ERF13 Antibody

What is ERF13 and why is it important in plant research?

ERF13 is an Arabidopsis transcription factor belonging to the B-3 ERF family subgroup (reclassified to group IX in Nakano et al., 2006). It plays a critical role in mediating plant responses to both biotic and abiotic stresses. Research has demonstrated that ERF13 acts as a negative regulator of defense against Pseudomonas syringae, with overexpression inducing susceptibility to bacterial pathogens . Additionally, ERF13 inhibits lateral root emergence by suppressing the expression of 3-KETOACYL-COA SYNTHASEs (KCSs) . Its position at the junction of multiple hormone signaling pathways (SA, JA, ET, and ABA) makes it a crucial component in understanding how plants coordinate defense responses and development .

What types of ERF13 antibodies are most commonly used in plant research?

While the search results don't specifically mention antibody types for ERF13, typical antibodies used in plant research include:

• Polyclonal antibodies: Used for general detection of ERF13 protein

• Monoclonal antibodies: Provide higher specificity for particular epitopes

• Phospho-specific antibodies: Especially valuable for detecting phosphorylated ERF13 at S168, which increases during early effector-triggered immunity

For ERF13 research, antibodies that can distinguish between phosphorylated and non-phosphorylated forms are particularly valuable, as phosphorylation at S168 has been shown to increase during RPM1-mediated effector-triggered immunity .

How can I verify the specificity of an ERF13 antibody?

To verify ERF13 antibody specificity:

• Run Western blots comparing wild-type plants with erf13 mutants or knock-down lines

• Include positive controls using tissue from ERF13 overexpression lines (e.g., Pro35S:GFP-ERF13)

• Perform pre-absorption tests with recombinant ERF13 protein

• Test cross-reactivity with closely related ERF family members, particularly those with high sequence homology

• Validate with orthogonal methods such as mass spectrometry identification of immunoprecipitated proteins

This validation is critical as ERF13 shares high sequence homology with other defense-related ERFs, which could lead to cross-reactivity .

How can ERF13 antibodies be used to study phosphorylation dynamics during plant immune responses?

ERF13 undergoes phosphorylation at S168 during early effector-triggered immunity . To study these dynamics:

• Use phospho-specific antibodies that recognize ERF13 phosphorylated at S168

• Implement time-course experiments following pathogen challenge or PAMP treatment

• Compare phosphorylation patterns between compatible and incompatible plant-pathogen interactions

• Use phosphatase treatments as controls to confirm specificity of phospho-antibodies

• Combine with kinase inhibitor treatments to identify responsible kinases

Proteome profiling has shown that S168-phosphorylated ERF13 increases concurrent with early effector-triggered immunity mediated by the R protein RPM1 . This approach can reveal how post-translational modifications regulate ERF13 function during immune responses.

What approaches can be used to study ERF13 degradation mechanisms using antibodies?

Research has shown that ERF13 is degraded through a ubiquitin-proteasome pathway involving the E3 ligases MAC3A and MAC3B . To study this process:

• Perform time-course experiments with auxin treatment and use antibodies to track ERF13 protein levels

• Compare degradation kinetics between wild-type plants and mac3a mac3b mutants

• Implement proteasome inhibitors (e.g., MG132) to prevent degradation

• Use co-immunoprecipitation with anti-FLAG antibodies to detect ubiquitinated forms of ERF13

• Design pulse-chase experiments to measure half-life of ERF13 protein in different genetic backgrounds

The following table summarizes observed degradation patterns of ERF13 variants:

These approaches reveal that auxin promotes ERF13 degradation through phosphorylation-dependent recruitment of MAC3A and MAC3B .

How can chromatin immunoprecipitation (ChIP) with ERF13 antibodies help identify target genes?

ChIP with ERF13 antibodies can reveal direct transcriptional targets:

• Fix plant tissue with formaldehyde to crosslink DNA-protein complexes

• Shear chromatin to appropriate fragment sizes (typically 200-500 bp)

• Immunoprecipitate with ERF13-specific antibodies

• Purify and analyze bound DNA by qPCR or sequencing

• Compare binding profiles during normal conditions versus pathogen challenge or hormone treatments

Potential targets to examine include JA/ET-inducible genes such as PDF1.2a, which has been shown to be stimulated by ERF13 overexpression . Additionally, KCS genes, which are suppressed by ERF13 during lateral root development, would be valuable targets to confirm direct binding .

What are the essential controls for immunoprecipitation experiments with ERF13 antibodies?

When performing immunoprecipitation (IP) with ERF13 antibodies:

• Input control: Reserve a portion of pre-cleared lysate before IP

• Negative control: Perform parallel IP with pre-immune serum or IgG from the same species

• Genetic control: Include erf13 mutant or knockdown lines

• Specificity control: Pre-absorb antibody with recombinant ERF13 protein

• Technical control: Use tagged versions (e.g., ERF13-MYC) and perform parallel IP with tag-specific antibodies

IP experiments have successfully identified ERF13 interacting partners, including the E3 ubiquitin ligases MAC3A and MAC3B, using Pro35S:ERF13-MYC seedlings coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS) .

