The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces

Abstract

The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence.

Keywords: Ralstonia solanacearum; biofilm; intercellular spaces; lectin; soil-borne vascular plant-pathogenic bacterium; virulence.

Figure 1

Colonization of intercellular spaces of tomato leaves by Ralstonia solanacearum strain OE1‐1. Fluorescence from green fluorescent protein (GFP)‐labelled R. solanacearum strain OE1‐1 (gOE1‐1) in intercellular spaces of tomato leaves 18 h after infiltration was observed under a fluorescent phase contrast microscope.

Figure 2

Formation of a microcolony (a) and a biofilm (b) produced by Ralstonia solanacearum strain OE1‐1 on the surfaces of tomato cells adjacent to intercellular spaces. Tomato leaves with the lower epidermis layers excised at 18 h (a) and 24 h (b) post‐infiltration with OE1‐1, and non‐inoculated leaf with the lower epidermis layers excised at 24 h (c), were observed under a scanning electron microscope. Arrows show planktonic OE1‐1 cells released from the biofilm structure. Scale bar, 5 μm.

Figure 3

Biofilm formation by Ralstonia solanacearum strains in vitro. (a) Biofilm formation by R. solanacearum strain OE1‐1 incubated in apoplast fluids and xylem fluids from tomato plants, and in ¼ × M63 medium in PVC plate wells stained with crystal violet. (b) Biofilm formation by strains OE1‐1, OE1‐1‐lecM::EZ‐Tn5 (lecM mutant) and the lecM mutant complemented with native lecM (lecM‐comp) incubated in ¼ × M63 medium in PVC plate wells stained with crystal violet. Asterisks indicate significant difference from wild‐type cells growing in apoplast fluids (P < 0.05, t‐test).

Figure 4

Observation of biofilm configurations produced by Ralstonia solanacearum strains under a scanning electron microscope. OE1‐1 was incubated in apoplast fluids (a–c) and xylem fluids (d, e) from tomato plants on nano‐percolators. OE1‐1‐lecM::EZ‐Tn5 (lecM mutant) (f, g) and the lecM mutant complemented with native lecM (lecM‐comp) (h) were incubated in apoplast fluids from tomato plants on nano‐percolators. Arrows show planktonic OE1‐1 cells released from biofilm structures. Scale bar, 5 μm.

Figure 5

Biofilm formation by strains OE1‐1 (a) and OE1‐1‐lecM::EZ‐Tn5 (lecM mutant, b) incubated in ¼ × M63 medium containing 1.0% arabinose (Ara), fructose (Fru), fucose (Fuc), galactose (Gal) or mannose (Man) in PVC plate wells stained with crystal violet. Asterisks indicate significant difference from wild‐type cells growing in apoplast fluids (P < 0.05, t‐test).

Figure 6

Population dynamics of Ralstonia solanacearum strains OE1‐1, OE1‐1‐lecM::EZ‐Tn5 (lecM mutant) and the lecM mutant complemented with native lecM (lecM‐comp) in leaves (a) and roots (b) of tomato plants inoculated by leaf infiltration and root dipping, respectively. Asterisks indicate significant difference from the wild‐type (P < 0.05, t‐test). CFU, colony‐forming unit.

Figure 7

Virulence of Ralstonia solanacearum strains on tomato plants. Bacterial wilt on 8‐week‐old tomato plants inoculated by leaf infiltration (a) and root dipping (b) with R. solanacearum strains OE1‐1, OE1‐1‐lecM::EZ‐Tn5 (lecM mutant) and the lecM mutant complemented with native lecM (lecM‐comp). Plants were rated on the following disease index scale: 0, no wilting; 1, 1%–25% wilting; 2, 26%–50% wilting; 3, 51%–75% wilting; 4, 76%–99% wilting; 5, dead.

Figure 8

Attachment ability and microcolony formation of Ralstonia solanacearum strains. Nitrocellulose membranes (2 × 2 cm²) prepared from Visking cellulose tubes (a) and glass slides (b–d) were soaked in suspensions of R. solanacearum strains OE1‐1 (a, b), OE1‐1‐lecM::EZ‐Tn5 (lecM mutant, c) and the lecM mutant complemented with native lecM (lecM‐comp, d).

Figure 9

Expression of lecM in Ralstonia solanacearum strain OE1‐1 cultured in apoplast fluids from tomato plants, harvested at an optical density at 600 nm (OD₆₀₀) of 0.01 and 0.3, and used for RNA extraction as described in Experimental procedures. The rpoD gene was used as an internal control for quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). The RNA levels of the analysed genes are expressed relative to the rpoD expression level. The experiment was performed at least twice using independent batches of samples with similar results. Results from a single representative sample are shown. The means ± standard deviation (SD) (error bars) of three determinations from cDNA from a single representative sample are shown. CFU, colony‐forming units.

Figure 10

Immunological quantification of exopolysaccharide I (EPS I) in supernatants using enzyme‐linked immunosorbent assay (ELISA) with anti‐R. solanacearum EPS I antibodies [optical density at 650 nm (OD₆₅₀)]. Asterisks indicate significant differences in OD₆₅₀ values from wild‐type cells (P < 0.05, t‐test).