Research Program
Cell Surface Interactions

Objectives
Microfluidic systems have been used as ‘artificial’ plant xylem vessels to study bacterial-plant relationships in ways that can not be accomplished in planta.  Optical assessment of both temporal and spatial activities of plant pathogenic bacteria, particularily Xylella fastidiosa and Xanthomonas campestris pv. campestris have been carried out.  Specific objectives were to: 1) design and develop microfluidic devices with valves (positioned close to the areas of interest) to better control presentation of fluorescent dyes and chemicals to bacteria, 2) assess relationships between bacterial pili and substrata, 3) obtain antibodies (MAb) to be used in studies aimed at disrupting pilus function and cell aggregation, and 4) evaluate influence of pathogenicity factors e.g. DSF molecules on cell migration and biofilm development.

Methods
Photolithography, deep RIE and/or SU-8 processes in were used to pattern silicon wafers, from which PDMS replicas were made and sandwiched between a glass microscope slide and coverslip to create ‘artificial’ xylem vessels.  Valves were incorporated into the devices in a double-PDMS sandwich.  Fluid flow was achieved through in and out ports with high precision syringe pumps.  Assessment of bacterial activities was accomplished principally through time-lapse light microscopy and image analysis software. 

Summary
Xylem vessels (water conduits) of plants are frequently colonized by plant pathogenic bacteria where they occlude the conduits, causing disease.  Xylella fastidiosa, a pathogen of grapevine, and Xanthomonas campestris pv. campestris, a pathogen of cruciferous crops were studied in the microfludic devices with respect to cell migration, cell aggregation, and colonization. 

Microfluidic devices with integrated valves were developed to provide introduction of cells, fluorophore stains, and signaling molecules to specific regions, followed by re-positioning of the cells into different chamber regions.  This work is continuing.  Monoclonal antibodies specific to pili and cell surface regions of Xylella fastidiosa were developed this past year.  They will be used in the microfluidic devices to assess pilus function in the bacterium.

Until recently all studies with Xylella fastidiosa in microfluidic devices were with cells grown in an artificial medium (PD2), primarily because X. fastidiosa had not been successfully grown in grapevine xylem sap in vitro, even though the pathogens grows in it in planta.  Using the microfluidic devices we were able to gradually recondition the bacterium to xylem sap conditions and can now routinely grow it in this ‘natural’ medium.  As a consequence we have observed very different biofilm characteristics than seen in PD2 medium, and quite possibly a very different migration rate by these bacteria.

These and other results are summarized in movies at:
http://www.nysaes.cornell.edu/pp/faculty/hoch/movies/

Accomplishments

• Developed ‘valved’ microfluidic to be used in studies with fluorophores, antibodies, and signaling molecules
• Obtained a panel of monoclonal antibodies toward surface epitopes of Xylella fastidiosa cells
• Demonstrated that grape xylem fluid promotes ‘classical’ biofilms in vitro.
• Demonstrated that a diffusible signal factor caused an rpfF-DIF2 Xylella fastidiosa mutant to increase twitching motility.