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<p>Contamination of produce by human pathogens threatens public health and is difficult to detect. Progress in the emerging field of synthetic biology has recently led to the development of "phytodetector" plants, which undergo a visible color change upon detection of certain dangerous chemicals. These plant-based detectors promise to provide a continuous and inexpensive means of specific chemical detection in the field. Although detector plants were originally conceived with the goal of detecting abiotic toxins and chemicals, they could potentially be adapted to detect molecules produced by human pathogens. </p>
<p>The goal of this project is to apply phytodetector technology to develop and characterize first-generation detector plants for detection of food safety pathogens. To achieve this goal, reporter bacterial strains and plant lines expressing candidate synthetic detection systems will be developed and tested for ability to detect small molecules produced by two bacterial species, Escherichia coli and Staphylococcus aureus. Experiments are designed to achieve the following objectives: first, to assess the level of sensitivity of the plant-compatible receptors to target bacteria; second, to determine the level of specificity of the detection circuits to target species; and third, to develop and test a series of transgenic plant lines for response to pathogen-specific molecules in greenhouse and growth chamber studies. The project timeline establishes January 2012 as a target date for development of dose response curves for substrate detection in bacterial reporter system, and July 2012 for determination of receptor specificity in this system. Transgenic plant lines will be developed by July 2012 and characterized by June 2013. By determining the sensitivity and specificity of the first receptor systems both in bacterial and plant reporter systems, the project will establish protocols and baseline performance levels for future generations of reporter systems, information needed to assess the feasibility and inform the long-term development of field- or greenhouse-based phytodetectors of plant-colonizing bacteria. In addition, the project will provide valuable career training for a postdoctoral-level plant bacteriologist in the areas of synthetic biology and food safety.</p>
<p>NON-TECHNICAL SUMMARY:<br/> Contamination of fruit and vegetable produce by human pathogenic bacteria causes hundreds of illnesses each year. Many outbreaks are suspected to originate from contamination in the field, where pathogenic bacteria may be introduced onto crop plants through tainted irrigation water or manure runoff. Preventing contaminated produce from entering the food chain is difficult because wash treatments may fail to kill bacteria internalized in plant tissues. Because specific detection methods are cost-prohibitive to implement on a wide scale, new technologies are needed to for detecting human pathogens on plants. In recent years biotechnology has been applied to develop chemical-detecting plants, called "phytodetectors", which undergo a color change upon detection of certain small molecules. Phytodetectors could continuously and inexpensively detect a
variety of chemicals of human importance, potentially including those produced by bacterial pathogens. This research project will apply phytodetector technology to the detection of molecules from two food safety organisms that may contaminate produce: Staphylococcus aureus and Escherichia coli. In this project, candidate sensing systems for these molecules will be introduced into plants. Experiments will be performed to determine how sensitive, strong, and specific these systems are in detecting the presence of plant-colonizing bacteria. The results will determine whether phytodetectors might be a promising method of detection of food safety organisms.<p>
APPROACH: <br/>Phytodetector technology detects extracellular small molecules using synthetic "detection circuits" that relay a signal to "readout circuits" to cause a response in plants. To develop candidate phytodetectors of food safety pathogens, detection circuits were designed to sense autoinducers, or small signaling molecules, secreted by two plant-colonizing human pathogens. This project will develop and assess these detection circuits in bacterial reporter systems and in first-generation phytodetector plants. Specificity studies will be conducted to determine whether the novel detection circuits respond to substrates from a selected panel of non-target bacteria, including environmental and plant pathogenic strains, and determine the detection threshold of any off-target bacteria. While the sensitivity and specificity assays are being conducted, a series of plant
lines will be developed and tested to determine the response of the detection circuits in plants. Transgenic Arabidopsis lines will be developed for each of two readout circuits: the degreening readout circuit, which provides a visible response, and the luciferase readout circuit, a single gene reporter that can be measured with a high degree of specificity. Transgenic lines will be assessed for strength of reporter gene response to substrate preparations: response of degreening lines will be quantified by measuring fluorescence yield, and luciferase expression lines will be screened by photon counting camera. Selected lines will be advanced and tested in depth for detection threshold, kinetics of response, and effects on bacterial population growth. Response kinetics will be determined by timepoint experiments, and detection thresholds and dose-response curves will be established at the
optimum timepoint. Finally, bacterial colonization studies will be performed to determine whether the first-generation phytodetectors can detect in-plant populations of bacteria, and whether the synthetic circuits have an effect on plant colonization.</p><p>
PROGRESS: <br/>2011/08 TO 2012/08 <br/>OUTPUTS: Two different strategies designed to achieve plant-based detection of human pathogens, specifically the efficacy of numerous candidate receptors for plant-based pathogen detection, were tested. Several fusions involving the quorum sensing receptors QseC and AgrC from two families of plant-colonizing human pathogens did not signal in a plate assay. Constructs based on the CviR signaling system from Chromobacterium violaceum have been developed and testing is in progress. I received training in microbial signaling and growth on plants through these research activities and through attendance at a workshop on Human Pathogens on Plants. Research related to the project was presented in a poster at this meeting and in an oral presentation at a conference on new technologies in agriculture. PARTICIPANTS: Lindsay
Triplett oversees all aspects of this project as PI. Jan Leach and June Medford serve as postdoctoral advisers. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.</p>