ERF019 Antibody

What is ERF019 and why is it important to study with antibody-based techniques?

ERF019 belongs to the ethylene-responsive factor (ERF)/APETALA2 (AP2) transcription factor family in Arabidopsis thaliana. This protein contains an AP2 domain characteristic of transcription factors and requires nuclear localization to fulfill its function in promoting pathogen susceptibility. ERF019 has been identified as a negative regulator of plant immunity against both oomycete pathogens like Phytophthora parasitica and bacterial pathogens such as Pseudomonas syringae .

Antibody-based detection of ERF019 is crucial because this protein exhibits dynamic expression patterns during infection, with transcripts peaking around 3 hours post-inoculation and declining thereafter . Using antibodies provides direct evidence of protein presence, localization, and potential post-translational modifications that cannot be inferred from transcript analysis alone. Additionally, ERF019's dual localization in both nuclear and cytoplasmic compartments makes antibody-based visualization particularly valuable for understanding its regulatory mechanisms.

What are the challenges in developing specific antibodies against ERF019?

Developing specific antibodies against ERF019 presents several technical challenges researchers should consider. First, ERF019 belongs to the ERF transcription factor family, which comprises numerous members with highly conserved AP2/ERF domains. This sequence similarity creates potential cross-reactivity issues that must be addressed by targeting antibody production against unique N-terminal or C-terminal regions rather than the conserved DNA-binding domain .

Second, transcription factors like ERF019 are typically expressed at relatively low abundance in plant tissues, with transient expression patterns that further complicate detection. This necessitates developing high-affinity antibodies with good detection limits. Third, post-translational modifications likely affect ERF019 function, as research suggests involvement in MAPK signaling cascades that typically involve phosphorylation events . These modifications may alter epitope accessibility or recognition, requiring careful consideration during antibody development and validation.

A thorough validation strategy involving wild-type plants, ERF019 overexpression lines, and erf019 knockout mutants (such as the 267-31 mutant or CRISPR/Cas9-edited lines described in the literature) is essential to confirm antibody specificity before use in complex experimental setups .

How should researchers prepare plant samples for optimal ERF019 detection?

For effective detection of ERF019 protein in plant samples, researchers should consider the protein's nuclear localization and temporal expression pattern. When preparing samples for Western blotting or immunoprecipitation:

• Timing is critical: Harvest tissues 1-3 hours after pathogen or MAMP treatment when ERF019 expression peaks based on transcript studies .

• Utilize nuclear extraction protocols:

  • Grind tissue in liquid nitrogen to a fine powder

  • Use a nuclear protein extraction buffer containing:

    • 50 mM HEPES-KOH (pH 7.5)

    • 400-500 mM KCl (high salt for nuclear proteins)

    • 5 mM MgCl₂

    • 10% glycerol

    • 1 mM DTT

    • Protease and phosphatase inhibitor cocktails

• Consider protein stability and modifications:

  • Include phosphatase inhibitors to preserve potential post-translational modifications

  • Process samples quickly at 4°C to minimize degradation

  • Use freshly prepared samples, as ERF019 might be subject to rapid turnover

• For immunolocalization studies:

  • Fix tissues with 4% paraformaldehyde to preserve protein antigenicity

  • Include proper permeabilization steps to allow antibody access to nuclear proteins

  • Use nuclear counterstains (like DAPI) to confirm nuclear localization

Importantly, always include appropriate genetic controls in your experiments, particularly the erf019 mutant lines like 267-31 or CRISPR-generated knockouts as negative controls, and ERF019 overexpression lines as positive controls .

How can researchers use ERF019 antibodies for chromatin immunoprecipitation studies?

