Competitiveness prediction for nodule colonization in Sinorhizobium meliloti through combined in vitro tagged strain characterization and genome-wide association analysis

<p>Associations between leguminous plants and symbiotic nitrogen-fixing rhizobia are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Although this symbiosis is in part species-specific, different rhizobial strains may colonise the same nodule. Some rhizobial strains are commonly known as better competitors than others, but detailed analyses that aim to predict rhizobial competitive abilities based on genomes are still scarce. Here, we performed a bacterial <em>genome-wide association (GWAS) analysis to define the </em>genomic determinants related to the competitive capabilities in the model rhizobial species <em>Sinorhizobium meliloti.</em> For this, 13 tester strains were GFP-tagged and assayed <i>vs.</i> 3 RFP-tagged reference competitor strains (<em>Rm1021, AK83, and BL225C) in a</em> <i>Medicago sativa</i> nodule occupancy test. Competition data and strain genomic sequences were employed to build a model for GWAS based on <i>k</i>-mers. Among the <i>k</i>-mers with the highest scores, 51 <i>k</i>-mers mapped on the genomes of four strains showing the highest competition phenotypes (&gt; 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) <i>vs.</i> BL225C. These <i>k</i>-mers were mainly located on the symbiosis-related megaplasmid pSymA, specifically on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (e.g., fatty acids). The same analysis was performed considering the sum of single and mixed nodules obtained in the competition assays <em>vs. </em>BL225C, retrieving <i>k</i>-mers mapped on the genes previously found and on <i>vir</i> genes. Therefore, the competition abilities seem to be linked to multiple genetic determinants and comprise several cellular components.</p>