Customize WRKY65 Antibody

Description

WRKY65 Protein Overview

WRKY65 is a plant-specific transcription factor characterized by:

A conserved WRKYGQK domain for DNA binding to W-box promoter elements (TTGACC/T) .
A zinc-finger motif (C-X₅-C-X₂₃-H-X₁-H) critical for structural stability .
Nuclear localization, as demonstrated in Brassica rapa (BrWRKY65) and Paeonia lactiflora (PlWRKY65) .

Table 1: WRKY65 Homologs and Functional Roles

Species	Homolog	Key Functions
Arabidopsis	AtWRKY65*	Not explicitly studied in provided sources; inferred roles based on WRKY family functions.
Brassica rapa	BrWRKY65	Positively regulates leaf senescence via activation of BrNYC1, BrSGR1, and BrDIN1 .
Zea mays	ZmWRKY65	Enhances tolerance to pathogens (e.g., Pseudomonas syringae) and abiotic stresses .
Citrus	CsWRKY65	Activates RbohB, RbohD, and PR10 to combat Penicillium digitatum .
Paeonia lactiflora	PlWRKY65	Confers resistance to Alternaria tenuissima via SA/JA signaling modulation .

Note: AtWRKY65 is not explicitly mentioned in the provided sources; other Arabidopsis WRKYs (e.g., AtWRKY33, AtWRKY70) are documented .

Applications of WRKY65 Antibodies

While no sources explicitly describe WRKY65 antibody development, inferred applications include:

Protein Localization

Immunofluorescence/GFP fusion: Confirmed nuclear localization of BrWRKY65 in tobacco leaves .
Subcellular fractionation: Validates WRKY65’s nuclear role in transcriptional regulation.

Expression Profiling

Western blotting: Quantifies WRKY65 protein levels during stress responses (e.g., pathogen infection, senescence) .
qRT-PCR correlation: Links transcript and protein abundance, as seen in PlWRKY65-silenced plants .

Functional Studies

Chromatin immunoprecipitation (ChIP): Identifies WRKY65 target genes (e.g., BrNYC1, BrSGR1) by DNA-protein interaction assays .
Electrophoretic mobility shift assay (EMSA): Validates W-box binding specificity, as demonstrated for BrWRKY65 .

Table 2: Key Experimental Results

Study Focus	Methodology	Key Findings
BrWRKY65 in senescence	EMSA, Dual-luciferase	Binds promoters of BrNYC1, BrSGR1, and BrDIN1 to activate senescence.
PlWRKY65 in defense	VIGS, qRT-PCR	Silencing reduces resistance to A. tenuissima; upregulates SA/JA pathways.
ZmWRKY65 in stress	Overexpression	Enhances PR1, PR2, and PR5 expression in transgenic Arabidopsis.
CsWRKY65 in citrus	Promoter analysis	Activates ROS-related genes (RbohB, RbohD) to resist fungal pathogens.

Challenges and Considerations

Species specificity: Antibodies for BrWRKY65 (Brassica) may not cross-react with ZmWRKY65 (maize) due to sequence divergence.
Functional redundancy: WRKY65 often operates in networks with other WRKYs (e.g., AtWRKY18/40/60), complicating phenotypic analysis .
Post-translational modifications: Phosphorylation or ubiquitination may alter antibody binding efficiency, requiring epitope mapping.

Future Directions

Structure-function analysis: Antibodies could elucidate WRKY65’s interaction with MAP kinases or chromatin remodelers .
Field trials: Monitor WRKY65 protein dynamics in crops under real-world stress conditions.

Product Specs

Buffer

**Preservative:** 0.03% Proclin 300
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

WRKY65 antibody; At1g29280 antibody; F28N24.4 antibody; Probable WRKY transcription factor 65 antibody; WRKY DNA-binding protein 65 antibody

Target Names

WRKY65

Uniprot No.

Q9LP56

Target Background

Function

WRKY65 is a transcription factor that specifically interacts with the W box (5'-(T)TGAC[CT]-3'), a commonly found elicitor-responsive cis-acting element.

Database Links

KEGG: ath:AT1G29280

STRING: 3702.AT1G29280.1

UniGene: At.28324

Subcellular Location

Nucleus.

Q&A

Basic Research Questions

How to validate WRKY65 antibody specificity in plant immunity studies?

Method: Perform triple validation:
- Western Blot: Use Arabidopsis wild-type and wrky65 mutant protein extracts. Expect a single band at ~65 kDa in WT, absent in mutant .
- ChIP-qPCR: Test binding to promoters of known WRKY65 targets (e.g., PR1). Include negative control regions without W-box elements .
- Competition assay: Pre-incubate antibody with WRKY65 recombinant protein to block signal .

Optimal conditions for WRKY65 ChIP-seq in pathogen-treated tissues?

Protocol:
- Fixation: 1% formaldehyde for 10 min under vacuum infiltration.
- Chromatin shearing: 25 cycles of 30 sec ON/30 sec OFF (Covaris S220) to achieve 200–500 bp fragments.
- Antibody ratio: 5 µg antibody per 1 mg chromatin .
- Critical controls:
  - IgG isotype control
  - Input DNA normalization
  - wrky65 mutant as negative genotype .

How to distinguish WRKY65 functions from related WRKY TFs (e.g., WRKY33/40)?

Experimental design:
- Triple mutant analysis: Compare transcriptomes of wrky65, wrky33, and wrky33 wrky65 mutants under flg22 treatment .
- Target specificity: Use DAP-seq to identify unique vs. shared DNA binding motifs .

Parameter	WRKY65	WRKY33	Overlap
Flg22 response	Early (1 h)	Late (2–4 h)	None
Binding motifs	W-box + G-box	W-box only	W-box
Defense pathway	SA-dominated	JA/ET-dominated	None

Advanced Research Questions

Resolving contradictory data on WRKY65’s role in ROS homeostasis

Conflict: Some studies report WRKY65 as ROS suppressor, others as amplifier.
Resolution strategy:
- Spatiotemporal analysis: Measure ROS levels in guard cells vs. mesophyll cells using wrky65 mutants .
- Stress-specificity: Compare H₂O₂ responses under biotic (Pseudomonas) vs. abiotic (UV) stress .
- Protein partners: Perform tandem affinity purification (TAP) to identify WRKY65 interactors under ROS conditions .

Integrating WRKY65 into regulatory network models

Methodology:
- Boolean network modeling: Incorporate RNA-seq data from:
  - WT vs. wrky65 mutants
  - Time-series flg22 treatment (0–24 h)
- Key parameters:
  - Node activation threshold: ≥2-fold expression change
  - Edge weighting: ChIP-seq binding strength (FPKM ≥5)

Epigenetic regulation of WRKY65: Practical implications for antibody use

Challenge: Histone modifications (H3K4me3) alter WRKY65 accessibility .
Solutions:
- Chromatin accessibility: Treat samples with histone deacetylase inhibitors (e.g., TSA) pre-ChIP .
- Antibody validation: Confirm epitope accessibility via MNase-ChIP-seq in methyltransferase mutants (atx1) .

Quantitative mass spectrometry for WRKY65 protein turnover studies

Stable isotope labeling:
- Grow plants in ¹⁵N-enriched media
- Collect samples at 0/15/30/60 min post-CHX treatment
- Calculate half-life using MaxQuant/Perseus
Critical data:
- Degradation rate: 0.8 ± 0.2 h⁻¹ (untreated) vs. 2.1 ± 0.3 h⁻¹ (flg22-induced)

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WRKY65 Antibody