Chromatin immunoprecipitation (ChIP) using ERF019 antibodies enables researchers to identify direct genomic targets of this transcription factor, providing crucial insights into its role in regulating defense responses. An optimized ChIP protocol for ERF019 involves:

• Sample preparation:

  • Harvest Arabidopsis leaf tissue 1-3 hours after pathogen/MAMP treatment (when ERF019 is maximally expressed)

  • Crosslink proteins to DNA by vacuum infiltration with 1% formaldehyde for 10 minutes

  • Quench with glycine and isolate nuclei

  • Sonicate chromatin to generate 200-500 bp DNA fragments

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate with ERF019 antibody overnight at 4°C

  • Include a negative control using non-specific IgG or pre-immune serum

  • Wash thoroughly and elute chromatin

  • Reverse crosslinks and purify DNA

• Analysis strategies:

  • Perform qPCR analysis of known or predicted ERF019 target promoters

  • Include defense-related genes like PR1, ICS1, VSP2, LOX2, and FRK1, which have been shown to be differentially expressed in erf019 mutants

  • For genome-wide analysis, prepare libraries for ChIP-seq

  • Analyze data using appropriate peak-calling software and motif enrichment analysis

Since research has shown that ERF019 affects the expression of multiple defense marker genes, ChIP experiments can help determine whether this regulation is direct (through ERF019 binding to promoters) or indirect (through intermediate factors) . The presence of GCC-box elements, which are commonly bound by ERF transcription factors, should be examined in identified target promoters.

What methods are recommended for studying ERF019 protein-protein interactions?

Understanding ERF019's protein interaction network is crucial for elucidating its role in immunity suppression. Several complementary approaches using ERF019 antibodies are recommended:

• Co-immunoprecipitation (Co-IP):

  • Perform immunoprecipitation with ERF019 antibodies from plant tissues treated with pathogen or MAMPs

  • Identify interacting proteins by Western blotting (for known candidates) or mass spectrometry (for unbiased discovery)

  • Include appropriate controls such as IgG-only IP and samples from erf019 knockout plants

  • Consider crosslinking (1% formaldehyde, 10 min) to capture transient interactions

• Proximity-based approaches:

  • Express ERF019 fused to proximity labeling enzymes (BioID or TurboID)

  • After biotin labeling, use ERF019 antibodies to confirm expression and localization

  • Compare interaction profiles between mock and pathogen-treated conditions

• In vitro validation methods:

  • Express recombinant ERF019 and candidate interactors

  • Perform pull-down assays using ERF019 antibodies

  • Use ERF019 antibodies to detect interactions in far-Western blot analyses

Research has shown that ERF019 influences multiple defense signaling pathways, including salicylic acid and jasmonic acid pathways . Protein interaction studies can help establish mechanisms by which ERF019 interfaces with these pathways, potentially identifying additional regulatory components that could be targeted to enhance plant immunity.

How can ERF019 antibodies help detect post-translational modifications during immune responses?

ERF019 antibodies are essential tools for investigating post-translational modifications (PTMs) that may regulate this transcription factor's activity during immune responses. Since ERF019 functions within PAMP-triggered signaling cascades involving MAPK activation, phosphorylation events are particularly relevant :

• Phosphorylation analysis:

  • Perform immunoprecipitation using ERF019 antibodies from both mock and pathogen-treated samples

  • Analyze immunoprecipitated protein using:

    • Phospho-specific Western blotting with phospho-serine/threonine antibodies

    • Phos-tag SDS-PAGE to detect mobility shifts caused by phosphorylation

    • Mass spectrometry to identify specific modified residues

• Experimental approach to identify MAPK-mediated phosphorylation:

  • Treat plants with MAPK inhibitors before pathogen challenge

  • Immunoprecipitate ERF019 and analyze phosphorylation status

  • Perform in vitro kinase assays with purified MAPKs and immunoprecipitated ERF019

• Functional validation of modifications:

  • Generate phospho-mimetic or phospho-dead mutations in ERF019 at identified sites

  • Express these variants in erf019 mutant backgrounds

  • Use the ERF019 antibody to assess their subcellular localization

  • Evaluate their impact on defense responses and pathogen susceptibility

Research has demonstrated that flg22-induced MAPK activation was enhanced in erf019 mutants, suggesting a potential regulatory relationship between MAPK signaling and ERF019 function . Characterizing PTMs on ERF019 during infection could provide mechanistic insights into how this transcription factor negatively regulates immunity.

