Lectins and polysaccharide EPS I have flow-responsive roles in the attachment and biofilm mechanics of plant pathogenic Ralstonia.

Bacterial biofilm formation and attachment to hosts are mediated by carbohydrate-binding lectins, exopolysaccharides, and their interactions in the extracellular matrix (ECM). During tomato infection Ralstonia pseudosolanacearum (Rps) GMI1000 highly expresses three lectins: LecM, LecF, and LecX. The latter two are uncharacterized. We evaluated the roles in bacterial wilt disease of LecF, a fucose-binding lectin, LecX, a xylose-binding lectin, and the Rps exopolysaccharide EPS I. Interestingly, single and double lectin mutants attached to tomato roots better and formed more biofilm under static conditions in vitro. Consistent with this finding, static bacterial aggregation was suppressed by heterologous expression of lecFGMI1000 and lecXGMI1000 in other Ralstonia strains that naturally lack these lectins. Crude ECM from a ΔlecF/X double mutant was more adhesive than the wild-type ECM, and LecF and LecX increased Rps attachment to ECM. The enhanced adhesiveness of the ΔlecF/X ECM could explain the double mutant's hyper-attachment in static conditions. Unexpectedly, mutating lectins decreased Rps attachment and biofilm viscosity under shear stress, which this pathogen experiences in plant xylem. LecF, LecX, and EPS I were all essential for biofilm development in xylem fluid flowing through cellulose-coated microfluidic channels. These results suggest that under shear stress, LecF and LecX increase Rps attachment by interacting with the ECM and plant cell wall components like cellulose. In static conditions such as on root surfaces and in clogged xylem vessels, the same lectins suppress attachment to facilitate pathogen dispersal. Thus, Rps lectins have a dual biological function that depends on the physical environment.

Development of a tomato xylem-mimicking microfluidic system to study Ralstonia pseudosolanacearum biofilm formation.

The bacterial wilt pathogen Ralstonia pseudosolanacearum (Rps) colonizes plant xylem vessels and blocks the flow of xylem sap by its biofilm (comprising of bacterial cells and extracellular material), resulting in devastating wilt disease across many economically important host plants including tomatoes. The technical challenges of imaging the xylem environment, along with the use of artificial cell culture plates and media in existing in vitro systems, limit the understanding of Rps biofilm formation and its infection dynamics. In this study, we designed and built a microfluidic system that mimicked the physical and chemical conditions of the tomato xylem vessels, and allowed us to dissect Rps responses to different xylem-like conditions. The system, incorporating functional surface coatings of carboxymethyl cellulose-dopamine, provided a bioactive environment that significantly enhanced Rps attachment and biofilm formation in the presence of tomato xylem sap. Using computational approaches, we confirmed that Rps experienced linear increasing drag forces in xylem-mimicking channels at higher flow rates. Consistently, attachment and biofilm assays conducted in our microfluidic system revealed that both seeding time and flow rates were critical for bacterial adhesion to surface and biofilm formation inside the channels. These findings provided insights into the Rps attachment and biofilm formation processes, contributing to a better understanding of plant-pathogen interactions during wilt disease development.