How should experiments be designed to study ERF13 phosphorylation in response to pathogen infection?

To study ERF13 phosphorylation dynamics:

• Design a time-course experiment with samples collected at multiple timepoints post-infection

• Include both virulent and avirulent pathogen strains (e.g., Pst DC3000 with and without AvrRpm1)

• Use phospho-specific antibodies targeting S168 of ERF13

• Implement controls with kinase inhibitors to block phosphorylation

• Compare phosphorylation patterns between wild-type plants and defense signaling mutants

Research has demonstrated that early AvrRpm1-triggered ETI signaling causes an increase in ERF13 phosphorylation at S168 . This experimental design allows for temporal resolution of post-translational modifications during plant immune responses.

What methods can be used to quantify ERF13 protein levels accurately?

To quantify ERF13 protein levels accurately:

• Western blot analysis with serial dilutions of recombinant ERF13 as standards

• Quantitative ELISA using purified ERF13 protein to generate standard curves

• Mass spectrometry-based quantification using labeled internal standards

• Fluorescence-based detection systems with GFP-tagged ERF13 constructs

• Compare protein levels with transcript levels using both Western blot and RT-qPCR

When studying protein degradation, it's crucial to include proteasome inhibitors like MG132 to prevent degradation during sample preparation . This approach has been successfully used to demonstrate that auxin promotes the degradation of ERF13 through the MPK14-MAC3A and MAC3B signaling module.

How can you overcome low detection sensitivity when working with ERF13 antibodies?

When facing low detection sensitivity:

• Implement signal amplification methods such as enhanced chemiluminescence (ECL) or tyramide signal amplification

• Concentrate proteins using immunoprecipitation prior to Western blotting

• Use tagged overexpression lines (e.g., Pro35S:ERF13-MYC or Pro35S:GFP-ERF13) for increased abundance

• Optimize extraction buffers to improve protein solubility and reduce proteolytic degradation

• Consider using more sensitive detection methods like proximity ligation assay (PLA) for in situ detection

Research groups have successfully used GFP-tagged ERF13 to track protein levels during lateral root development, demonstrating that as MAC3A gradually accumulates in the lateral root primordium, ERF13 levels decrease .

What strategies can address cross-reactivity with other ERF family members?

To minimize cross-reactivity:

• Generate antibodies against unique regions of ERF13 rather than conserved domains

• Perform extensive pre-absorption tests with recombinant proteins of related ERFs

• Validate specificity using erf13 mutants and other erf mutant lines

• Consider using epitope-tagged ERF13 constructs in transgenic plants

• Implement sequential immunoprecipitation to improve specificity

Cross-reactivity is a particular concern as AtERF13 shares a high degree of homology with Pti4 and other defense-related ERFs . Careful antibody design and validation are essential to ensure specificity.

How can researchers distinguish between different post-translationally modified forms of ERF13?

To distinguish between different post-translationally modified forms:

• Use phospho-specific antibodies that recognize phosphorylated S168

• Employ 2D gel electrophoresis to separate different phosphorylated species

• Implement Phos-tag SDS-PAGE to enhance mobility shifts of phosphorylated proteins

• Apply mass spectrometry to identify and quantify specific modifications

• Compare protein mobility with phospho-mimic (ERF13 DDD) and non-phosphorylatable (ERF13 AAA) variants

Research has shown that phosphorylation enhances the interaction between ERF13 and the E3 ligases MAC3A and MAC3B, leading to more rapid degradation . These techniques help elucidate how post-translational modifications regulate ERF13 stability and function.

How can ERF13 antibodies be used to study hormone crosstalk in plant immunity?

ERF13 functions at the intersection of multiple hormone signaling pathways . To study this crosstalk:

• Apply different hormone treatments (SA, JA, ET, ABA) and track ERF13 protein levels and modifications

• Use ERF13 antibodies in co-IP experiments to identify hormone-specific interacting partners

• Perform ChIP-seq after hormone treatments to identify condition-specific DNA binding patterns

• Compare ERF13 protein dynamics in hormone signaling mutants

• Implement biosensor approaches with tagged ERF13 to visualize real-time responses to hormones

This approach can reveal how ERF13 negotiates the crosstalk between hormones that shapes and tailors plant defense responses, as it has been shown that ERF13 lies at a junction in the signaling pathways of SA, JA, ET, and ABA .

What are the potential applications of ERF13 antibodies in studying the molecular basis of crop resistance?

ERF13 antibodies can advance crop resistance research by:

• Comparing ERF13 protein dynamics between resistant and susceptible crop varieties

• Tracking ERF13 expression and modification patterns during pathogen infection in economically important crops

• Screening for breeding lines with optimal ERF13 expression or modification patterns

• Validating the conservation of ERF13-mediated defense mechanisms across different plant species

• Developing diagnostic tools to assess plant immune status based on ERF13 modification patterns

As food security is directly related to plant health, understanding ERF13's role in immunity can help develop crops with enhanced disease resistance . The negative regulatory role of ERF13 in defense against P. syringae suggests that modulating its activity could improve crop protection strategies.