In Vitro Analytical Approaches to Study Plant Ligand-Receptor Interactions

Jiménez‐Sandoval, Pedro; Santiago, Julia

doi:10.1104/pp.19.01396

Cited by 24 publications

(14 citation statements)

References 132 publications

(128 reference statements)

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“…However, none of these MAMPs/DAMPs, including cello-, laminari-, xyloglucan-, mannan-and arabinoxylo-oligosaccharides, have a single candidate PRR for their perception, whose elucidation will require the combination of heavy biochemical and genetic approaches. In particular, biomolecular interaction assays with pure ECDs of putative receptors have been shown to be the golden standard to allow quantification of potential MAMP/DAMP-ECD binding (Sandoval and Santiago, 2020). However, these approaches are time consuming and resource demanding, thus high-throughput screenings based on these technologies are of high risk and not always affordable.…”

Section: Discussionmentioning

confidence: 99%

“…In view of these results and to further clarify this issue, we expressed CERK1 and LYK4 Arabidopsis ECDs in insect cells and purified them by affinity chromatography (Figure S4). Then, isothermal titration calorimetry (ITC) experiments (Sandoval and Santiago, 2020) were carried out to confirm the in silico binding predictions of both CERK1-dependent ligands 1,4-b-D-(GlcNAc) 6 and 1,3-b-D-(Glc) 6 . Our ITC results proved direct interactions of CERK1 with 1,4-b-D-(GlcNac) 6 (K d values of 37.5 AE 10.0 µM; Figure 5).…”

Section: Computational Prediction Methods For the Identification Of Prmentioning

confidence: 99%

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Computational prediction method to decipher receptor–glycoligand interactions in plant immunity

et al. 2021

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Microbial and plant cell walls have been selected by the plant immune system as a source of microbe-and plant damage-associated molecular patterns (MAMPs/DAMPs) that are perceived by extracellular ectodomains (ECDs) of plant pattern recognition receptors (PRRs) triggering immune responses. From the vast number of ligands that PRRs can bind, those composed of carbohydrate moieties are poorly studied, and only a handful of PRR/glycan pairs have been determined. Here we present a computational screening method, based on the first step of molecular dynamics simulation, that is able to predict putative ECD-PRR/ glycan interactions. This method has been developed and optimized with Arabidopsis LysM-PRR members CERK1 and LYK4, which are involved in the perception of fungal MAMPs, chitohexaose (1,4-b-D-(GlcNAc) 6) and laminarihexaose (1,3-b-D-(Glc) 6). Our in silico results predicted CERK1 interactions with 1,4-b-D-(GlcNAc) 6 whilst discarding its direct binding by LYK4. In contrast, no direct interaction between CERK1/laminarihexaose was predicted by the model despite CERK1 being required for laminarihexaose immune activation, suggesting that CERK1 may act as a co-receptor for its recognition. These in silico results were validated by isothermal titration calorimetry binding assays between these MAMPs and recombinant ECDs-LysM-PRRs. The robustness of the developed computational screening method was further validated by predicting that CERK1 does not bind the DAMP 1,4-b-D-(Glc) 6 (cellohexaose), and then probing that immune responses triggered by this DAMP were not impaired in the Arabidopsis cerk1 mutant. The computational predictive glycan/PRR binding method developed here might accelerate the discovery of protein-glycan interactions and provide information on immune responses activated by glycoligands.

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Section: Discussionmentioning

confidence: 99%

Section: Computational Prediction Methods For the Identification Of Prmentioning

confidence: 99%

Computational prediction method to decipher receptor–glycoligand interactions in plant immunity

et al. 2021

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“…AtDORN1, an L-type lectin receptor kinase that mediates the perception of extracellular ATP in Arabidopsis, binds radiolabeled ATP after recombinant expression in Escherichia coli bacteria (Choi et al, 2014). Many other techniques to study biomolecular interactions have been applied in plant receptor research, such as surface plasmon resonance (SPR), microscale thermophoresis (MST), and grating-coupled interferometry (GCI), all of which have their specific advantages and disadvantages (Sandoval and Santiago, 2020). While such methods allow quantification of binding properties, many of them require large amounts of purified receptor protein or ligand labelling, as well as specialized facilities, costly equipment or expensive consumables.…”

Section: Introductionmentioning

confidence: 99%

“…using RLK/RLP loss-or gain-of-function approaches, are instrumental in identifying potential ligand-receptor pairs, but additional methods are required to prove a direct interaction between a ligand and its receptor. Various different methods have been used to assess ligand binding to plant receptors (Sandoval and Santiago, 2020). The flg22-FLS2 interaction has been demonstrated, for example, by radioligand binding assay and chemical cross-linking using immunoprecipitated receptors from Arabidopsis and AtFLS2-exressing tomato cells (Chinchilla et al, 2006).…”

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

Ranf

Liu

Schaeffer

et al. 2021

Preprint

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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.

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“…Therefore, assessing ligand-receptor interactions requires more integrative and quantitative approaches, including biophysical methods to measure membrane receptor dynamics and oligomerization state upon ligand binding. Sandoval and Santiago (2020) overview the state-ofthe-art in vitro ligand-binding assays used to investigate receptor-ligand interactions. Among these, new structure-based ligand-binding technologies have emerged as powerful methods to uncover key residues and conformational changes of the receptors upon ligand binding.…”

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confidence: 99%

Update on Receptors and Signaling

Cheung

Russinova

et al. 2020

Plant Physiol.

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In Vitro Analytical Approaches to Study Plant Ligand-Receptor Interactions

Abstract: An overview of state-of-the-art in vitro methods details receptor-ligand interactions, highlighting experiment recommendations, as well as advantages and limitations.

Cited by 24 publications

References 132 publications

Computational prediction method to decipher receptor–glycoligand interactions in plant immunity

Computational prediction method to decipher receptor–glycoligand interactions in plant immunity

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

Update on Receptors and Signaling

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