Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in an intracellular manner. These nodules are formed by reprogramming differentiated root cells. The model legume Medicago truncatula forms indeterminate nodules with a meristem at their apex. This organ grows by the activity of the meristem that adds cells to the different nodule tissues. In Medicago sativa it has been shown that the nodule meristem is derived from the root middle cortex. During nodule initiation, inner cortical cells and pericycle cells are also mitotically activated. However, whether and how these cells contribute to the mature nodule has not been studied. Here, we produce a nodule fate map that precisely describes the origin of the different nodule tissues based on sequential longitudinal sections and on the use of marker genes that allow the distinction of cells originating from different root tissues. We show that nodule meristem originates from the third cortical layer, while several cell layers of the base of the nodule are directly formed from cells of the inner cortical layers, root endodermis and pericycle. The latter two differentiate into the uninfected tissues that are located at the base of the mature nodule, whereas the cells derived from the inner cortical cell layers form about eight cell layers of infected cells. This nodule fate map has then been used to re-analyse several mutant nodule phenotypes. This showed, among other things, that intracellular release of rhizobia in primordium cells and meristem daughter cells are regulated in a different manner.

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.

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Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital ''in situ''. This digital ''in situ'' offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.