Video: https://youtu.be/oQjUYKyfR_E
Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. This video showcases a 3D volumetric rendering of a GFP-SIMK line, a genetically modified plant expressing Green Fluorescent Protein (GFP), interacting with E. meliloti expressing monomeric Red Fluorescent Protein (mRFP). This dual-fluorescence approach allows for simultaneous visualization of both plant and bacterial components within the root system.
To characterize the GFP–SIMK localization pattern during early nodulation stages in vitro, live-cell imaging of alfalfa roots stably expressing GFP-tagged SIMK, co-cultivated with mRFP-labelled E. meliloti, was performed by LSFM at 3–4 dpi. This model allows the simultaneous and independent study of root hairs symbiotically interacting with rhizobia, but also root hairs untouched by rhizobia that were exposed to air inside the Petri dish. Both types of root hairs are present on the same root exposed to the same conditions and treatments. Therefore, 3D rendering of symbiotically infected GFP-SIMK root enabled us to distinguish not only non-interacting alfalfa root hairs from those that interact with rhizobia but also the position of their nuclei with respect to infection.
Keywords: Alfalfa, Ensifer meliloti, infection pocket, immunolocalization, infection thread, light-sheet fluorescence microscopy, MAPKs, root hairs, SIMK, subcellular localization
Citation: Kateřina Hlaváčková, Olga Šamajová, Miroslava Hrbáčková, Jozef Šamaj, Miroslav Ovečka, Advanced microscopy resolves dynamic localization patterns of stress-induced mitogen-activated protein kinase (SIMK) during alfalfa root hair interactions with Ensifer meliloti, Journal of Experimental Botany, Volume 74, Issue 12, 27 June 2023, Pages 3729–3748, https://doi.org/10.1093/jxb/erad111
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