Studying the biochemical function of the pea receptor-like kinases sym10, sym37 and k1, required for the legume-rhizobia symbiosis development
- Authors: Dolgikh E.A.1, Kirienko A.N.1, Kovaleva O.D.1, Tikhonovich I.A.1
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Affiliations:
- All-Russian Research Institute for Agricultural Microbiology
- Issue: Vol 15, No 4 (2017)
- Pages: 4-12
- Section: Genetic basis of ecosystems evolution
- URL: https://journals.rcsi.science/ecolgenet/article/view/6164
- DOI: https://doi.org/10.17816/ecogen1544-12
- ID: 6164
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Abstract
Background. Rhizobial Nod factors (NFs), the key regulators of legume-rhizobia symbiosis, act in low concentrations and their biological activity depends on structural features, that suggests the presence of specific receptors in plants. Putative receptors, LysM-receptor-like kinases (LysM-RLKs), were found in model legumes L. japonicus and M. truncatula. However, binding capacity with NFs was only studied for L. japonicus LysM-RLKs. In pea a few candidates for NF receptors like Sym10, Sym37 and K1 were found. Analysis of mutants revealed the importance of these proteins for symbiosis development. However, the biochemical function of these receptors has not been studied.
Materials and methods. Sequences encoding extracellular domains (ECDs) of LysM-RLKs Sym10, Sym37, and K1 were cloned in the pRSETa vector. Constructs were introduced in E. coli strain C41 to produce proteins with His6 residues on either the amino or carboxyl terminus. Protein purification was carried out using metal chelate affinity chromatography. The binding capacity with ligand was evaluated using ProteonXPR36 biosensor.
Results. To study binding capacity with NFs, we have developed approaches for the synthesis of LysM-RLK Sym10, Sym37 and K1 in soluble form in heterologous system. The high level of protein synthesis was achieved at +28 °C using 0,5 mM IPTG in 2-16 hours. Analysis of binding capacity of ECDs with NFs revealed the low affinity using the surface plasmon resonance.
Conclusion. The possibility of recombinant receptor synthesis in soluble state in E. coli at high level was demonstrated. Analysis of binding capacity with NFs showed the potential interaction, but with low affinity.
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##article.viewOnOriginalSite##About the authors
Elena A. Dolgikh
All-Russian Research Institute for Agricultural Microbiology
Author for correspondence.
Email: dol2helen@yahoo.com
ORCID iD: 0000-0003-3433-2102
SPIN-code: 4453-2060
Group leader, Laboratory of molecular and cell biology
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608Anna N. Kirienko
All-Russian Research Institute for Agricultural Microbiology
Email: kirienkoann@yandex.ru
Laboratory of molecular and cell biology
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608Oksana D. Kovaleva
All-Russian Research Institute for Agricultural Microbiology
Email: meriones@list.ru
PhD student, Laboratory of molecular and cell biology
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608Igor A. Tikhonovich
All-Russian Research Institute for Agricultural Microbiology
Email: arriam2008@yandex.ru
Professor, Director
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608References
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