Plant-Based Ligand-Depletion Binding Assay for Assessing Pattern Recognition Receptor-Ligand Interactions

Area of Science:

• Plant molecular biology
• Plant immunology
• Biochemistry

Background:

• Cell-surface receptor-ligand interactions are vital for plant signal transduction and immune responses mediated by pattern recognition receptors (PRRs).
• Conventional biophysical methods for assessing these interactions are precise but require specialized equipment, expensive reagents, and large protein quantities.

Purpose of the Study:

• To introduce an accessible, plant-based ligand-depletion binding assay for studying PRR-ligand interactions.
• To provide a cost-effective and scalable alternative to conventional biophysical methods for medium-throughput screening.

Main Methods:

• Transient expression of PRR extracellular domains (ECDs) in the apoplast of *Nicotiana benthamiana* using *Agrobacterium*-mediated transformation.
• Ligand-binding assay utilizing ultrafiltration to separate ECD-ligand complexes from unbound ligands.
• Detection of ligands via *Arabidopsis thaliana* Ca²⁺ signaling responses as a biosensor readout.

Main Results:

• Demonstrated a workflow using the LRR receptor-like kinase (LORE) ECD and its ligand 3-hydroxydecanoic acid (3-OH-C10:0).
• Successfully employed *A. thaliana* seedlings as biosensors to detect ligand binding.
• Established a cost-effective and scalable pipeline for PRR-ligand interaction assessment.

Conclusions:

• The developed plant-based assay offers a practical alternative for studying diverse PRR-ligand interactions.
• This approach facilitates research in plant immunity by enabling medium-throughput screening of receptor variants and ligand candidates.
• The method is broadly adaptable for investigating various plant immune signaling pathways.