AHL quorum sensing regulates T6SS and volatiles production in rice root-colonizing <i>Enterobacter asburiae</i> AG129

Pseudomonadota (formerly Proteobacteria) commonly use a contact independent cell-cell communication system known as quorum sensing (QS) mediated by N-acyl-homoserine lactone (AHL) signal molecules. The canonical AHL QS system involves a luxI-family gene, which encodes an AHL synthase, and a luxR-family gene, which encodes a transcriptional regulator responsive to the cognate AHL(s). This study involves the AHL QS system of Enterobacter asburiae AG129, a root associated strain isolated from rice (Oryza sativa). Enterobacter asburiae AG129 produces the N-butanoyl homoserine lactone (C4-AHL) signal molecule. Genome sequencing of strain AG129 revealed the presence of a canonical AHL QS system, comprising genetically adjacent easI-like and easR-like genes. A genomic easI knockout mutant was no longer able to produce AHLs, but the in-trans complementation with a plasmid carrying the easI gene restored the AHL production. QS mediated by AHLs in AG129 was found to influence rice root colonization, and secretome analysis highlighted a significant regulatory role in the expression of Type VI secretion system (T6SS) proteins. Gas chromatography-mass spectrometry analysis identified 16 volatile organic compounds (VOCs) that were more abundantly emitted by the wild-type strain compared to the easI mutant. Overall, our findings suggest that AHL-based QS in E. asburiae AG129 positively regulates T6SS expression and VOC production, while negatively affecting root colonization and motility. This study is among the first to explore the role of QS signaling in a bacterial root-endophyte, providing evidence of a connection between QS activity and the ability of the bacterium to inhabit, compete and colonize the plant root endosphere.