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Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures
Author(s) -
Goossens Jonas,
De Geyter Nathan,
Walton Alan,
Eeckhout Dominique,
Mertens Jan,
Pollier Jacob,
FiallosJurado Jennifer,
De Keyser Annick,
De Clercq Rebecca,
Van Leene Jelle,
Gevaert Kris,
De Jaeger Geert,
Goormachtig Sofie,
Goossens Alain
Publication year - 2016
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13258
Subject(s) - medicago truncatula , legume , tandem , isolation (microbiology) , medicago , biology , botany , chemistry , biochemistry , microbiology and biotechnology , symbiosis , genetics , engineering , bacteria , gene , aerospace engineering
Summary Tandem affinity purification coupled to mass spectrometry ( TAP ‐ MS ) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP ‐ MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes ‐mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta . Subsequently, binary protein–protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two‐hybrid screen. Together, by establishing a M. truncatula TAP ‐ MS platform, we extended the molecular toolbox of this model species.