| The symbiosome membrane in Medicago truncatula nodules is a specialized, plasmalemma-derived membrane that surrounds each nitrogen-fixing bacteroid. Functions attributed to the symbiosome membrane include serving as a structural barrier between the host and internalized microbe and controlling metabolite exchange between the symbionts. Symbiosome membrane biogenesis and maintenance demands a considerable amount of membrane and lipid material; however, little is known about how proteins are selectively targeted to the membrane and how they function. To better understand Medicago truncatula symbiosome membrane formation and function, a proteomic approach was taken to identify proteins that facilitate biochemical specialization of this membrane. Using 2D SDS-PAGE followed by HPLC-tandem mass spectrometry, 51 putative symbiosome membrane proteins were identified. Plant proteins identified were categorized into functional classes to more clearly define their biochemical functions. Using this information, a model that hypothesizes four potential pathways involved in symbiosome membrane formation was devised.; In depth analysis of individual proteins identified in the proteomic study followed. To analyze two proteins identified, MtSYP132 and MtENOD16, a peptide specific polyclonal antibody was generated for each and used as a biochemical marker in determining their subcellular localization. Biochemical data suggests that MtSYP132, a syntaxin protein, is a component of the symbiosome membrane. MtSYP132 subcellular distribution within the symbiosome membrane was confirmed using immunomicroscopy. A second protein, MtENOD16, is a phytocyanin-related protein hypothesized to be GPI-anchored to the symbiosome membrane. Biochemical data and immunomicroscopy confirms that MtENOD16 is abundant in the symbiosome membrane. Immunomicroscopy also demonstrates that MtENOD16 is present in the infection thread matrix, suggesting possible cleavage of the GPI anchor and protein secretion into the extracellular matrix. Transgenic plants generated using RNAi to reduce MtENOD16 gene expression demonstrated that MtENOD16 has a role in infection thread growth and a second role in bacterial release from the infection thread.; Collectively, the results of this work demonstrate the unique, biochemical nature of the symbiosome membrane and describe two symbiosome membrane proteins not previously reported in the literature. The identification and subcellular localization of MtSYP132 and MtENOD16 shed insight into specific mechanisms that may function in membrane specialization in nodules. |
