Dual-Transcriptome Dissection of the Mechanisms Underlying Alfalfa Phenotypic Differences Induced by Two Rhizobial Isolates

Different rhizobial strains can lead to distinct symbiotic phenotypes in alfalfa, yet molecular differences at the mature nodule stage remain unclear. Here, we analyzed 21-day post-inoculation (dpi) nodules induced by strains <i>WE2</i> and <i>WWL2</i>. We measured nitrogenase activity (acetylene reduction assay, ARA) and performed dual RNA-seq to compare gene expression in both the alfalfa host and the rhizobia. On the host side, <i>WE2</i>-induced nodules showed higher expression of mature nodule marker genes (<i>ENOD93</i> and leghemoglobin (Lb) genes) and higher expression of genes encoding SWEET transporters and amino acid and peptide transporters. Host differentially expressed genes were enriched in pathways related to transmembrane transport, redox and heme-related functions, and processes linked to maintaining microaerobic conditions. On the rhizobial side, <i>WE2</i> nodules showed higher expression of genes involved in microaerobic respiration and nitrogen fixation (e.g., <i>nif</i>/<i>fix</i> and key respiratory chain genes), whereas <i>WWL2</i> nodules showed higher expression of genes linked to transport, chemotaxis/motility, and environmental information processing. Together, these host and rhizobia expression patterns suggest coordinated differences between host pathways related to resource supply and microaerobic conditions and rhizobial expression programs for respiration and nitrogen fixation. Based on these associations, we propose a working model and provide candidate genes and pathways for functional validation and inoculant screening.