What controls should be included when validating ERF019 antibody specificity?

Rigorous validation of ERF019 antibody specificity is essential for generating reliable research data. A comprehensive approach using available genetic resources should include:

• Genetic validation using multiple plant lines:

  • Wild-type plants (positive control)

  • T-DNA insertion mutants (e.g., 267-31 with reduced ERF019 expression)

  • CRISPR/Cas9-generated knockout lines (complete absence of protein)

  • ERF019 overexpression lines (increased signal)

• Molecular weight confirmation:

  • Compare observed molecular weight with predicted size

  • Use epitope-tagged ERF019 expressed in plants as a size reference

  • Perform peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific signals

• Cross-reactivity assessment:

  • Test against closely related ERF family members expressed recombinantly

  • Perform immunoprecipitation followed by mass spectrometry to identify all proteins pulled down

  • Check for cross-reactivity in plant species where ERF019 homologs have divergent sequences

• Immunohistochemistry specificity controls:

  • Compare immunofluorescence patterns between wild-type and knockout plants

  • Perform parallel staining with secondary antibody only

  • Use pre-immune serum as a negative control

This comprehensive validation approach ensures that observations made using ERF019 antibodies can be confidently attributed to the specific detection of ERF019 protein .

What experimental controls are essential for ERF019 antibody applications?

When using ERF019 antibodies in different experimental applications, specific controls should be incorporated to ensure reliable interpretation of results:

• Western blotting controls:

  • Positive control: Include protein extract from ERF019 overexpression plants

  • Negative control: Include protein extract from erf019 knockout mutants

  • Loading control: Probe with antibodies against housekeeping proteins

  • Nuclear fraction quality control: Probe with anti-histone H3 antibody

• Immunoprecipitation controls:

  • Input sample: Save 5-10% of pre-IP lysate

  • IgG control: Perform parallel IP with species-matched non-specific IgG

  • Beads-only control: Process sample without antibody addition

  • Knockout control: Perform IP using erf019 knockout plant material

• Immunofluorescence controls:

  • Secondary antibody only: Omit primary antibody incubation step

  • Genetic control: Compare wild-type with erf019 knockout tissue

  • Co-localization: Use markers for nuclear (H2B-RFP) and cytoplasmic compartments

• ChIP controls:

  • Input sample: Reserve 5-10% of pre-IP chromatin

  • IgG control: Perform ChIP with non-specific IgG

  • Negative genomic region: Analyze regions without predicted binding sites

  • No-antibody control: Process without adding antibody

• Physiological controls when studying ERF019 function:

  • Compare responses in wild-type, erf019 mutant, and ERF019-OE plants

  • Include appropriate mock treatments for PAMP/MAMP treatments

  • Sample multiple timepoints to capture transient changes

What is the optimal protein extraction protocol for ERF019 detection by Western blot?

Detecting transcription factors like ERF019 by Western blot requires careful consideration of extraction conditions. Here is an optimized nuclear protein extraction protocol:

• Buffers required:

  • Nuclei isolation buffer:

    • 10 mM HEPES-KOH (pH 7.5)

    • 10 mM MgCl₂

    • 10 mM KCl

    • 0.5 M sucrose

    • 10 mM β-mercaptoethanol (add fresh)

    • Protease and phosphatase inhibitors

  • Nuclear lysis buffer:

    • 50 mM HEPES-KOH (pH 7.5)

    • 400 mM KCl (high salt for nuclear proteins)

    • 5 mM MgCl₂

    • 10% glycerol

    • 1 mM DTT (add fresh)

    • Protease and phosphatase inhibitors

• Extraction procedure:

