WRKY70 Antibody
Description
Functional Role of WRKY70 in Plant Immunity
Research using WRKY70 antibodies has revealed its dual role in pathogen defense:
Key Findings
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Pathogen-Specific Regulation:
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SA-JA Crosstalk: Acts as a node integrating SA and JA pathways, with overexpression favoring SA-mediated defenses and suppressing JA responses .
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ROS and Cell Wall Defenses: WRKY70 deficiency elevates hydrogen peroxide (H₂O₂) and promotes cell wall fortification via protein cross-linking, enhancing necrotroph resistance .
Experimental Applications of WRKY70 Antibody
The antibody has been instrumental in:
Protein Localization Studies
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Immunolocalization assays show WRKY70 accumulates in nuclei during pathogen infection, consistent with its role as a transcription factor .
Gene Expression Regulation
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Chromatin immunoprecipitation (ChIP) confirms WRKY70 binds promoters of SA-responsive genes (e.g., PR2, PAD4) and JA-repressed genes .
Mutant Characterization
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Western blot analyses of wrky54wrky70 double mutants revealed compensatory upregulation of WRKY54 in wrky70 single mutants, highlighting functional redundancy .
Comparative Analysis of WRKY70 and Homologs
Data from antibody-based studies distinguish WRKY70 from its closest homolog, WRKY54:
| Feature | WRKY70 | WRKY54 |
|---|---|---|
| Pathogen Response | Stronger role in SA-JA crosstalk | Modulates SA biosynthesis |
| Mutant Phenotype | Enhanced necrotroph resistance | Mild impact on defense |
| Protein Interaction | Forms heterodimers with WRKY54 | Binds WRKY70 but not other group III WRKYs |
| Expression Pattern | Induced by SA, repressed by JA | Constitutively low expression |
Limitations and Challenges
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Specificity Issues: Despite high specificity, cross-reactivity with WRKY54 may occur due to sequence homology in the WRKY domain .
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Context-Dependent Effects: WRKY70’s role varies between pathogens; antibody-based assays require pathogen-specific controls .
Future Research Directions
Product Specs
Target Background
WRKY70 is a transcription factor implicated in the regulation of senescence, and responses to biotic and abiotic stresses. It achieves this by modulating various phytohormone signaling pathways. WRKY70 exhibits specific interaction with the W box (5'- (T)TGAC[CT]-3'), a common elicitor-responsive cis-acting element, and binds to the 5'-[CT]GACTTTT-3' motif in target gene promoters to induce their expression. While playing an important role, it is not essential for jasmonate and salicylic acid signaling. It positively regulates the salicylic acid (SA)-mediated pathway but negatively regulates the jasmonic acid (JA)-mediated pathway, thus influencing the balance between these antagonistic pathways.
In conjunction with WRKY46, WRKY53, and WRKY54, WRKY70 inhibits defense responses against necrotrophic pathogens (e.g., Pectobacterium carotovorum and Botrytis cinerea), but promotes defense responses (including SA-induced pathogenesis-related (PR) gene expression) against biotrophic/hemibiotrophic SA-monitored pathogens (e.g., Pseudomonas syringae, Erwinia carotovora subsp. carotovora SCC3193, and Erwinia cichoracearum). This is likely achieved by negatively regulating the JA/ethylene (ET) pathway and positively regulating the SA pathway. It contributes to the suppression of jasmonic acid (MeJA)-induced expression of JA-responsive genes (e.g., PDF1.2) and promotes susceptibility to JA-monitored pathogens (e.g., Alternaria brassicicola), possibly by facilitating SA-mediated suppression of JA-mediated defense. Furthermore, WRKY70 represses the biosynthesis of camalexin and indol-3-ylmethyl glucosinolate (IGS), and represses both SA and JA/ET-mediated defense marker gene expression. It functions as a negative regulator of SA biosynthesis and EDS1-dependent defense against Erwinia amylovora.
WRKY70 is required for RPP4-mediated disease resistance and basal defense against Hyaloperonospora parasitica, potentially through late up-regulation (LURP) of resistance genes (e.g., CML10/CaBP22 and LURP1). It is also likely involved in defense responses to insects (e.g., Plutella xylostella and Brevicoryne brassicae). In collaboration with WRKY54, it negatively regulates developmental senescence, likely via the regulation of senescence-associated marker genes. With WRKY46 and WRKY54, it promotes brassinosteroid (BR)-regulated plant growth but inhibits drought response through gene expression modulation. It also prevents stomatal closure with WRKY54, consequently affecting osmotic stress tolerance. Finally, WRKY70 regulates Bacillus cereus AR156-induced systemic resistance (ISR) to P. syringae pv. tomato DC3000.
