Ralstonia pseudosolanacearum PhcQ Controls Quorum Sensing-Dependent Phenotypes by Binding PhcA and Maintaining Its...

The quorum-sensing (QS) system plays a crucial role in regulating the virulence of the Ralstonia solanacearum species complex (RSSC). It is controlled by the phcBSR operon and phcA genes. PhcQ may contribute to the activation of PhcA and regulates the expression of QS-dependent genes. Here, we genetically demonstrate that PhcQ functions as a key component of the QS regulatory pathway by maintaining the protein stability of PhcA in R. pseudosolanacearum OE1-1. Deletion of phcQ significantly increased the expression of genes encoding the type III secretion system but decreased the expression of genes for the synthesis of exopolysaccharide, consistent with that of PhcA. The phcQ mutants exhibited enhanced growth at early stages and retained weak virulence on tobacco plants, although they are nonvirulent on tomato plants. Using an overexpression system, we positioned PhcQ downstream of PhcB and upstream of PhcA in the QS regulatory cascade. PhcQ did not affect phcA transcription, while proteomic analysis and western blot revealed that PhcQ maintains PhcA protein stability, with PhcA protein levels reduced to approximately 41% in phcQ mutants. Further structural analysis revealed a disordered tail within the C-terminus of PhcA that is dispensable for PhcA function yet critical for protein stability. Using bimolecular fluorescence complementation and GST pull-down assay, we demonstrated that PhcQ binds directly to PhcA both in vitro and in planta, and deletion of this C-terminus tail impaired their binding affinity. These findings reveal a novel regulatory mechanism where PhcQ binds to PhcA and maintains its protein stability to ensure proper QS-dependent gene regulation in RSSC.