Source: COLORADO STATE UNIVERSITY submitted to
PLANT-BASED DETECTION OF FOOD SAFETY PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0226119
Grant No.
2011-67012-30670
Project No.
COL0-2010-05115
Proposal No.
2010-05115
Multistate No.
(N/A)
Program Code
A7201
Project Start Date
Aug 15, 2011
Project End Date
Aug 14, 2014
Grant Year
2011
Project Director
Triplett, L.
Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Bioagricultural Sciences and Pest Management
Non Technical Summary
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.
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
(N/A)
Developmental
80%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7122499104050%
7124010104050%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
2499 - Plant research, general; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
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. 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.
Project Methods
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.

Progress 08/15/11 to 08/14/14

Outputs
Target Audience: This project reached our target audience of the scientific community studying bacterial-plant interactions and bacterial diagnostics and detection. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The postdoctoral research fellow supported by this project received training in the theory and methodology of synthetic biology and in human pathogens on plants, as stated in the project objectives. In addition, the project provided training in the areas of plant pathogen diversity and diagnostics, genomic analysis, andseveral biochemical techniques. Finally, the project provided important professional development for the postdoc in the skills ofproject management and budgeting, mentoring, manuscript preparation, presentation, and networking. How have the results been disseminated to communities of interest? The results ofthe research supported by this grant have been disseminatedin the form of 5 formal research presentations at professional conferences,6 peer-reviewed studies, and 1 book chapter, alltargeted toward the researchcommunityofplant-bacterial interaction researchers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Toward the stated goals, a series ofsyntheticreceptors were rationally designed based on predicted domain architecture, synthesized, and tested for responses to thetarget small molecules produced by S. aureus and E. coli under a variety of experimental conditions and treatment doses. Additional variants were then created by site-directed mutagenesiscreating insertions or deletions that might increase the likelihood of signal transduction in the E. coli synthetic reporter system, creating a total of 10 novel synthetic receptors among the two target molecules.Ultimately, these receptors did notproduce a response to either target small molecule, and so we were not able toachieve the goalof producing transgenic phytodetector plants for food safety.However, the goal of training a postdoctoral plant bacteriologist in synthetic biology and food safety was achieved. In addition, concurrently duringthe long period of iteratively testing the receptors,the trainee supported by thisproject was able to develop and test bacterial detection tools using the more established detection technique of LAMP and conventional PCR.The trainee assembled computational tasks into a rapid genomic primer-design pipeline, providing valuable computational skills training. Novel diagnosticprotocols for two species of plant-associated bacteriawerethoroughly tested for sensitivity, specificity, and detection limit,and published. The diagnostic tools weredesigned based on newly-generated genomic data,which allowed the trainee to assist in the comparative genomics analysis yielding several other insights.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: P.A. Fory, L. Triplett, C. Ballen, J. F. Abello, J. Duitama, M. G. Aricapa, G. A. Prado, F. Correa, J. Hamilton, J. E. Leach, J. Tohme, and G. M. Mosquera. 2014. Comparative analysis of two emerging rice seed bacterial pathogens. Phytopathology 104: 436-444
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu, W., Liu, J., L. Triplett, J.E. Leach, and G.-L. Wang. 2014. Novel insights into rice innate immunity against fungal and bacterial pathogens. Annual Review of Phytopathology 52: 213-241.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Triplett, L., V. Verdier, R. Koebnik, and J.E. Leach. Genomics of Xanthomonas oryzae. Genomics of Plant-Associated Bacteria. Eds. D. Gross, A. Lichens-Park, and C. Kole. Springer International, 2014. 127-150.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Verdier, V.*, L.R. Triplett*, A.W. Hummel, R. Corral, R. Andres Cernadas, C.L. Schmidt, A.J. Bogdanove, and J.E. Leach. 2012. TAL effectors targeting OsSWEET genes enhance virulence on diverse rice varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae. New Phytologist 196: 1197-1207.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ash, G.J., J.M. Lang, L.R. Triplett, B.J. Stodart, V. Verdier, C. Vera Cruz, P. Rott, and J.E. Leach. 2014. Development of a genomics-based LAMP (Loop-mediated isothermal amplification) assay for detection of Pseudomonas fuscovaginae from rice. Plant Disease 98: 909-919.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Lang, J.M., P. Langlois, M.H.R. Nguyen, L.R. Triplett, L. Purdie, T.A. Holton, A. Djikeng, C.M. Vera Cruz, V. Verdier, and J.E. Leach, 2014. Sensitive detection of Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola by Loop-Mediated Isothermal Amplification. Applied and Environmental Microbiology, Online ahead of print doi:10.1128/AEM.00274-14.
  • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: L. Triplett, V. Verdier, T. Campillo, C. Van Malderghem, I. Cleenwerk, M. Maes, L. Deblais, R. Corral, O. Koita, B. Cottyn, and J. Leach. 2014. Characterization of a novel clade of Xanthomonas isolated from rice leaves in Mali and proposal of Xanthomonas maliensis sp. nov.


Progress 08/15/11 to 08/14/12

Outputs
OUTPUTS: 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: 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.

Impacts
OUTCOMES/IMPACTS: This proof-of-concept project is aimed at generating transgenic sentinel plants that detect colonization by human pathogens with a measurable response. The proposed strategy required design of chimeric receptors fusing the signaling component of an established PhoR-based plant sentinel system to the receptor moiety of quorum sensing signaling receptors QseC and AgrC from two families of plant-colonizing human pathogens. Sequence alignment and secondary structural prediction were used to identify the likeliest putative chimera to achieve signal transduction between the pathogen and PhoR receptors. Restriction cloning, artificial gene synthesis, and site-directed mutagenesis techniques were used to generate five candidate AgrC-derived plant compatible receptors and four QseC-derived receptors. Receptors were introduced into a B-galactosidase bacterial reporter system to test signal transduction. AgrC-derived reporter strains were tested using cultures of a Staphylococcus aureus strain known to produce the corresponding signaling peptide, or with highly concentrated supernatant extracts. QseC-derived reporters were tested using E. coli supernatants or a synthetic signaling analogue, commercially available norepinephrine. None of the reporter strains produced a measurable response when treated with the pathogen-derived compounds, indicating that we were not able to achieve signal transduction through this route. In a second, alternate strategy, we designed a plant-optimized signaling pathway based on the CviR signaling system from Chromobacterium violaceum. Expressed in plants, this system will result in increased expression of luciferase in the presence of C6-HSL, a small signaling molecule produced by gram-negative bacteria including produce contaminants Yersinia enterocolitica and Y. pseudotuberculosis. The components of the plant based CviR system have been synthesized and cloned into a binary expression vector for introduction into plants. The resulting transgenic plants will be tested with synthetic C6-HSL and bacterial colonization studies, allowing us to address the goals of this proposal, i.e., to determine the specificity and sensitivity of transgenic detector plants to signals produced by human pathogenic bacteria

Publications

  • No publications reported this period