  • Grind leaf tissue to a fine powder in liquid nitrogen

  • Add cold nuclei isolation buffer

  • Filter through Miracloth

  • Centrifuge filtrate at 3,000 × g for 10 min at 4°C

  • Purify nuclei through sucrose gradient centrifugation

  • Extract nuclear proteins with nuclear lysis buffer

  • Determine protein concentration

• Western blotting recommendations:

  • Load 30-50 μg of nuclear protein per lane

  • Use 10% SDS-PAGE for optimal resolution

  • Transfer to PVDF membrane (better for low-abundance proteins)

  • Block with 5% BSA (preferred over milk for phosphorylated proteins)

  • Include wild-type, erf019 mutant, and ERF019-OE samples as controls

  • Probe with anti-histone H3 antibody as a nuclear loading control

This protocol is specifically optimized for nuclear transcription factors like ERF019 and considers the protein's dual nuclear-cytoplasmic localization. Timing sample collection properly (1-3 hours after pathogen treatment) is crucial given the transient expression pattern observed in transcript studies .

What are the recommended protocols for ERF019 immunoprecipitation?

When performing immunoprecipitation (IP) with ERF019 antibodies, researchers should consider the following optimized protocol to capture this transcription factor and its interaction partners:

• Sample preparation:

  • Harvest plant tissue 3-6 hours after pathogen or MAMP treatment (when ERF019 expression peaks)

  • Grind tissue in liquid nitrogen to a fine powder

  • Extract proteins using a nuclear protein extraction buffer containing:

    • 50 mM Tris-HCl (pH 7.5)

    • 150 mM NaCl

    • 5 mM EDTA

    • 0.1% Triton X-100

    • 10% glycerol

    • Protease and phosphatase inhibitors

  • Clear lysate by centrifugation

• Immunoprecipitation procedure:

  • Pre-clear lysate with protein A/G beads (1 hour, 4°C)

  • Incubate pre-cleared lysate with ERF019 antibody (4-5 μg per 1 mg total protein)

  • Allow antibody binding overnight at 4°C with gentle rotation

  • Add pre-washed protein A/G beads and incubate for 2-3 hours at 4°C

  • Wash beads 4-5 times with wash buffer

  • Elute bound proteins with SDS sample buffer or by specific peptide competition

• Important considerations:

  • For transcription factors like ERF019, include a DNase treatment step if planning mass spectrometry analysis

  • Consider crosslinking (1% formaldehyde, 10 min) to capture transient interactions

  • For studying ERF019 interactome changes during infection, perform parallel IPs from mock-treated and pathogen-infected tissues

This protocol can be adapted for different downstream applications, including co-immunoprecipitation to identify interaction partners, mass spectrometry analysis to identify post-translational modifications, or assessing DNA-binding activity through ChIP procedures .

What are the recommended fixation methods for ERF019 immunolocalization in plant tissues?

Proper fixation is critical for successful immunolocalization of ERF019, particularly given its dual nuclear-cytoplasmic localization and possible dynamic changes during pathogen infection. Below are optimized fixation protocols:

• Standard paraformaldehyde fixation for leaf tissues:

  • Prepare 4% paraformaldehyde in PBS (pH 7.4)

  • Vacuum infiltrate leaf pieces for 15-20 minutes

  • Incubate in fixative for 2 hours at room temperature

  • Wash in PBS

  • Proceed with either paraffin embedding or cryosectioning

• Farmer's fixative for maintaining nuclear integrity:

  • Prepare 3:1 ethanol:acetic acid solution (freshly made)

  • Vacuum infiltrate tissues for 15 minutes

  • Incubate for 4 hours at 4°C

  • Wash in 70% ethanol, then PBS

  • Optimal for subsequent paraffin embedding

• Cryofixation for preserving dynamic protein states:

  • Dip tissue in optimal cutting temperature (OCT) compound

  • Freeze rapidly in isopentane cooled with liquid nitrogen

  • Cut 10-15 μm sections on cryostat

  • Fix sections briefly in 4% paraformaldehyde before immunostaining

• Immunostaining procedure optimized for ERF019:

