A Simplified Density Gradient-Free Method for Isolating Functional Symbiosomes From Legume Nodules

Symbiosomes are plant membrane-bound, organelle-like structures that enclose single or multiple bacteroids (differentiated rhizobia) within legume cells, serving specifically as the site for N₂ fixation. Although isolated symbiosomes have been used to study nitrogen fixation, ion transport, and metabolite translocation, conventional density gradient-based isolation methods for their isolation are labor-intensive. Here, we describe a simplified, density gradient-free protocol for rapid enrichment of symbiosome-containing fractions from mature legume nodules using the Medicago truncatula-Sinorhizobium meliloti symbiosis as an example. The method combines razor blade homogenization, filtration through a 40 μm cell strainer, and sequential low-speed centrifugation to remove debris and enrich symbiosome-associated fractions. The resulting material is compatible with downstream mass spectrometry-based proteomics and mutant phenotypic analyses. Compared with conventional approaches, this protocol is rapid, simple, and suitable for higher-throughput biochemical and proteomic studies of symbiosomes. Key features • Density gradient-free enrichment of symbiosome-containing fractions from mature legume nodules. • Rapid workflow using homogenization, filtration, and low-speed centrifugation with minimal specialized equipment. • Compatible with downstream mass spectrometry-based proteomic analysis of symbiosome-associated proteins. • Suitable for comparative analysis of wildtype and mutant nodules in symbiotic nitrogen fixation studies.