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

Abstract

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 'depletion'). Subsequently, protein-bound 3-OH-C10:0 retained in the high molecular weight fraction in the depletion step is released and detected (step 'binding'). 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.

LORE receptor homomerization is required for 3‐hydroxydecanoic acid‐induced immune signaling and determines the natural variation of immunosensitivity within the Arabidopsis genus

“…1d). Although receptor complex formation should be rapid, this timeframe was suggested for comparable FLIM studies (Somssich et al, 2015). Generally, plants may maintain receptor complexes in a stable, preformed steady-state as a strategy to ensure rapid signaling upon ligand binding that is subsequently fine-tuned by multiple regulators in higher-order complexes.…”

“…As described previously (Shu et al ., 2021), ECDs were transiently expressed in N. benthamiana apoplasts and harvested via AWFs, which were concentrated and incubated with 3‐OH‐C10:0 (9 : 1, v/v). Unbound elicitor was separated by filtration (Vivaspin 500 centrifugal concentrator, 30 kDa MWCO; Sartorius).…”

“…Five microliters concentrated AWF (1.5 mg ml −1 total protein concentration) was analyzed for protein expression by immunoblot. Ligand‐binding assay was performed as described previously (Shu et al ., 2021). Purified ECDs (500 nM) were incubated with 3‐OH‐C10:0 (72 : 8 μl).…”

“…Only 3‐OH‐FAs/HAAs with acyl chains comprising 8 to 12 carbon atoms activate At LORE signaling. 3‐hydroxydecanoic acid (3‐OH‐C10:0) is the strongest elicitor in A. thaliana and binds to the At LORE‐ECD (Kutschera et al ., 2019; Shu et al ., 2021). LORE possesses a strong RD‐type kinase (Johnson et al ., 1996; Ranf et al ., 2015), which auto‐phosphorylates At LORE upon 3‐OH‐C10:0 sensing and activates the receptor‐like cytoplasmic kinase PBS1‐LIKE 34/35/36 ( At PBL34/35/36; Luo et al ., 2020), RPM1‐INDUCED PROTEIN KINASE ( At RIPK) and the LORE‐ASSOCIATED PROTEIN PHOSPHATASE (LOPP; Wang et al ., 2023).…”

Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms

“…Depletion Binding Assay. Protein expression and ligand depletion were performed as described (84) with slight modifications. Briefly, LORE extracellular domain mCherry fusion protein (eLORE-mCherry) or mCherry control fused with N-terminal LORE signal peptide were transiently expressed in N. benthamiana leaf apoplast using a 1:1 mixture of Agrobacterium tumefaciens GV3101 carrying the respective expression plasmid or the p19 silencing suppressor (total optical density [OD] 600 0.5).…”