Functionalized microchannels as xylem-mimicking environment: Quantifying X. fastidiosa cell adhesion

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

“…We validated both experimentally and computationally the laminar flow profile within the channel ( Figures 4A–C ; Supplementary Figure S5 ) that emulated the natural flow within the plant xylem ( Michelle Holbrook and Zwieniecki, 2011 ). Our simulation further demonstrated the linear relationship of flow shear stress and drag force applied on a bacterial cell model ( Figures 4C–E ), which was consistent with other works studying the flow effect on xylem-inhabiting pathogens ( De La Fuente et al, 2007 ; Monteiro et al, 2021 ). Then, to mimic the chemical component of xylem cell wall, CMC was coated on the channel surfaces through polydopamine surface chemistry ( Lee et al, 2007 ; Lee et al, 2019 ; Barclay et al, 2017 ) which provided a robust stable coating material for multiple substrates including glass and PDMS ( Figures 3A–D ) while retaining both CMC and DOPA unique properties ( Figure 2 ).…”

“…First, our system contained 20 parallel channels allowing multiple replicates in one single experiment, similarly to other high-throughput lab-on-chip systems ( De La Fuente et al, 2007 ; Monteiro et al, 2021 ) ( Figures 1A, B ). Each channel had a square cross section of 50 × 50 µm ( Figure 1B ), comparable with the majority of tomato-xylem cross areas ( Ingel et al, 2021 ).…”

“…The xylem environment was generated by introducing xylem sap collected directly from tomato cultivars to the channels using a syringe pump. While numerous studies employed syringe pumps to regulate flow within microfluidic channels ( De La Fuente et al, 2007 ; Cogan et al, 2013 ; Monteiro et al, 2021 ), only few have integrated xylem-like media into their systems ( Cogan et al, 2013 ; Harting et al, 2021 ) or explored the use of actual xylem sap, owing to its less defined properties ( Kandel et al, 2016 ; Khokhani et al, 2017 ; Gerlin et al, 2021 ) and its less favorable conditions for bacterial biofilm formation in vitro ( Supplementary Figure S2 ; Figures 3E–H ). We, therefore, analyzed the viscosity of various culture media and xylem saps, showing their similarity, which should not alter the flow behavior ( Supplementary Figure S4 ).…”

“…Previous studies on Rps biofilm (including our own work) relied on culturing bacterial biofilm in defined media (CPG or minimal medium) on glass and plastic surfaces, or imaging Rps in fixed plant tissue, which may limit the biological relevance of the findings (Mori et al, 2016;Tran et al, 2016;Khokhani et al, 2017;Tancos et al, 2018). First, our system contained 20 parallel channels allowing multiple replicates in one single experiment, similarly to other highthroughput lab-on-chip systems (De La Fuente et al, 2007;Monteiro et al, 2021) (Figures 1A, B). Each channel had a square cross section of 50 × 50 µm (Figure 1B), comparable with the majority of tomato-xylem cross areas (Ingel et al, 2021).…”

“…Additionally, more sophisticated designs introduced interactive channels and pectin-rich simulated xylem media, enabling investigations into the dynamics between Pseudomonas protegens and the Verticillium sp., a fungal pathogen causing vascular wilt diseases ( Harting et al, 2021 ). The system’s complexity was further achieved by adding cell wall components and bacterial adhesins, like the Xylella fastidiosa adhesin XadA, as a coating, enabling studies of both plant and bacterial factors in the biofilm formation process of xylem pathogens ( Monteiro et al, 2021 ).…”