Mutations in legume symbiosis genes create a complex selective landscape for natural genetic variation in rhizobia during multi-strain inoculations

In the mutualism between leguminous plants and rhizobia bacteria, rhizobia live inside root nodules, creating the potential for host genes to shape the rhizobial selective environment. Single-strain screens have identified many host genes influencing symbiosis. However, it’s unknown whether these genes influence which rhizobial strains colonize and thrive inside nodules during multi-strain inoculations. In this study, we inoculated 18 Medicago truncatula symbiotic mutants (including mutations that alter NCR peptide production, plant defence, and nodule number regulation) with a mixture of 86 Sinorhizobium meliloti strains. In multi-strain inoculations, most mutations led to reduced host benefits but widely varying effects on host investment and rhizobial benefit (i.e., strain relative fitness), revealing widespread host gene by strain fitness interactions. Genome-wide association studies identify genetic variants on rhizobial replicons pSymA and pSymB as important in mediating strain fitness responses to host mutations. While most top variants only affected rhizobial fitness when one host gene was disrupted, we identified ten variant groups with pervasive effects across six or more host mutations. These variants occurred primarily on pSymA, the symbiotic replicon, and include fixL and a few metabolic genes. In contrast to the limited-effect variants, variants with pervasive positive effects in mutants tended to adversely affect strain fitness in wild-type hosts. Our results reveal how host symbiosis genes perturb the selective landscape and symbiotic outcomes for rhizobia and set the stage for improving rhizobial inoculants and breeding legume hosts better adapted for multi-strain environments.