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Title: Detection of bacterial aggregation in cell suspensions treated with pathogenic bacteria

item Baker, Con
item Owens, Robert
item Whitaker, Bruce
item Mock, Norton
item Deahl, Kenneth
item Roberts, Daniel
item ORLANDI, ELIZABETH - University Of Pennsylvania
item AVERYANOV, A - University Of Moscow

Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/2011
Publication Date: 6/15/2011
Citation: Baker, C.J., Owens, R.A., Whitaker, B.D., Mock, N.M., Deahl, K.L., Roberts, D.P., Orlandi, E., Averyanov, A.A. 2011. Detection of bacterial aggregation in cell suspensions treated with pathogenic bacteria. Physiological and Molecular Plant Pathology. 75:170-175.

Interpretive Summary: Plant diseases cause major losses to farmers each year. Better understanding of the biochemical basis for plant resistance to disease will lead to improved strategies to improve plant health and reduce losses. In this paper we report a new finding using a plant ‘cell suspension’ model system that focuses on the very early events in the plant-bacterial interaction. A newly adapted technique, flow cytometry, which can detect slight changes in the bacterial concentration, detected that within a few hours after bacteria contact the plant cells, the bacteria aggregate together. This is considered to be the first stage in bacterial binding and infection of the plant cell. If we can use this model system to find the specific biochemicals or signals that trigger the bacterial binding we may be able to devise methods or plants to resist the bacterial infection. This information will be of use to plant scientists who are devising new strategies to improve disease resistance in plants.

Technical Abstract: The early interaction between plant cells and pathogenic bacteria were studied using tobacco cell suspensions treated with pathogenic and nonpathogenic Pseudomonas species. Previous studies of this system have documented that interactions with pathogens that cause a hypersensitive response on whole plants will cause a series of physiological effects including both an increase in oxygen uptake as well as an alteration of apoplastic phenolics. Here we describe first reports of bacterial aggregation which also correlates with the oxygen uptake response. A flow cytometer and fluorescent stains were used to monitor bacterial concentrations during the interaction. A decrease in planktonic bacterial numbers occurred 2-4 hr, depending on the specific interaction, after inoculation. This decrease was followed by an increase in bacterial multiplication for at least 12 hr. Bacterial aggregates were observed in unfiltered samples containing both plant and bacterial cells viewed with a fluorescent microscope. The addition of a DNA binding stain allowed detection and both plant and bacterial cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These incapacitated bacterial cells appear to be removed along with the plant cells by the filtering step prior to the flow cytometer measurement, causing the decrease in bacterial. This phenomenon was also observed with other P. syringae isolates including the compatible P. tabaci pathogen, but was not observed with plant cells inoculated with the nonpathogen, P. fluorescens. These results suggest that the oxygen uptake response observed early during plant pathogen interactions may involve the production of a biofilm, which could facilitate binding and onset of Type III secretion associated with pathogenicity.