Contractor's Quarterly Progress, Status and Management Report
9th Quarter 2008 Reporting Period
Contractor's Name and Address:
William B. Gurley, professor (sub-Project Director)
University of Florida
Microbiology and Cell Science
Bldg. 981, P. O. Box 110700
Gainesville, FL 32611-0700
Phone: (352) 392-1568
FAX: (52) 392-5922
Email: wgurley@ufl.edu
Eva Czarnecka-Verner, professor/Associate Scientist (sub-Project
Co-Director)
Email: evaczar@ufl.edu
Contract Number: W911SR-07-C-0084
Subagreement Number: 6415-1012-69-A
Report Date: Jan 27, 2010
Period Covered by Report: Oct 1, 2009- Dec 31, 2009
Title of Report: Florida Biodefense Research Consortium, Task Area 2, Subtask
10/27: A strategy for Elimination of Human Pathogen-
Contaminated Raw Produce from the Food Supply
CDRL Item Number: A003
Security Classification: Unclassified Sensitive
Issuing Government Activity:
AMC Acquisition Center - Edgewood
AMSSB-ACC/Bldg. E04455
5183 Blackhawk Road
Aberdeen Proving Ground, MD 21010-5424
1
Item b. Description of Progress Made Against Milestones During Reporting Period.
Abstract
The recent outbreaks of pathogenic E. coli strain 0157:H7 in lettuce and spinach in 2006 caused
human death and illness and has drawn attention to the vulnerability of the Nation’s food supply
to attack by those wishing us harm. The proposed experiments represent the beginning steps in
developing crop plants that can sense pathogenic bacteria and report their presence to the grower,
marketer and consumer. The experimental approach outlined in this proposal builds on the innate
ability of plants to sense bacteria present on their surfaces, a property shared by most eukaryotes
in the form of innate immunity. In order to be effective, the endogenous sensing networks must
be enhanced in their sensitivity and in their ability to discriminate between pathogenic and
nonpathogenic bacteria (specificity). Hence, the experimental approach has two major aims: 1)
amplification of the sensing signal, and 2) increasing the specificity of detection. Due to the lack
of DNA sequence information for leafy produce, such as lettuce and spinach, these initial
experiments will be conducted in Arabidopsis where complete genomic information is available.
The goal is to engineer regulatory networks termed “modules” that can be combined to achieve
enhanced sensitivity and specificity. The longer range goal to develop plants as biosensors
requires that plants not only be able to sense pathogenic organisms, but also report their
presence. In terms of technical difficulty, the reporting function appears to be a much easier task,
so this aspect will be not be addressed in this proposal due to time limitations. However, future
plans call for the sensing signal output to be directed towards causing the plant to manifest very
obvious signs of bacterial contamination, such as turning red/purple by activation of the
anthocyanin synthesis pathway, or by causing the leaves to die.
This project fits into the larger task of engineering plants to take on roles as passive
biosensors with the ability to detect and report harmful organisms, novel substances and
environmental indicators. The specific goal of developing cultivars of lettuce and spinach that
can detect and report pathogenic E. coli supports the general mission of protecting our homeland
food supply.
2
Table of Contents
Cover page ...............................................................................................................1
Abstract ....................................................................................................................2
Table of Contents .....................................................................................................3
Acknowledgements ..................................................................................................4
Summary ..................................................................................................................5
Introduction ..............................................................................................................5
Methods, Assumptions and Procedures ............................................................... 5-6
Results and Discussion ............................................................................................7
Items c through p ................................................................................................. 7-9
Conclusion .............................................................................................................10
Appendix A-1 Literature Cited ..............................................................................11
Appendix A-2 Figures.........................................................................................N/A
3
Acknowledgements
This work was supported by Department of Defense Contract Number W911SR-07-C-0084. We
would like to thank the USF Center for Biological Defense and Diana McCluskey for their
support and encouragement in this project.
4
Summary
th
The 8 quarter research was focused on investigating flagellin22 elicitor capacity to induce
the ATX promoter in an autofeedback loop scheme.
1. Objective 1C. Development of a transcriptional auto-feedback loop. A series of flagellin
inducible promoters have been cloned and evaluated in an Arabidopsis-based transient assay
system. The highest levels of induction were obtained by fine tuning of the assay and with
overexpression of the flagellin receptor (AtFLS2). Therefore, the AtFLS2 receptor protein was
coexpressed in all assays reported below.
