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

Abstract

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 ralfuranone productivity-deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces compared with strain OE1-1, losing its virulence. To analyse the 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 a scanning electron microscope. The ΔralA strain formed significantly fewer microcolonies and mBFs than strain OE1-1. Supplementation of ralfuranones A, B, J and K, but not L, significantly enhanced the development of mBF formation by ΔralA. Furthermore, a phcB- and ralA-deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1-1, although a QS-deficient, phcB-deleted mutant formed mBFs similar to OE1-1. Supplementation with 3-OH MAME significantly reduced the formation of mBFs by ΔphcB/ralA. The application of each ralfuranone significantly increased the formation of mBFs by ΔphcB/ralA supplied with 3-OH MAME. Together, our findings indicate that ralfuranones are implicated not only in the development of mBFs by strain OE1-1, but also in the suppression of QS-mediated negative regulation of mBF formation.

Keywords: Ralstonia solanacearum; mushroom-type biofilm; ralfuranones; virulence.

Figure 1

Behaviour and virulence of Ralstonia solanacearum strain OE1–1, the ralfuranone‐deficient mutant ΔralA and the complemented ΔralA mutant ralA‐comp in tomato plants. (a) The population of R. solanacearum strains in the roots of tomato plants inoculated by root dipping was analysed using Hara–Ono medium. CFU, colony‐forming unit. (b) Ralstonia solanacearum strains in roots (I) and stems (II and III) of tomato plants at 10 days after inoculation by root dipping were detected using the plate‐printing assay. (c) Bacterial wilt on tomato plants inoculated with R. solanacearum strains by root dipping was assayed. Plants were rated on a 0–5 disease index scale: 0, no wilting; 1, 1%–25% wilting; 2, 26%–50% wilting; 3, 51%–75% wilting; 4, 76%–99% wilting; 5, dead. Bars indicate standard errors. Asterisks indicate a significant difference from the wild‐type (P < 0.05, t‐test).

Figure 2

The numbers of microcolonies of 1–5 μm in diameter, immature mushroom‐type biofilms (mBFs) of 5–10 μm in diameter and mature mBFs of >10 μm in diameter produced by cells of Ralstonia solanacearum strain OE1–1 incubated in tomato apoplast fluid on nano‐percolators for 24 or 32 h. Bars indicate the standard errors. Asterisks indicate a significant difference from OE1–1 incubated for 24 h (P < 0.05, t‐test).

Figure 3

Influence of ralfuranones on the formation of microcolonies of 1–5 μm in diameter (a), immature mushroom‐type biofilms (mBFs) of 5–10 μm in diameter (b) and mature mBFs of >10 μm in diameter (c) by Ralstonia solanacearum strains. Cells of R. solanacearum strain OE1–1, the ralfuranone‐deficient mutant (ΔralA), with or without application of ralfuranones A, B, J, K or L at concentrations of 20 μm, and the complemented ΔralA mutant (ralA‐comp) incubated in tomato apoplast fluid on nano‐percolators were observed under a scanning electron microscope. Bars indicate the standard errors. Asterisks indicate a significant difference from ΔralA (P < 0.05, t‐test).

Figure 4

Influence of phc quorum sensing (QS) on the formation of microcolonies of 1–5 μm in diameter (a), immature mushroom‐type biofilms (mBFs) of 5–10 μm in diameter (b) and mature mBFs of >10 μm in diameter (c) by Ralstonia solanacearum strains. Cells of R. solanacearum strain OE1–1, the phc QS‐deficient mutant (ΔphcB), and the phcB‐ and ralA‐deficient mutant (ΔphcB/ralA), with or without application of methyl 3‐hydroxymyristate (3‐OH MAME) at a concentration of 100 nm, incubated in tomato apoplast fluid on nano‐percolators were observed under a scanning electron microscope. Bars indicate the standard errors. Asterisks indicate a significant difference from ΔralA (P < 0.05, t‐test).

Figure 5

Effect of phc quorum sensing and ralfuranones on the formation of microcolonies of 1–5 μm in diameter (a), immature mushroom‐type biofilms (mBFs) of 5–10 μm in diameter (b) and mature mBFs of >10 μm in diameter (c) by Ralstonia solanacearum strains. Cells of R. solanacearum strain OE1–1 and the phcB‐ and ralA‐deficient mutant ΔphcB/ralA, with application of methyl 3‐hydroxymyristate (3‐OH MAME) at a concentration of 100 nm and with or without application of ralfuranones A, B, J, K or L at a concentration of 20 μm, incubated in apoplast fluids from tomato plants on nano‐percolators were observed under a scanning electron microscope. Bars indicate the standard errors. Asterisks indicate a significant difference from ΔphcB/ralA supplemented with 3‐OH MAME (P < 0.05, t‐test).

Figure 6

Production of exopolysaccharide I (EPS I) and cell aggregation by the epsB‐deleted ΔepsB mutant of Ralstonia solanacearum. (a) Immunological quantification of EPS I in the supernatants of the phc quorum sensing (QS)‐deficient mutant ΔphcB and ΔepsB of R. solanacearum was analysed using enzyme‐linked immunosorbent assay (ELISA) with anti‐R. solanacearum EPS I antibodies. EPS I productivity was quantified by the absorbance at 650 nm (A ₆₅₀). (b) Cell aggregation by R. solanacearum strains incubated in 1/4 × M63 medium in polyvinylchloride (PVC) plate wells was stained with crystal violet and its absorbance at 550 nm (A ₅₅₀) was assayed. Bars indicate the standard errors. Asterisks indicate significant difference from wild‐type strain OE1–1 (P < 0.05, t‐test).

Figure 7

Influence of exopolysaccharide I (EPS I) on the formation of microcolonies of 1–5 μm in diameter (a), immature mushroom‐type biofilms (mBFs) of 5–10 μm in diameter (b) and mature mBFs at >10 μm in diameter (c) by Ralstonia solanacearum strains. Cells of R. solanacearum strains OE1–1 and the epsB‐deleted ΔepsB mutant incubated in apoplast fluids from tomato plants on nano‐percolators were observed under a scanning electron microscope. Bars indicate the standard errors. Asterisks indicate a significant difference from ΔphcB/ralA supplemented with methyl 3‐hydroxymyristate (3‐OH MAME) (P < 0.05, t‐test).