Description: This article discusses multiple domains in MtENOD8 protein. Symbiotic nitrogen fixation occurs in nodules, specialized organs on the roots of legumes. Within nodules, host plant cells are infected with rhizobia that are encapsulated by a plant-derived membrane forming a novel organelle, the symbiosome. In Medicago truncatula, the symbiosome consists of the symbiosome membrane (SymM), a single rhizobium, and the soluble space between them, called the symbiosome space (Syms). The SymS is enriched with plant-derived proteins, including the MtENOD8 protein. Here, the authors present evidence from GFP fusion experiments that the MtENOD8 protein contains at least three symbiosome targeting domains, including its N-terminal signal peptide (SP). When ectopically expressed in non-nodulated root tissue, the MtENOD8 SP delivers GFP to the vacuole. During the course of nodulation, there is a nodule-specific re-direction MtENOD8-SP-GFP from the vacuole to punctate intermediates and subsequently to symbiosomes, with re-direction of MtENOD8-SP-GFP from the vacuole to punctate intermediates preceding intracellular rhizobial infection. Experiments with Medicago mutants having defects in rhizobial infection and symbiosome development demonstrated that the MtNIP/LATD gene is required for re-direction of the MtENOD8-SP-GFP from the vacuoles to punctate intermediates in nodules. The authors' evidence shows that MtENOD8 has evolved redundant targeting sequences for symbiosome ...