Disruption of putrescine export in experimentally evolved <i>Ralstonia pseudosolanacearum</i> enhances symbiosis with <i>Mimosa pudica</i>

Polyamines are essential molecules across all domains of life, but their role as signaling molecules in host-microbe interactions is increasingly recognized. However, because they are produced by both the host and the microbe, their dual origin makes their functional dissection challenging. The plant pathogen <i>Ralstonia pseudosolanacearum</i> GMI1000 secretes large amounts of putrescine both <i>in vitro</i> and in the xylem sap of host plants. In this study, we investigated the genetic changes underlying its experimental evolution into a legume symbiont. We showed that the <i>paeA</i> gene (RSc2277), which was repeatedly mutated during this process, encodes a putrescine exporter. Mutations in <i>paeA</i> completely abolished putrescine excretion <i>in vitro</i> and enhanced bacterial proliferation within nodules during interaction with the legume <i>Mimosa pudica</i>. When these mutations occurred in symbionts already capable of intracellular infection, they further increased bacterial load in nodules and allowed the detection of nitrogenase activity. In addition, <i>paeA</i>-mutated symbionts modulated host gene expression toward a more functional symbiotic state by repressing defense-related genes and inducing nodule development genes. These nodule development genes include genes encoding leghemoglobins and an arginine decarboxylase, a key enzyme in plant putrescine biosynthesis. These results indicate that bacterial and plant putrescine have distinct functions in legume symbiosis and highlight the complex role of polyamines in plant-microbe interactions.IMPORTANCERhizobia, the nitrogen-fixing symbionts of legumes, emerged through repeated and independent horizontal transfers of some essential symbiotic genes. However, these transfers alone are often insufficient to convert the recipient bacterium into a functional legume symbiont. In a laboratory experiment, we evolved the plant pathogen <i>Ralstonia pseudosolanacearum</i> into a nodulating and intracellularly infecting symbiont of <i>Mimosa pudica</i>. This transition required genomic modifications in the recipient bacterium to activate its acquired symbiotic potential. Here, we demonstrated that one of these key adaptive modifications is the inactivation of bacterial putrescine export. This polyamine, when produced by the microsymbiont, appears to act as a negative signal for the plant. This study provides new insights into the distinct roles of bacterial- and plant-derived putrescine in plant-microbe interactions, highlighting their functional divergence despite being produced by both organisms.