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.