Proteomic and metabolomic profiling depicts the functional landscape of the Medicago truncatula symbiosome.

The symbiosome, a temporary plant organelle enabling nitrogen fixation in legume-rhizobia symbiosis, consists of a plant-derived symbiosome membrane (SM), symbiosome space (SS), and enclosed bacteroid. Here, we isolate and purify symbiosomes from Medicago truncatula-Sinorhizobium meliloti root nodules and perform label-free quantitative mass spectrometry to profile protein abundances in the symbiosomes. We identify 1,018 M. truncatula proteins, including 829 in the SM and 457 in the SS. Combined with transport assays, our data reveal multiple dicarboxylate transporters in the SM that potentially deliver carbon sources to bacteroids. The SM is enriched in membrane trafficking proteins, lipid raft-associated components, and receptor-like proteins, together with numerous cell wall-associated proteins, highlighting the extracellular properties of the symbiosome. Proteomic and metabolomic analyses reveal the SS as a metabolically active compartment enriched in both plant and rhizobial proteins involved in carbon and amino acid metabolism. These findings offer insights into the molecular basis of symbiotic nitrogen fixation.