Abstract
One of the mechanisms mediating the legume–rhizobium symbiosis is the molecular recognition at the plasma membrane of signaling molecules called Nod factors . In the model legume Medicago truncatula , two Nod factor receptor kinases have been described: NFP and LYK3 . These plasma membrane-localized proteins are present at very low amounts in root tissues, and even using antibodies specific against them, detection remains challenging. In the current work, we tested several methods for the extraction, enrichment, and immunodetection of LYK3 in root extracts using M. truncatula lines expressing a tagged version of LYK3, LYK3-HAST. Upon differential centrifugation, a significant fraction of LYK3 was found to precipitate at speed at which plasma membrane markers remain soluble. The sedimentation pattern of LYK3 was similar to that of markers for the endoplasmic reticulum (ER) and vacuole, suggesting that LYK3 may be also present in larger endomembrane structures. Affinity columns were shown to be effective for the enrichment of the protein, leading to an increase in the signal detected. Interestingly, LYK3 (or at least the tagged N-terminal fraction of the protein) was reproducibly identified at a range of molecular sizes, which are likely to correspond to the monomer, dimer, and a truncated form. This observation opens up the possibility of the existence of post-translational regulation mechanisms (e.g., cleavage) with likely implications in signaling.
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Acknowledgments
M.M.C. received funding from the Public University of Navarra, and E.L. is the recipient of a Juan de la Cierva fellowship. This work has been financed by the Marie Curie IOF “SymBioSignal” project, the MINECO AGL2011-30386-C02-01/AGR project, and the Fundación Caja Navarra project CAN2015-70497.
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Chroni, M.M., Larrainzar, E. (2016). Optimization of the Immunodetection of Tagged-LYK3 Nod Factor Receptor in Medicago truncatula Root Extracts. In: González-Andrés, F., James, E. (eds) Biological Nitrogen Fixation and Beneficial Plant-Microbe Interaction. Springer, Cham. https://doi.org/10.1007/978-3-319-32528-6_8
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DOI: https://doi.org/10.1007/978-3-319-32528-6_8
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