References:
- PMID: 26433201: Bacillus cereus AR156 treatment significantly stimulated WRKY70 transcription but suppressed WRKY11 transcription in Arabidopsis leaves, highlighting their roles in AR156-enhanced cellular defense responses.
- PMID: 25339349: Lower WRKY70 expression leads to significantly higher MYC2 expression through salicylic acid (SA)-jasmonic acid (JA) cross-talk.
- PMID: 23603962: AtMYB44 regulates defense responses by transcriptionally activating the downstream gene WRKY70.
- PMID: 24104863: Data indicate that the nucleotides CGAC are essential for WRKY70-activated gene expression.
- PMID: 23067202: AtMYB44 modulates antagonistic interactions by activating SA-mediated defenses and repressing JA-mediated defenses through direct control of WRKY70.
- PMID: 20841424: WRKY70 is required for snc2-1D-mediated resistance.
- PMID: 16623907: WRKY70 plays a pivotal role in balancing SA-dependent and JA-dependent defense pathways.
- PMID: 17310369: AtWRKY70, a negative regulator of developmental senescence, was characterized to better understand transcriptional control during leaf senescence.
- PMID: 18713432: Inactivation of the WRKY70 gene in the wrky70-1 mutant does not alter responses to jasmonic acid (JA) and salicylic acid (SA), and the wrky70 mutation cannot restore JA responses in the coi1 mutant.
Q&A
Here’s a structured FAQ collection for researchers working with WRKY70 antibodies, synthesized from peer-reviewed studies and technical methodologies:
What are the primary experimental applications of WRKY70 antibodies in plant immunity studies?
WRKY70 antibodies are critical for:
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Western blotting: Detecting endogenous WRKY70 protein levels in mutant/wild-type comparisons (e.g., snc2-1D vs wrky54 wrky70 mutants) .
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Immunoprecipitation (IP): Studying protein-protein interactions, such as WRKY70’s binding to OsMPK3/OsMPK6 in MAP kinase cascades .
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Electrophoretic Mobility Shift Assay (EMSA): Confirming WRKY70’s DNA-binding activity to W-box motifs in promoters (e.g., SARD1, CBP60g) .
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Immunolocalization: Tracking subcellular localization via BiFC assays in Nicotiana benthamiana .
Methodological Tip: Optimize antibody dilution (1:1,000–1:5,000) in TBST with 5% nonfat milk to reduce background noise in western blots .
How to validate WRKY70 antibody specificity in Arabidopsis and rice systems?
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Knockout controls: Compare signal intensity in wild-type vs wrky70 mutants (e.g., snc2-1D wrky70 lines) .
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Cross-reactivity tests: Validate using homologs like WRKY54; true-specific antibodies show no signal in wrky54 wrky70 double mutants .
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Peptide competition assays: Pre-incubate antibodies with immunizing peptides (e.g., CVYYASRAKDEPRDD) to confirm epitope specificity .
How to resolve contradictory data on WRKY70’s role in SA/JA signaling across pathosystems?
Case Example: WRKY70 enhances SA-mediated resistance to biotrophs (e.g., Pseudomonas syringae) but increases susceptibility to necrotrophs (e.g., Botrytis cinerea) .
| Pathogen Type | WRKY70 Function | Mechanism | Citation |
|---|---|---|---|
| Biotrophs | Positive regulator | Activates PR genes via SARD1/CBP60g loop | |
| Necrotrophs | Negative regulator | Suppresses JA/ET defenses, reduces ROS accumulation |
Methodological Approach:
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Use tissue-specific promoters (e.g., WRKY70p::GUS) to spatiotemporally dissect signaling outputs.
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Combine wrky70 mutants with sid2 (SA-deficient) or coi1 (JA-insensitive) backgrounds to isolate pathway crosstalk .
What protocols ensure reliable detection of post-translationally modified WRKY70?
WRKY70 is phosphorylated by OsMPK3/OsMPK6, which enhances its transactivation activity without altering DNA binding .
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Phospho-specific antibodies: Develop antibodies targeting phosphorylated Ser/Thr residues in WRKY70’s MAPK docking domain.
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In vitro kinase assays: Combine immunoprecipitated WRKY70 with active MAPKs (e.g., OsMPK3 DD) and ATP-γ-S for thiophosphorylation .
Critical Controls:
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Include catalytically inactive MAPK mutants (e.g., OsMPK3 KR) to confirm phosphorylation dependency.
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Validate with Phos-tag™ SDS-PAGE to separate phosphorylated/non-phosphorylated isoforms .
How to address cross-reactivity between WRKY70 and homologs (e.g., WRKY54) in EMSA?
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Competitive DNA probes: Include excess unlabeled W-box DNA to outcompect non-specific binding .
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Mutant WRKY70 proteins: Use D/E-substituted WRKY70 (impaired DNA binding) as negative controls in supershift assays .
