Treffer: Plant-Based Ligand-Depletion Binding Assay for Assessing Pattern Recognition Receptor-Ligand Interactions.
Title:
Plant-Based Ligand-Depletion Binding Assay for Assessing Pattern Recognition Receptor-Ligand Interactions.
Authors:
Source:
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2026; Vol. 3012, pp. 35-56.
Publication Type:
Journal Article
Language:
English
Journal Info:
Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE
Imprint Name(s):
Publication: Totowa, NJ : Humana Press
Original Publication: Clifton, N.J. : Humana Press
Original Publication: Clifton, N.J. : Humana Press
MeSH Terms:
Receptors, Pattern Recognition*/metabolism , Receptors, Pattern Recognition*/genetics , Arabidopsis*/metabolism , Arabidopsis*/genetics , Nicotiana*/metabolism , Nicotiana*/genetics , Arabidopsis Proteins*/metabolism , Arabidopsis Proteins*/genetics, Biosensing Techniques/methods ; Seedlings/metabolism ; Seedlings/genetics ; Ligands ; Protein Binding ; Plants, Genetically Modified
References:
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Gómez-Gómez L, Boller T (2000) FLS2: an LRR receptor–like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5:1003–1011. (PMID: 10.1016/S1097-2765(00)80265-810911994)
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Ranf S, Gisch N, Schäffer M, Illig T, Westphal L, Knirel YA, Sánchez-Carballo PM, Zähringer U, Hückelhoven R, Lee J, Scheel D (2015) A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana. Nat Immunol 16:426–433. (PMID: 10.1038/ni.312425729922)
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Bigeard J, Colcombet J, Hirt H (2015) Signaling mechanisms in pattern-triggered immunity (PTI). Mol Plant 8:521–539. (PMID: 10.1016/j.molp.2014.12.02225744358)
Sandoval PJ, Santiago J (2020) In vitro analytical approaches to study plant ligand-receptor interactions1. Plant Physiol 182:1697–1712. (PMID: 10.1104/pp.19.01396320340537140929)
Xiao Y, Stegmann M, Han Z, DeFalco TA, Parys K, Xu L, Belkhadir Y, Zipfel C, Chai J (2019) Mechanisms of RALF peptide perception by a heterotypic receptor complex. Nature 572:270–274. (PMID: 10.1038/s41586-019-1409-731291642)
Kato H, Nemoto K, Shimizu M, Abe A, Asai S, Ishihama N, Matsuoka S, Daimon T, Ojika M, Kawakita K, Onai K, Shirasu K, Yoshida M, Ishiura M, Takemoto D, Takano Y, Terauchi R (2022) Recognition of pathogen-derived sphingolipids in Arabidopsis. Science 376:857–860. (PMID: 10.1126/science.abn065035587979)
Liu T, Liu Z, Song C, Hu Y, Han Z, She J, Fan F, Wang J, Jin C, Chang J, Zhou J-M, Chai J (2012) Chitin-induced dimerization activates a plant immune receptor. Science 336:1160–1164. (PMID: 10.1126/science.121886722654057)
Shu L-J, Schäffer M, Eschrig S, Ranf S (2021) Low cost, medium throughput depletion-binding assay for screening S-domain-receptor ligand interactions using in planta protein expression. bioRxiv. https://doi.org/10.1101/2021.06.16.448648.
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. (PMID: 10.3389/fmicb.2014.00172248605554029002)
van Oers MM, Pijlman GP, Vlak JM (2015) Thirty years of baculovirus–insect cell protein expression: from dark horse to mainstream technology. J Gen Virol 96:6–23. (PMID: 10.1099/vir.0.067108-025246703)
Schillberg S, Raven N, Spiegel H, Rasche S, Buntru M (2019) Critical analysis of the commercial potential of plants for the production of recombinant proteins. Front Plant Sci 10:720. (PMID: 10.3389/fpls.2019.00720312448686579924)
Martin R, Liu F, Staskawicz B (2022) Isolation of protein complexes from tobacco leaves by a two-step tandem affinity purification. Curr Protoc 2:e572. (PMID: 10.1002/cpz1.57236205456)
Lawson AW, Macha A, Neumann U, Gunkel M, Chai J, Behrmann E, Schulze-Lefert P (2025) Purifying recombinant proteins from Nicotiana benthamiana for structural studies. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01249-2.
