .

  • Investigate whether ERF114 activates distinct sets of genes in response to different pathogen classes.

  • Assess if ERF114 interacts with different co-factors depending on the challenging pathogen.

• Cross-protection experiments:

  • Pre-treat plants with PevD1 to induce ERF114 expression .

  • Challenge with various pathogens to determine the spectrum of PevD1/ERF114-mediated protection.

  • Use gene expression analysis to identify common and pathogen-specific responses.

• Combinatorial approaches:

  • Create double mutants between erf114 and other immunity pathway components.

  • Test for additive, synergistic, or redundant effects on disease resistance.

  • Develop plants with engineered ERF114 variants (constitutively active, tissue-specific expression) to enhance broad-spectrum resistance.