Spatiotemporal analysis of cell division during symbiotic root nodule development in the model legume Medicago truncatula

This protocol combines rhizobial spot inoculation with deep-tissue imaging to capture cellular processes during early nodule development in the legume Medicago truncatula . We describe steps to visualize DNA replication activity and cell geometry as indicators of cell proliferation and cell expansion processes. We detail steps for rhizobial spot inoculation of seedlings, incubation in 5-ethynyl-2′-deoxyuridine (EdU) medium, sample fixation and labeling of replicated DNA, clearing, staining of the cell walls, followed by confocal imaging. For complete details on the use and execution of this protocol, please refer to Lee et al. 1 • Instructions on preparing Rhizobia bacteria and Medicago seedlings for spot inoculation • Guidance on root zone marking and the spot application of the bacterial droplet • Steps for DNA labeling, tissue clearing, cell wall staining, and imaging of nodules Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. This protocol combines rhizobial spot inoculation with deep-tissue imaging to capture cellular processes during early nodule development in the legume Medicago truncatula . We describe steps to visualize DNA replication activity and cell geometry as indicators of cell proliferation and cell expansion processes. We detail steps for rhizobial spot inoculation of seedlings, incubation in 5-ethynyl-2′-deoxyuridine (EdU) medium, sample fixation and labeling of replicated DNA, clearing, staining of the cell walls, followed by confocal imaging.