Eschrig S, Schäffer M, Shu L-J, Illig T, Eibel S, Fernandez A, Ranf S (2024) LORE receptor homomerization is required for 3-hydroxydecanoic acid-induced immune signaling and determines the natural variation of immunosensitivity within the Arabidopsis genus. New Phytol 242:2163–2179. (PMID: 10.1111/nph.1971538532564)
Ranf S, Grimmer J, Poschl Y, Pecher P, Chinchilla D, Scheel D, Lee J (2012) Defense-related calcium signaling mutants uncovered via a quantitative high-throughput screen in Arabidopsis thaliana. Mol Plant 5:115–130. (PMID: 10.1093/mp/ssr06421859959)
Kaseniit KE, Katz N, Kolber NS, Call CC, Wengier DL, Cody WB, Sattely ES, Gao XJ (2023) Modular, programmable RNA sensing using ADAR editing in living cells. Nat Biotechnol 41(4):482–487. (PMID: 10.1038/s41587-022-01493-x36198772)
Lawrenson T, Youles M, Chhetry M, Clarke M, Harwood W, Hundleby P (2023) Efficient targeted mutagenesis in Brassica crops using CRISPR/Cas systems. In: Yang B, Harwood W, Que Q (eds) Plant genome engineering: methods and protocols. Springer, New York, pp 253–271. (PMID: 10.1007/978-1-0716-3131-7_16)
Jay F, Brioudes F, Voinnet O (2023) A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19. Plant J 113:186–204. (PMID: 10.1111/tpj.1603236403224)
Robert S, Khalf M, Goulet M-C, D’Aoust M-A, Sainsbury F, Michaud D (2013) Protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of Nicotiana benthamiana. PLoS One 8:e70203. (PMID: 10.1371/journal.pone.0070203238946183720903)
Alonso Villela SM, Kraïem H, Bouhaouala-Zahar B, Bideaux C, Aceves Lara CA, Fillaudeau L (2020) A protocol for recombinant protein quantification by densitometry. MicrobiologyOpen 9(6):1175–1182. (PMID: 10.1002/mbo3.102732255275)
Kwanyuen P, Wilson RF (2000) Optimization of coomassie staining for quantitative densitometry of soybean storage proteins in gradient gel electrophoresis. J Amer Oil Chem Soc 77:1251–1254. (PMID: 10.1007/s11746-000-0196-0)
Shimomura O, Kishi Y, Inouye S (1993) The relative rate of aequorin regeneration from apoaequorin and coelenterazine analogues. Biochem J 296:549–551. (PMID: 10.1042/bj296054982800501137732)
Inouye S, Shimomura O (1997) The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate. Biochem Biophys Res Commun 233:349–353. (PMID: 10.1006/bbrc.1997.64529144537)
Ngou BPM, Ding P, Jones JDG (2022) Thirty years of resistance: zig-zag through the plant immune system. Plant Cell 34:1447–1478. (PMID: 10.1093/plcell/koac041351676979048904)
Shu L-J, Kahlon PS, Ranf S (2023) The power of patterns: new insights into pattern-triggered immunity. New Phytol 240:960–967. (PMID: 10.1111/nph.1914837525301)
Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18:265–276. (PMID: 10.1046/j.1365-313X.1999.00265.x10377992)
Gómez-Gómez L, Boller T (2000) FLS2: an LRR receptor–like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5:1003–1011. (PMID: 10.1016/S1097-2765(00)80265-810911994)
Cao Y, Liang Y, Tanaka K, Nguyen CT, Jedrzejczak RP, Joachimiak A, Stacey G (2014) The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife 3:e03766. (PMID: 10.7554/eLife.03766253409594356144)
Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H, Shibuya N (2007) CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci U S A 104:19613–19618. (PMID: 10.1073/pnas.0705147104180427242148337)
