Expression and purification of Nod factor receptors - Initial characterization of ligand binding

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

  • Angelique Broghammer, Danmark
Carbohydrate signals have been shown to regulate defence, growth and development in plants. Decorated chitin molecules, lipochitooligosaccharides, synthesized and secreted by rhizobia are the major signal molecules initiating the plant processes establishing legume-rhizobia symbiosis. Lipochitooligosaccharides also serve as signals in the mutually beneficial interactions between arbuscular mycorrhiza (AM) and most land plants. In the model legume Lotus japonicus the Nod factor receptors, LjNFR1 and LjNFR5, two LysM receptor like kinases (LysM-RLK), are responsible for perceiving the rhizobial lipochitooligosaccharide, also called Nod factor, which is synthesized
by its symbiont Mesorhizobium loti.
To characterize the Nod factor receptors biochemically, expression and purification of recombinant LjNFR1 and LjNFR5 has been attempted in various heterologous expression systems. Two plant-based expression systems were optimized and LjNFR1- and LjNFR5-GFP/YFP fusion proteins were successfully expressed in A. thaliana and N. benthamiana and subsequently assayed for function. Analysis of post translational processing of the expressed receptors showed that: 1) The N-terminal signal peptides were removed; 2) LjNFR1 and LjNFR5 ectodomains were glycosylated; 3) LjNFR1 retained its in vitro kinase activity and 4) LjNFR1 and LjNFR5 were localized to the plasma membrane. In depth mass spectroscopy analysis of the N-glycan structure of LjNFR5 resulted in identification of two different glycan structures with identical chemical composition, Man3XylFucGlcNAc4.
In an early attempt to investigate the Nod factor perception mechanism, direct binding of Nod factor was visualized in a bead assay with fluorescently labeled Nod factor and GFP-tagged fusion protein purified from the membrane fraction of leaf material, expressing recombinant LjNFR1 or LjNFR5. By subsequent use of quantitative solid phase and free solution techniques, binding constants in the nanomolar range for both LjNFR1 and LjNFR5 were determined. Structure dependent ligand specificity was shown using chitooligosaccharides. Finally, two emerging techniques, Microscale Thermophoresis and Glycochips, are presented together with preliminary data demonstrating their future relevance. The established ligand binding assays can be applied to characterization of other LysM-RLKs and their respective ligands.
ForlagAarhus Universitet
Antal sider193
StatusUdgivet - feb. 2012

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