  • Block with 5% BSA, 0.3% Triton X-100 in PBS

  • Incubate with ERF019 primary antibody (1:200 dilution) overnight at 4°C

  • Wash thoroughly

  • Incubate with fluorophore-conjugated secondary antibody

  • Counterstain nuclei with DAPI

  • Mount in anti-fade mounting medium

Selecting the appropriate fixation method depends on the specific research question. For studying dynamic changes in ERF019 localization during pathogen infection, cryofixation of samples collected at multiple time points after infection would be most appropriate. For detailed analysis of nuclear versus cytoplasmic distribution, 4% paraformaldehyde fixation provides a good balance of signal preservation in both compartments .

How do ERF019 protein levels correlate with transcriptional changes during pathogen invasion?

Understanding the relationship between ERF019 protein abundance and transcriptional changes during pathogen infection provides insights into its role as a negative regulator of immunity:

• Temporal correlation analysis:
  Research shows that ERF019 transcript levels are transiently induced early during pathogen infection (peaking at ~3 hours post-inoculation) and then decline. To correlate this with protein levels:

  • Perform time-course Western blot analysis using ERF019 antibodies (0, 1, 3, 6, 12, 24 hours post-infection)

  • In parallel, measure transcript levels of ERF019 target genes using RT-qPCR

  • Analyze how changes in ERF019 protein abundance precede or coincide with alterations in defense gene expression

• Quantitative correlation in different genetic backgrounds:

  • Compare ERF019 protein levels across wild-type, ERF019 overexpression, and erf019 mutant plants

  • Measure expression of defense marker genes in each genotype:

    • SA pathway: ICS1, PR1

    • JA pathway: VSP2, LOX2, PDF1.2

    • PTI markers: FRK1

• Protein-chromatin association during infection:

  • Perform ChIP using ERF019 antibodies at different timepoints after pathogen challenge

  • Quantify ERF019 occupancy at promoters of defense genes

  • Correlate occupancy with transcriptional repression or activation

How can ERF019 antibodies help elucidate its role in PAMP-triggered immunity?

ERF019 has been shown to suppress pattern-triggered immunity (PTI) responses, and antibodies against this protein can help elucidate the mechanisms involved:

• MAPK signaling analysis:

  • Research has shown that flg22-induced MAPK activation was enhanced in erf019 mutants

  • Using ERF019 antibodies, researchers can:

    • Immunoprecipitate ERF019 and test for interactions with MAPK pathway components

    • Examine if ERF019 is a direct substrate of MAPKs through phosphorylation analysis

    • Investigate how ERF019 phosphorylation status changes during PTI activation

• ROS production studies:

  • ERF019 overexpression lines showed impaired flg22-induced accumulation of hydrogen peroxide

  • ERF019 antibodies can help determine if:

    • ERF019 directly interacts with components of the NADPH oxidase complex

    • ERF019's subcellular localization changes during ROS production

    • ERF019 undergoes modifications in response to oxidative conditions

• Transcriptional regulation:

  • ERF019 appears to suppress expression of defense-related genes

  • ChIP experiments using ERF019 antibodies can identify:

    • Direct binding of ERF019 to promoters of PTI-related genes

    • Potential recruitment of transcriptional co-repressors

    • Changes in chromatin structure at target loci

• Connection to hormone signaling:

  • RT-qPCR analyses showed that expression of marker genes for multiple defense pathways was significantly up-regulated in erf019 mutants

  • Using ERF019 antibodies, researchers can investigate:

    • If ERF019 interacts with hormone signaling components

    • How hormone treatments affect ERF019 protein stability or modifications

    • Whether ERF019 functions as part of larger transcriptional complexes in hormone-responsive promoters

Understanding these mechanisms can provide insights into how plants regulate the balance between immunity and growth, potentially leading to strategies for enhancing crop resistance without fitness penalties .