Low cost, medium throughput depletion-binding assay for screening S-domain-receptor ligand interactions using in planta protein expression

Background: Plant cell-surface receptors sense various ligands to regulate physiological processes. Matching ligand-receptor pairs requires evidence of their direct interaction and is often a bottleneck in functional receptor studies. The S-domain-type (SD) pattern recognition receptor LORE senses medium chain-3-hydroxy fatty acids (mc-3-OH-FAs) such as 3-hydroxy decanoic acid (3-OH-C10:0) via its extracellular domain (ECD) They are perceived as signals of danger from Gram-negative bacteria and activate immune responses in Arabidopsis thaliana. LORE is found in low levels in planta and is poorly expressed in heterologous systems. Furthermore, chemical modifications of the mc-3-OH-FA ligand affect its biological activity. Taken together, this makes LORE-mc-3-OH-FA binding studies particularly challenging. Results: To investigate the LORE-mc-3-OH-FA interaction, we have developed a sensitive assay system based on protein expression in planta. The ECDs of LORE and other proteins of interest were transiently expressed as soluble, apoplastic mCherry fusion proteins in Nicotiana benthamiana and collected in apoplastic washing fluids. Protein-ligand complexes and unbound ligand were separated according to their molecular weight. In a two-step procedure, we first investigated whether the ECD-mCherry fusion protein depletes 3-OH-C10:0 from the low molecular weight fraction (step 9depletion9). Subsequently, protein-bound 3-OH-C10:0 retained in the high molecular weight fraction in the depletion step is released and detected (step 9binding9). Both the unbound and the released 3-OH-C10:0 ligand are detected by a sensitive bioassay using LORE loss- and gain-of-function Arabidopsis plants. Using the depletion-binding assay, we show that the ECD of AtLORE and its ortholog from Capsella rubella, CrubLORE, bind 3-OH-C10:0. The ECD of AtSD1-23, the closest paralog of AtLORE, and mCherry did not bind 3-OH-C10:0 and are suitable negative controls. Conclusion: The depletion-binding assay is a simple method for reliably detecting interactions between plant-expressed SD-type receptor ectodomains and mc-3-OH-FAs. It does not require special equipment or expensive consumables and is suitable for medium throughput screening. The assay is very flexible and can be easily adapted to investigate ligand interactions of other extracellular receptor domains.