Eschrig, S. | bioRxiv Science

bioRxiv2021

LORE homomerization is required for 3-OH-C10:0 induced immune signaling

O_LIThe S-domain-type receptor-like kinase (SD-RLK) LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) from Arabidopsis thaliana is a pattern recognition receptor that senses medium-chain 3-hydroxy fatty acids, such as 3-hydroxydecanoic acid (3-OH-C10:0), to activate pattern-triggered immunity. Here, we show that LORE homomerization is required to activate 3-OH-C10:0-induced immune signaling. C_LIO_LIFluorescence lifetime imaging in Nicotiana benthamiana demonstrated that AtLORE homomerizes via the extracellular and transmembrane domains. Co-expression of AtLORE truncations lacking the intracellular domain exerts a dominant negative effect on AtLORE signaling in both N. benthamiana and A. thaliana, highlighting that homomerization is essential for signaling. C_LIO_LIScreening for 3-OH-C10:0-induced reactive oxygen species production revealed natural variation within the Arabidopsis genus. Arabidopsis lyrata and Arabidopsis halleri do not respond to 3-OH-C10:0, although both possess a putative LORE orthologue. Both LORE orthologues have defective extracellular domains that bind 3-OH-C10:0 to a similar level but lack the ability to homomerize. Thus, ligand binding is independent of LORE homomerization. Analysis of AtLORE and AlyrLORE chimera suggests that the loss of AlyrLORE homomerization is caused by several amino acid polymorphisms across the extracellular domain. C_LIO_LIOur findings shed light on the activation mechanism of LORE and the loss of 3-OH-C10:0 perception within the Arabidopsis genus. C_LI

plant biology↗

bioRxiv2021

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

BackgroundPlant 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. ResultsTo 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. ConclusionThe 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.

plant biology↗