# Contribution of a Novel TetR/AcrR Family Transcriptional Regulator RalT of Ralstonia pseudosolanacearum Strain OE1‐1 to the Fine‐Tuning of Its Virulence

## TL;DR

The study identifies a new regulator, RalT, that fine-tunes the virulence of a plant pathogen by controlling gene expression related to quorum sensing and ralfuranone.

## Contribution

Discovery of RalT, a TetR/AcrR family regulator, as a key player in fine-tuning virulence gene regulation in Ralstonia pseudosolanacearum.

## Key findings

- RalT negatively regulates ralT and QS/Ral-dependent genes like ralA and xpsR.
- Deletion of ralT increases EPS I production and enhances virulence in the pathogen.
- Ralfuranone biosynthesis genes are regulated via positive feedback through RalT.

## Abstract

During the quorum‐sensing (QS)‐active state, the Gram‐negative phytopathogenic Ralstonia pseudosolanacearum strain OE1‐1 activates the transcriptional regulator PhcA, regulating the QS‐dependent genes including ralfuranone production‐related genes such as ralA and major exopolysaccharide EPS I production‐related genes such as xpsR encoding the transcriptional regulator XpsR, which are responsible for OE1‐1 virulence. Ralfuranone affects the regulation of more than 80% (QS/Ral‐dependent genes) of QS‐dependent genes, indicating the ralfuranone‐mediated feedback regulation of QS. To elucidate the mechanisms underlying the regulation of QS/Ral‐dependent genes, we analyzed the transcriptomes of phcA‐deletion (ΔphcA) and ralfuranone‐deficient (ΔralA) mutants, as well as strain OE1‐1 by RNA‐sequencing. We found a novel TetR/AcrR family transcriptional regulator (RalT)‐encoding gene (ralT); the expression level of ralT reduced significantly in ΔralA but not ΔphcA relative to expression level in strain OE1‐1, and RalT negatively regulated ralT. A transcriptome analysis of the ralT‐deletion mutant (ΔralT) showed that the ralT‐deletion reduced the expression levels of 89.4% of positively QS/Ral‐dependent genes including ralA and xpsR, while enhanced 44.6% of negatively QS/Ral‐dependent genes. The transcript levels of these genes were positively correlated between ΔralT and ΔphcA or ΔralA, suggesting contribution of RalT to the regulation of some QS/Ral‐dependent genes. However, the ralT‐deletion enhanced EPS I production, suggesting that RalT represses the XpsR‐independent factor(s), which is regulated PhcA and contributes to the EPS I production. Furthermore, ΔralT exhibited enhanced virulence, compared with strain OE1‐1. Collectively, results of the present genetic study suggest that RalT contributes to the exquisite fine‐tuning of OE1‐1 virulence.

Predicted regulation of quorum sensing‐, ralfuranone‐, and RalT‐dependent genes (QS/Ral/p0599‐dependent genes) via a novel TetR/AcrR family transcriptional regulator RalT in Ralstonia pseudosolanacearum strain OE1‐1 was shown here. The regulation of ralT is dependent on ralfuranone, and contribution of RalT is popular with the regulation of some quorum sensing and ralfuranone‐dependent genes (QS/Ral/p0599‐dependent genes). Furthermore, ralfuranone biosynthesis‐related genes are regulated via positive feedback through RalT, suggesting that the regulation of QS/Ral/p0599‐dependent genes is exquisitely fine‐tuned through RalT.