# Disruption of putrescine export in experimentally evolved Ralstonia pseudosolanacearum enhances symbiosis with Mimosa pudica

## TL;DR

Disrupting putrescine export in a plant pathogen helps it become a better symbiont for a legume, showing how polyamines affect plant-microbe interactions.

## Contribution

The study identifies that inactivating the paeA gene, which encodes a putrescine exporter, is key to enhancing symbiosis in Ralstonia pseudosolanacearum with Mimosa pudica.

## Key findings

- Mutations in paeA abolished putrescine excretion and increased bacterial proliferation in legume nodules.
- paeA-mutated bacteria induced functional symbiosis by modulating host gene expression toward nodule development.
- Bacterial-derived putrescine acts as a negative signal for the plant, while plant-derived putrescine has distinct roles in symbiosis.

## Abstract

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 Ralstonia pseudosolanacearum GMI1000 secretes large amounts of putrescine both in vitro 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 paeA gene (RSc2277), which was repeatedly mutated during this process, encodes a putrescine exporter. Mutations in paeA completely abolished putrescine excretion in vitro and enhanced bacterial proliferation within nodules during interaction with the legume Mimosa pudica. 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, paeA-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.

Rhizobia, 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 Ralstonia pseudosolanacearum into a nodulating and intracellularly infecting symbiont of Mimosa pudica. 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.