Ranf S, Gisch N, Schäffer M, Illig T, Westphal L, Knirel YA, Sánchez-Carballo PM, Zähringer U, Hückelhoven R, Lee J, Scheel D (2015) A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana. Nat Immunol 16:426–433. (PMID: 10.1038/ni.312425729922)
Kutschera A, Dawid C, Gisch N, Schmid C, Raasch L, Gerster T, Schäffer M, Smakowska-Luzan E, Belkhadir Y, Vlot AC, Chandler CE, Schellenberger R, Schwudke D, Ernst RK, Dorey S, Hückelhoven R, Hofmann T, Ranf S (2019) Bacterial medium-chain 3-hydroxy fatty acid metabolites trigger immunity in Arabidopsis plants. Science 364:178–181. (PMID: 10.1126/science.aau127930975887)
Schellenberger R, Crouzet J, Nickzad A, Shu LJ, Kutschera A, Gerster T, Borie N, Dawid C, Cloutier M, Villaume S, Dhondt-Cordelier S, Hubert J, Cordelier S, Mazeyrat-Gourbeyre F, Schmid C, Ongena M, Renault JH, Haudrechy A, Hofmann T, Baillieul F, Clement C, Zipfel C, Gauthier C, Deziel E, Ranf S, Dorey S (2021) Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms. Proc Natl Acad Sci U S A 118:e2101366118. (PMID: 10.1073/pnas.2101366118345613048488661)
Bigeard J, Colcombet J, Hirt H (2015) Signaling mechanisms in pattern-triggered immunity (PTI). Mol Plant 8:521–539. (PMID: 10.1016/j.molp.2014.12.02225744358)
Sandoval PJ, Santiago J (2020) In vitro analytical approaches to study plant ligand-receptor interactions1. Plant Physiol 182:1697–1712. (PMID: 10.1104/pp.19.01396320340537140929)
Xiao Y, Stegmann M, Han Z, DeFalco TA, Parys K, Xu L, Belkhadir Y, Zipfel C, Chai J (2019) Mechanisms of RALF peptide perception by a heterotypic receptor complex. Nature 572:270–274. (PMID: 10.1038/s41586-019-1409-731291642)
Kato H, Nemoto K, Shimizu M, Abe A, Asai S, Ishihama N, Matsuoka S, Daimon T, Ojika M, Kawakita K, Onai K, Shirasu K, Yoshida M, Ishiura M, Takemoto D, Takano Y, Terauchi R (2022) Recognition of pathogen-derived sphingolipids in Arabidopsis. Science 376:857–860. (PMID: 10.1126/science.abn065035587979)
Liu T, Liu Z, Song C, Hu Y, Han Z, She J, Fan F, Wang J, Jin C, Chang J, Zhou J-M, Chai J (2012) Chitin-induced dimerization activates a plant immune receptor. Science 336:1160–1164. (PMID: 10.1126/science.121886722654057)
Shu L-J, Schäffer M, Eschrig S, Ranf S (2021) Low cost, medium throughput depletion-binding assay for screening S-domain-receptor ligand interactions using in planta protein expression. bioRxiv. https://doi.org/10.1101/2021.06.16.448648.
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. (PMID: 10.3389/fmicb.2014.00172248605554029002)
van Oers MM, Pijlman GP, Vlak JM (2015) Thirty years of baculovirus–insect cell protein expression: from dark horse to mainstream technology. J Gen Virol 96:6–23. (PMID: 10.1099/vir.0.067108-025246703)
Schillberg S, Raven N, Spiegel H, Rasche S, Buntru M (2019) Critical analysis of the commercial potential of plants for the production of recombinant proteins. Front Plant Sci 10:720. (PMID: 10.3389/fpls.2019.00720312448686579924)
Martin R, Liu F, Staskawicz B (2022) Isolation of protein complexes from tobacco leaves by a two-step tandem affinity purification. Curr Protoc 2:e572. (PMID: 10.1002/cpz1.57236205456)
Lawson AW, Macha A, Neumann U, Gunkel M, Chai J, Behrmann E, Schulze-Lefert P (2025) Purifying recombinant proteins from Nicotiana benthamiana for structural studies. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01249-2.
