Ralfuranones secreted by Ralstonia solanacearum function as intercellular second messenger molecules during its biofilm formation
1Fac. of Agri., Kochi Univ., 2Osaka Pref. Univ., 3Osaka Univ., 4Kobe Univ., 5RIMG, Kochi Univ.
After invasion into intercellular spaces of tomato plants, cells of Ralstonia solanacearum strain OE1-1 (OE1-1) attach on tomato cells and produce biofilm structures. This biofilm formation is required for its virulence. OE1-1 cells produce mushroom-type biofilms when incubated in apoplast fluids but not xylem fluids from tomato plants in a cell-density dependent manner, quorum sensing (QS). OE1-1 produces and extracellularly secretes ralfuranones including ralfuranone A, ralfuranone B, ralfuranone I, ralfuranone J, ralfuranone K and ralfuranone L, dependently on a furanone synthetase encoded by the ralA gene, of which expression is positively regulated by PhcA activated in QS. The deletion mutant of ralA (ΔralA) loses its virulence on tomato plants. In this study, we analyzed involvement of ralfuranones in biofilm formation by OE1-1 cells. The analysis on biofilm formation using the crystal violet staining showed that the ΔralA produced biofilms significantly less than OE1-1. Observation under a scanning electron microscope showed that the ΔralA did not form any mushroom-type biofilm. The application with ralfuranones partially recovered production of mushroom-type biofilms by the ΔralA. The transcriptome analysis with the RNA-seq of R. solanacearum strains showed that the activated PhcA was implicated in positive regulation of expression of many virulence-related genes including extracellular polysaccharide (EPS) production-related genes. The deletion of ralA gene resulted in significantly reduced expression of about 80 % of PhcA-positively regulated genes including EPS production-related genes. The deletion of ralA gene resulted in significantly reduced EPS production. The ralfuranones application recovered expression of EPS production-related genes. Taken together, during biofilm formation, ralfuranones may function as second messenger molecules in intercellular signaling systems of OE1-1 cells, involved in biofilm formation.
keywords:R. solanacearum,biofilm,ralfuranones,second messenger molecules