Abstract
The biogenesis and functionality of pattern recognition receptors (PRRs) are critical for robust plant immune responses. Here, we present methods to determine the N-glycosylation state and ligand-induced activity of these receptors for comparative quantitative analysis. These techniques can be used to identify mutants and chemical inhibitors affecting PRR biogenesis and functionality. When combined, these techniques can provide useful insights on biological processes necessary to synthesize a properly membrane-localized and ligand-responsive PRR.
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Aebi M, Bernasconi R, Clerc S, Molinari M (2010) N-Glycan structures: recognition and processing in the ER. Trends Biochem Sci 35:74–82
Strasser R (2014) Biological significance of complex N-glycans in plants and their impact on plant physiology. Front Plant Sci 5:363
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
Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JDG, Boller T, Felix G (2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125:749–760
Saijo Y (2010) ER quality control of immune receptors and regulators in plants. Cell Microbiol 12:716–724
Li J, Zhao-Hui C, Batoux M, Nekrasov V, Roux M, Chinchilla D et al (2009) Specific ER quality control components required for biogenesis of the plant innate immune receptor EFR. Proc Natl Acad Sci U S A 106:15973–15978
Lu X, Tintor N, Mentzel T, Kombrink E, Boller T, Robatzek S et al (2009) Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidease II allele. Proc Natl Acad Sci U S A 106:22522–22527
Nekrasov V, Li J, Batoux M, Roux M, Chu Z-H, Lacombe S, Bougon A et al (2009) Control of the pattern-recognition receptor EFR by an ER protein complex in plant immunity. EMBO J 28:3428–3438
Saijo Y, Tintor N, Lu X, Rauf P, Pajerowska-Mukhtar K, Häweker H et al (2009) Receptor quality control in the endoplasmic reticulum for plant innate immunity. EMBO J 28:3439–3449
von Numers N, Survila M, Aalto M, Batoux M, Heino P, Palva ET et al (2010) Requirement of a homolog of glucosidease II beta-subunit for EFR-mediated defense signaling in Arabidopsis thaliana. Mol Plant 3:740–750
Häweker H, Rips S, Koiwa H, Salomon S, Saijo Y, Chinchilla D, Robatzek S, von Schaewen A (2010) Pattern recognition receptors require N-glycosylation to mediate plant immunity. J Biol Chem 285:4629–4636
Sun W, Cao Y, Jansen KL, Bittel P, Boller T, Bent AF (2012) Probing the Arabidopsis flagellin receptor: FLS2-FLS2 association and the contributions of specific domains to signaling function. Plant Cell 24:1096–1113
Chinchilla D, Bauer Z, Regenass M, Boller T, Felix G (2006) The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18:465–476
Elbein AD (1987) Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu Rev Biochem 56:497–534
Martínez IM, Chrispeels MJ (2003) Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes. Plant Cell 15:561–576
Iwata Y, Koizumi N (2005) An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants. Proc Natl Acad Sci U S A 102:5280–5285
Lozoya E, Block A, Lois R, Hahlbrock K, Scheel D (1991) Transcriptional repression of light-induced flavonoid synthesis by elicitor treatment of cultured parsley cells. Plant J 1:277–234
Lo SC, Nicholson RL (1998) Reduction of light-induced anthocyanin accumulation in inoculated sorghum mesocotyls. Implications for a compensatory role in the defense response. Plant Physiol 116:979–989
McLusky SR, Bennett MH, Beale MH, Lewis MJ, Gaskin P, Mansfield JW (1999) Cell wall alterations and localized accumulation of feruloyl-3’-methoxytyramine in onion epidermis at sites of attempted penetration by Botrytis allii are associated with actin polarisation, peroxidase activity and suppression of flavonoid biosynthesis. Plant J 17:523–534
Serrano M, Kanehara K, Torres M, Yamada K, Tintor N, Kombrink E et al (2012) Repression of sucrose/ultraviolet B light-induced flavonoid accumulation in microbe-associated molecular pattern-triggered immunity in Arabidopsis. Plant Physiol 158:408–422
Korasick DA, McMichael C, Walker KA, Anderson JC, Bednarek SY (2010) Novel functions of Stomatal Cytokinesis-Defective 1 (SCD1) in innate immune responses against bacteria. J Biol Chem 285:23342–23350
Smith JM, Leslie ME, Robinson SJ, Korasick DA, Zhang T, Backues SK et al (2014) Loss of Arabidopsis thaliana dynamin-related protein 2B reveals separation of innate immune signaling pathways. PLoS Pathog 10:e1004578
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Mancinelli AL, Schwartz OM (1984) The photoregulation of the anthocyanin synthesis IX. The photosensitivity of the response in dark and light-grown tomato seedlings. Plant Cell Physiol 25:93–105
Acknowledgments
This work was supported by T32 GM007499 (to S.A.L.) and Yale University Elizabeth Brown Postdoctoral Fellowship (to T.C.).
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Lawrence, S.A., Ceserani, T., Clay, N.K. (2017). Assays to Investigate the N-Glycosylation State and Function of Plant Pattern Recognition Receptors. In: Shan, L., He, P. (eds) Plant Pattern Recognition Receptors. Methods in Molecular Biology, vol 1578. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6859-6_6
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DOI: https://doi.org/10.1007/978-1-4939-6859-6_6
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