Incompatible symbiotic interactions between Sinorhizobium meliloti strain Rm41 and the ecotypes of the host Medicago truncatula

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Legumes develop a symbiosis with nitrogen-fixing soil bacteria called rhizobia. In this relationship, legumes provide an optimal environment and energy source for rhizobia. In return, rhizobia fix atmospheric nitrogen into ammonia for the plants. A significant characteristic of legume-rhizobia symbiosis is its high level of specificity. Each rhizobial species/strain can form successful symbiosis with only a few or even single legume species/genotype(s), and vice versa. Such specificity can exist at multiple stages during the nodule development and functioning and failure to suit the requirements results in different phenotypes. It can occur at early stages associated with bacterial infection and nodulation, leading to Nod- phenotype. It also may occur at later stages of nodule development associated with nitrogen fixation, leading to Fix- phenotype. Even when nitrogen-fixing symbiosis is formed successfully, the efficiency of nitrogen fixation differs between different legume-rhizobia pairings. Our research group screened several ecotypes from Medicago truncatula using different Sinorhizobium meliloti and Sinorhizobium medicae strains as inoculants and identified incompatibility between S. meliloti strain Rm41 and M. truncatula ecotypes F83005 and Jemalong. To identify those existing or missing bacterial gene(s) that caused the incompatibilities, we created an ORFeome library carrying all predicted genes of S. meliloti strain 1021, a large insert size genomic library from S. meliloti strain FSM-MA, insertion and chemically induced mutant populations of strain Rm41 and inoculated transconjugant and mutant pools of strain Rm41 on F83005 and Jemalong. We identified 6 insertion mutants which restored the compatibility between F83005 and Rm41 and carried the insertion mutation in the same gene annotated as BN406_06091. The gene BN406_06091 is located on the second symbiotic megaplasmid and encodes a protein of unknown function. It is surrounded by a number of genes coding for proteins involved in sugar and polysaccharide synthesis and modifications, implicating its role in polysaccharide production. We suppose that the BN406_06091 gene containing region is responsible for the production of the strain-specific O-antigen of lipopolysaccharide (LPS) and created mutants that might be affected in the LPS structure. Unfortunately, none of the created mutants could restore the compatibility with F83005. In future work, we will delete the whole strain-specific and alternatively the whole genus-specific LPS regions and check the phenotype of the mutants as well as that of the mutants carrying the BN406_06091 gene. We will also explore the natural variations in Sinorhizobium-specific polysaccharide production related genes: we identified strains with some missing genes in the region with or without a BN406_06091 homologue and also with different core structure. We will introduce either the whole or delimited regions with or without mutation in the BN406_06091 gene and the phenotype of the strains and their derivatives will be investigated.

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