Multiple domains in MtENOD8 protein including the signal peptide target it to the symbiosome

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, a single rhizobium, and the soluble space between them, called the symbiosome space. The symbiosome space is enriched with plant-derived proteins, including the M. truncatula EARLY NODULIN8 (MtENOD8) protein. Here, we present evidence from green fluorescent protein (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 nonnodulated root tissue, the MtENOD8 SP delivers GFP to the vacuole. During the course of nodulation, there is a nodule-specific redirection of MtENOD8-SP-GFP from the vacuole to punctate intermediates and subsequently to symbiosomes, with redirection of MtENOD8-SP-GFP from the vacuole to punctate intermediates preceding intracellular rhizobial infection. Experiments with M. truncatula mutants having defects in rhizobial infection and symbiosome development demonstrated that the MtNIP/LATD gene is required for redirection of the MtENOD8-SP-GFP from the vacuoles to punctate intermediates in nodules. Our evidence shows that MtENOD8 has evolved redundant targeting sequences for symbiosome targeting and that intracellular localization of ectopically expressed MtENOD8-SP-GFP is useful as a marker for monitoring the extent of development in mutant nodules.