The model legume, Medicago truncatula, is able to enter into a symbiotic relationship with soil bacteria, known as rhizobia. This relationship involves a carbon for nitrogen exchange in which the plant provides reduced carbon from photosynthesis in exchange for reduced, or “fixed” atmospheric nitrogen, which allows the plant to thrive in nitrogen depleted soils. Rhizobia infect and enter plant root organs, known as nodules, where they reside inside the plant cell in a novel organelle, known as the symbiosome where nitrogen fixation occurs. the symbiosome is enriched in plant proteins, however, little is known about the mechanisms that direct plant proteins to the symbiosome. Using the M. truncatula ENOD8 (MtENOD8) protein as a model to explore symbiosome protein targeting, 3-cis domains were identified within MtENOD8 capable of directing green fluorescent protein (GFP) to the symbiosome, including its N-terminal signal peptide (SP). the SP delivered GFP to the vacuole in the absence of nodules suggesting that symbiosome proteins share a common targeting pathway with vacuolar proteins. a time course analysis during nodulation indicated that there is a nodule specific redirection of MtENOD8-SP from the vacuole to the symbiosome in a MtNIP/LATD dependent manner. GFP expression by the MtENOD8 promoter revealed spatial discrepancy between promoter activity and protein localization. in situ localization of MtENOD8 mRNA showed localization to infected cells, where the protein is found, suggesting mRNA cell-to-cell movement. Expression of MtENOD8 in Arabidopsis showed that the SP did not direct GFP to the vacuole indicating that vacuolar targeting of MtENOD8’s SP may be legume specific. Taken together, the research presented here indicates that the MtENOD8 symbiosome protein has evolved redundant domains for targeting, which has part of a common pathway with vacuolar proteins. Observed spatial discrepancy between the MtENOD8 promoter and protein shows additional mechanisms of gene regulation through cell-to-cell mRNA movement, previously unknown in nodules.

To elaborate on the function(s) of the ENOD8 gene in the nodules of M. truncatula, several different experimental approaches were used. A census of the ENOD8 genes was first completed indicating that only ENOD8.1 (nt10554-12564 of GenBank AF463407) is highly expressed in nodule tissues. A maltose binding protein-ENOD8 fusion protein was made with an E. coli recombinant system. A variety of biochemical assays were undertaken with the MBP-ENOD8 recombinant protein expressed in E. coli, which did not yield the esterase activity observed for ENOD8 protein nodule fractions purified from M. sativa, tested on general esterase substrates, α-naphthyl acetate, and p-nitrophenylacetate. Attempts were also made to express ENOD8 in a Pichia pastoris system; no ENOD8 protein could be detected from Pichia pastoris strains which were transformed with the ENOD8 expression cassette. Additionally, it was shown that the ENOD8 protein can be recombinantly synthesized by Nicotiana benthamiana in a soluble form, which could be tested for activity toward esterase substrates, bearing resemblance to nodule compounds, such as the Nod factor. Transcription localization studies using an ENOD8 promoter gusA fusion indicated that ENOD8 is expressed in the bacteroid-invaded zone of the nodule. The ENOD8 protein was also detected in that same zone by immunolocalization. Confocal immunomicroscopy with an affinity-purified anti-ENOD8 oligopeptide antibody showed that the ENOD8 protein localizes at the interface between the plant and the bacteroid-differentiated rhizobia, in the symbiosome membrane or symbiosome space. This suggests a possible link between ENOD8 protein and bacteroid differentiation, nitrogen fixation, or plant defense. These possible functions for ENOD8 could be tested with an ENOD8-RNAi transgenic line devoid of detectable ENOD8 proteins.