P-420 - Mechanisms behind nodule infection and bacteroid dedifferentiation in Rhizobium leguminosarum

Rhizobia form nitrogen-fixing symbioses with legumes, a process initiated by the perception of plant-derived flavonoids in the rhizosphere and culminating in their differentiation into bacteroids within nodules. While some rhizobia undergo terminal bacteroid differentiation and lose viability upon nodule senescence, others retain the ability to revert to a free-living state. We used time course TnSeq and RNAseq analyses to investigate the genetic requirements of Rhizobium leguminosarum during infection of determinate nodules and subsequent bacteroid dedifferentiation. We identified 704 genes essential for nodule infection and bacteroid development, several of which are nonessential for infection of indeterminate nodules, including those involved in lipopolysaccharide modification and de novo NAD⁺ biosynthesis. Genes required for dedifferentiation include those involved in iron toxicity and ribonucleotide synthesis, with mutants exhibiting reduced viability or delayed regrowth. RNAseq analysis revealed a staged dedifferentiation process: initially, a rapid loss of bacteroid identity and upregulation of translation-related genes within 1.5 hours, followed by activation of DNA biosynthesis genes, and eventually the resumption of cell division and motility over a time course of 8 hours. These findings provide new insights into the genetic regulation of nodule infection and for the first time bacteroid dedifferentiation, elucidating the mechanisms of rhizobial lifestyle changes.