# Two ferric uptake regulator proteins in Ralstonia pseudosolanacearum strain OE1-1 function cooperatively in response to the extracellular iron level under ferrous iron-rich conditions

## TL;DR

This study shows that two iron-regulating proteins in a plant pathogen work together to control iron uptake and disease-causing ability in tomato plants.

## Contribution

The study reveals cooperative function of two Fur proteins in regulating iron response and virulence in Ralstonia pseudosolanacearum.

## Key findings

- Fur1 and Fur2 repress siderophore-related genes under Fe2+-rich conditions.
- The double deletion of Fur1 and Fur2 significantly reduces bacterial virulence in tomato plants.
- Fur1 and Fur2 control extracellular Fe3+-chelating activity and growth under iron-rich conditions.

## Abstract

Soil-borne Gram-negative bacteria in the Ralstonia solanacearum species complex (RSSC) cause bacterial wilt symptoms in diverse crop plants. Although iron is an important metal ion for most organisms, the regulatory effect of iron on soil-borne RSSC gene expression remains unknown. Thus, we identified two ferric uptake regulator (Fur) proteins, Fur1 and Fur2, in R. pseudosolanacearum strain OE1-1. All four RSSC phylotype strains had two Fur homologs, a conserved Fur (Fur1) and an alternative Fur (Fur2). We also analyzed the transcriptomes of a fur1 deletion mutant, fur2 deletion mutant, and fur1⁄fur2 double deletion mutant, revealing that Fur1 and Fur2 cooperatively repress siderophore-related gene expression under Fe2+-rich conditions. Furthermore, extracellular Fe3+-chelating activity was cooperatively controlled by Fur1 and Fur2 under Fe2+-rich conditions. Additionally, nitrate metabolism-related gene expression levels were upregulated only in the fur1⁄fur2 double deletion mutant under Fe2+-rich conditions. This double deletion mutant had a lower growth level than either single deletion mutant. Virulence assays involving tomato plants revealed that single deletions (fur1 or fur2) moderately decreased virulence, whereas the double deletion (fur1 and fur2) resulted in a significant decrease in virulence. Considered together, these results suggest that the two Fur homologs cooperatively regulate gene expression under Fe2+-rich conditions to control strain OE1-1 iron uptake, growth, and virulence.

The Ralstonia solanacearum species complex (RSSC) comprising soil-borne Gram-negative phytopathogenic bacteria causes bacterial wilt diseases of diverse crop plants. Considering that phylotype I strain OE1-1 enters iron-rich roots from iron-deficient soil during an infection of tomato plants, the mechanisms controlling strain OE1-1 gene expression in response to extracellular iron levels should be clarified. In this study, RSSC was revealed to have two ferric uptake regulator homologs (Fur1 and Fur2). Notably, Fur1 and Fur2 cooperatively repress the expression of genes related to siderophores (Fe3+-chelating compounds) as well as the extracellular Fe3+-chelating activity in the presence of sufficient amounts of extracellular Fe2+. Additionally, Fur1 and Fur2 contribute to the virulence of strain OE1-1 in tomato plants. These findings suggest that RSSC uses two Fur proteins to modulate extracellular Fe3+-chelating activities in response to extracellular iron levels to maintain virulence in crop plants.