4D spatial transcriptomics reveals nodule identity emerges through stacked parallel developmental programs

Legume root nodules enable symbiotic nitrogen fixation through the development of specialized cells that accommodate nitrogen-fixing bacteria intracellularly and support bacterial nitrogenase activity. Here, we present a 4D (3D space and time) spatial transcriptomic atlas of Medicago truncatula nodules and lateral roots, revealing specialized symbiotic cell types that develop alongside a conserved lateral-root-derived program that underpins vascularization. Spatial profiling of both plant and bacterial transcripts resolves distinct transcriptional states and previously unrecognized cell states. Spatial analysis of developmental regulator mutants uncovers a cascading series of cell-type-specific programs during nodule maturation. LSH1/LSH2 are central regulators of these programs, and lsh1/lsh2 mutants exhibit collapse of hormonal gradients and nodule identity. Strikingly, loss of nodule identity collapses to a primary-root identity rather than lateral-root fate. This work reveals how tissue complexity emerges through stacked developmental programs sustained in distinct cellular compartments, allowing the emergence of cell types specialized for harboring nitrogen-fixing bacteria. HIGHLIGHTSO_LI>136,000 cells define a 4D spatial atlas of nodules and lateral roots C_LIO_LI3D spatial and dual-species analysis resolves dynamic host and rhizobial cell states C_LIO_LIA shared meristem generates 3 spatially coordinated symbiotic, non-symbiotic, and vascular cell programs C_LIO_LILSH1/LSH2 are critical for regulatory programs underlying nodule development and identity C_LIO_LINodule identity loss collapses toward a primary root-associated state, not a lateral root fate C_LI