Ralfuranones contribute to mushroom‐type biofilm formation by Ralstonia solanacearum strain OE1‐1

SUMMARY After invasion into intercellular spaces of tomato plants, the soil-borne plant pathogenic Ralstonia solanacearum strain OE1-1 forms mushroom-shaped biofilms (mushroom-type biofilms, mBFs) on tomato cells, leading to its virulence. The strain OE1-1 produces aryl-furanone secondary metabolites, ralfuranones (A, B, J, K and L), dependent on the quorum sensing (QS) system with methyl 3-hydroxymyristate (3-OH MAME) synthesized by PhcB as a QS signal. Ralfuranones are associated with the feedback loop of the QS system. A ralfuranones productivity-deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces than strain OE1-1, losing its virulence. To analyse function of ralfuranones in mBF formation by OE1-1 cells, we observed cell aggregates of R. solanacearum strains statically incubated in tomato apoplast fluids on filters under the scanning electron microscope. The ΔralA strain formed microcolonies and mBFs significantly less than strain OE1-1. Supplement of ralfuranones A, B, J or K but not L significantly enhanced development of mBF formation by the ΔralA. Furthermore, a phcB- and ralA-deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1-1, though a QS-deficient phcB-deleted mutant formed mBFs similar to OE1-1. Supplementation with 3-OH MAME significantly reduced formation of mBFs by the ΔphcB/ralA. Application of each ralfuranone significantly increased formation of mBFs by the ΔphcB/ralA supplied with 3-OH MAME. Together, our findings indicate that ralfuranones are implicated in not only development of mBFs by strain OE1-1 but also suppression of the QS-mediated negative regulation of mBF formation. This article is protected by copyright. All rights reserved.