From intracellular bacteria to differentiated bacteroids: transcriptome and metabolome analysis in Aeschynomene nodules using the Bradyrhizobium sp. ORS285 bclA mutant

Soil bacteria called rhizobia trigger the formation of root nodules on legume plants. The rhizobia infect these symbiotic organs and adopt an intracellular lifestyle within the nodule cells where they differentiate into nitrogen-fixing bacteroids. Several legume lineages enforce their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this process in Bradyrhizobium sp. ORS285, a symbiont of Aeschynomene spp.. In the absence of BclA, the bacteria proceed until the intracellular infection of nodule cells but they cannot differentiate into enlarged polyploid and functional bacteroids. The bclA nodule bacteria constitute thus an intermediate stage between the free-living soil bacteria and the nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied with a first transcriptome switch involving several hundreds of upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving less genes but ones that are expressed to extremely elevated levels. The transcriptomes further suggested a dynamic role for oxygen and redox regulation of gene expression during nodule formation and a non-symbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids. Importance The legume-rhizobium symbiosis is a major ecological process fueling the biogeochemical nitrogen cycle with reduced nitrogen. It represents also a promising strategy to cut down the use of chemical nitrogen fertilizers in agriculture, thereby improving its sustainability. This interaction leads to the intracellular accommodation of rhizobia within plant cells of symbiotic organs where they differentiate into nitrogen-fixing bacteroids. In specific legume clades, this differentiation process requires the bacterial transporter BclA to counteract antimicrobial peptides produced by the host. Transcriptome analysis of Bradyrhizobium wild-type and bclA mutant bacteria in culture and in symbiosis with Aeschynomene host plants dissected the bacterial transcriptional response in distinct phases and highlighted functions of the transporter in the free-living stage of the bacterial life cycle.