Eschrig S, Schäffer M, Shu L-J, Illig T, Eibel S, Fernandez A, Ranf S (2024) LORE receptor homomerization is required for 3-hydroxydecanoic acid-induced immune signaling and determines the natural variation of immunosensitivity within the Arabidopsis genus. New Phytol 242:2163–2179. (PMID: 10.1111/nph.1971538532564)
Ranf S, Grimmer J, Poschl Y, Pecher P, Chinchilla D, Scheel D, Lee J (2012) Defense-related calcium signaling mutants uncovered via a quantitative high-throughput screen in Arabidopsis thaliana. Mol Plant 5:115–130. (PMID: 10.1093/mp/ssr06421859959)
Kaseniit KE, Katz N, Kolber NS, Call CC, Wengier DL, Cody WB, Sattely ES, Gao XJ (2023) Modular, programmable RNA sensing using ADAR editing in living cells. Nat Biotechnol 41(4):482–487. (PMID: 10.1038/s41587-022-01493-x36198772)
Lawrenson T, Youles M, Chhetry M, Clarke M, Harwood W, Hundleby P (2023) Efficient targeted mutagenesis in Brassica crops using CRISPR/Cas systems. In: Yang B, Harwood W, Que Q (eds) Plant genome engineering: methods and protocols. Springer, New York, pp 253–271. (PMID: 10.1007/978-1-0716-3131-7_16)
Jay F, Brioudes F, Voinnet O (2023) A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19. Plant J 113:186–204. (PMID: 10.1111/tpj.1603236403224)
Robert S, Khalf M, Goulet M-C, D’Aoust M-A, Sainsbury F, Michaud D (2013) Protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of Nicotiana benthamiana. PLoS One 8:e70203. (PMID: 10.1371/journal.pone.0070203238946183720903)
Alonso Villela SM, Kraïem H, Bouhaouala-Zahar B, Bideaux C, Aceves Lara CA, Fillaudeau L (2020) A protocol for recombinant protein quantification by densitometry. MicrobiologyOpen 9(6):1175–1182. (PMID: 10.1002/mbo3.102732255275)
Kwanyuen P, Wilson RF (2000) Optimization of coomassie staining for quantitative densitometry of soybean storage proteins in gradient gel electrophoresis. J Amer Oil Chem Soc 77:1251–1254. (PMID: 10.1007/s11746-000-0196-0)
Shimomura O, Kishi Y, Inouye S (1993) The relative rate of aequorin regeneration from apoaequorin and coelenterazine analogues. Biochem J 296:549–551. (PMID: 10.1042/bj296054982800501137732)
Inouye S, Shimomura O (1997) The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate. Biochem Biophys Res Commun 233:349–353. (PMID: 10.1006/bbrc.1997.64529144537)
Contributed Indexing:
Keywords: 3-Hydroxy fatty acid; Agrobacterium-mediated transient protein expression; Apoplastic washing fluid; Ca2+ signaling; LORE; Ligand-depletion binding assay; PAMP/MAMP; Pattern recognition receptor
Substance Nomenclature:
0 (Receptors, Pattern Recognition)
0 (Ligands)
0 (Arabidopsis Proteins)
0 (Ligands)
0 (Arabidopsis Proteins)
Entry Date(s):
Date Created: 20260205 Date Completed: 20260205 Latest Revision: 20260205
Update Code:
20260206
DOI:
10.1007/978-1-0716-5138-4_3
PMID:
41642514
Database:
MEDLINE
Weitere Informationen
The assessment of cell-surface receptor-ligand interactions is of fundamental importance for understanding plant signal transduction, particularly in immune responses mediated by pattern recognition receptors (PRRs). Iconic biophysical methods such as microscale thermophoresis, isothermal titration calorimetry, and surface plasmon resonance provide precise quantification of binding properties but require specialized equipment, costly reagents, and large quantities of purified proteins or labeled ligands. To offer a more accessible and plant-based approach, this chapter introduces a ligand-depletion binding assay using Nicotiana benthamiana to express PRR extracellular domains (ECDs) and Arabidopsis thaliana seedlings as sensitive biosensors for detecting ligands. Using the ECD of the PRR LORE and its ligand, 3-hydroxydecanoic acid (3-OH-C10:0), as an example, we demonstrate a workflow in which recombinant PRR ECDs are transiently expressed in the apoplast of N. benthamiana via Agrobacterium-mediated transformation. The ligand-binding assay utilizes ultrafiltration by concentrator columns to separate ECD-ligand complexes and unbound ligands by molecular weight. A. thaliana Ca<sup>2+</sup> signaling responses serve as a robust readout for ligand detection. This cost-effective and scalable pipeline provides an alternative to conventional methods, enabling medium-throughput screening of receptor variants and ligand candidates. The approach can be broadly adapted to study diverse PRR-ligand interactions, facilitating research in plant immunity.
(© 2026. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)