At2g16250 Antibody

Target Protein Characterization

AT2G16250 encodes a 967-amino acid transmembrane protein with:

• Domain architecture:

  • N-terminal signal peptide (residues 1-22)

  • 15 leucine-rich repeats (LRRs; residues 43-384)

  • Transmembrane domain (residues 705-727)

  • C-terminal serine/threonine kinase domain (residues 752-967)

Phosphorylation sites identified through mass spectrometry:

This receptor belongs to the ARM3 clade of RG-I recognition receptors, distinguished by larger extracellular domains (ECDs) compared to ARM1/ARM2 clades .

Functional Role in Plant Immunity

Key findings from recent studies:

RG-I Recognition Mechanism

• Binds RG-I fragments with K_D = 92.4 ± 18.7 nM (SPR analysis)

• Requires intact RG-I backbone structure for binding (endo-RG-I hydrolase sensitivity)

• Triggers MAPK cascade activation (phosphorylation of MPK3/6 within 5-15 mins)

Genetic Evidence

• At2g16250 knockout plants show:

  • 63% increased susceptibility to Pseudomonas syringae DC3000

  • Delayed ROS burst (28% reduction at 30 min post-treatment)

  • Impaired callose deposition (42% fewer deposits vs wild type)

Antibody Applications

The antibody has been critical in demonstrating ARM3a's role in:

• RG-I-induced transcriptional reprogramming (303 DEGs shared with OGDP10-15 elicitor)

• Formation of receptor complexes with CERK1 and FLS2

• Phosphorylation-dependent signal amplification (Y285F mutant loses 78% activity)

Validation Data

Following the "five pillars" of antibody characterization :

1. Genetic validation: No signal in at2g16250 T-DNA mutants

2. Orthogonal confirmation: Co-localization with RG-AuNPs (r = 0.89 vs immunofluorescence)

3. Independent antibody correlation: 92% concordance with HA-tagged ARM3a lines

4. MS verification: Identified 8 unique peptides covering 41% sequence

5. **Application-specific testing: Works in IF, WB, IP but not ELISA