Promoters showing the highest levels of induction by the elicitor peptide flg22 were
tested using an auto-feedback loop to achieve transcriptional amplification of the signal. The
feedback loop was shown to significantly improve flg22 inducibility in Arabidopsis protoplasts.
Promoter structure was evaluated and basal activity, as well as flg22-inducibility, tested by a
series of strategic deletions that removed blocks of W boxes. Soybean transcriptional repressor
lowered basal activity of the feedback loop, but improved elicitor inducibility.
Introduction
The goal of this project is to keep contaminated lettuce out of the food supply by developing
regulatory circuits that allow producers and consumers know if potentially pathogenic bacteria
are present. The 12 month funding period provides time to evaluate simple strategies to increase
the sensitivity (objective 1) and specificity (objective 2) of detection by the plant. The
experimental system will rely heavily on transient assays in protoplasts from the model plant,
Arabidopsis, and the testing of successful constructs in lettuce. A two-pronged approach is being
used to increase the sensitivity of the plant to the presence of E. coli and other potential
pathogens: 1-an autofeedback loop based on transcriptional activation (objective 1C), and 2- a
feedback system based on production of an elicitor peptide by the plant (objective 1B).
Increasing the specificity of recognition will employ a plant two-hybrid approach to reduce the
non-specific activation of the recognition system. In this strategy’s simplest form, the plant will
mount a strong hypersensitive response when it detects endophytic E. coli. At present, plants are
removed from the food chain if they appear infected by plant pathogens, most of which do little
harm to humans. Likewise, the engineered lettuce will appear diseased and, thus, be removed
from the food chain when colonized by potential human, as well as plant pathogens.
Methods, Assumptions and Procedures
1. Methods and Procedures:
A. Preparation of Protoplasts and DNA transformation. The protocol for protoplasts and
transformation is based largely on the published method of Yoo et al (2007). Arabidopsis and
lettuce mesophyll cells are used to prepare protoplasts by first digesting away the cell wall
overnight and removing the debris by filtration through a fine mesh nylon screen. Plasmid DNA
is introduced using a PEG-based procedure where the protoplasts are transformed on the second
day of the protoplasting and the cells harvested on day 3. Treatment with elicitor peptides is
conducted approximately 16 hr after transformation, and the cells are harvested and assayed for
luciferase activity after an additional 3-4 hr.
B. Dual luciferase reporter system. Transient assays are conducted using protoplasts
prepared from the leaves (mesophyll cells) of Arabidopsis and lettuce. Promoters under
5
evaluation will be used to drive expression of the firefly luciferase (F-Luc2) reporter according
to the Promega Dual Luciferase Reporter System. The maize ubiquitin promoter (or CaMV 35S)
will drive expression of the Renilla luciferase (R-Luc) reporter that will serve as the internal
standard. Firefly luciferase activity will be divided by Renilla luciferase activity as a way to
normalize protoplast transformation efficiencies and viability. This approach has provided us
with reproducible data in the Arabidopsis transient assays, and it has been used extensively by
others (De Sutter et al., 2005). Our plan is to conduct the initial experiments in Arabidopsis
protoplasts and then select the most successful constructs for transformation into Arabidopsis
and lettuce where the evaluation process will be repeated.
2. Assumptions:
A. Our first assumption is that a transcription-based auto-feedback loop can be
constructed that can be tightly regulated so that it is not activated when no E. coli is present and
can be highly induced by the presence of relatively low numbers of endophytic bacteria.
Achieving high levels of activity is not a problem; however, getting the system shut off in the
absence of E. coli will be difficult.
B. We also assume that it is possible to express a fragment of the flagellin protein fused
to GFP (or another protein) in a way that triggers the innate immunity system of the plant. If,
however, this assumption is proven false, there are other proteins in plants that act as endogenous
elicitors of innate immunity that can be used instead (Bergey et al., 1996; Huffaker et al., 2006;
Huffaker and Ryan, 2007; Ryan and Pearce, 2003).
6
Results and Discussion
Specific Milestones:
1. Develop a signal amplification regulatory circuit to increase the sensitivity of
Arabidopsis to bacteria present on leaves.
1A. Determine which promoters show the best on/off induction pattern by E. coli and
elicitor peptide (flagellin 22; flg22) using a luciferase reporter system in transient
assays.
1B. Amplification of signal by induction of elicitor synthesis by the plant.
1C. Assess the feasibility of employing an engineered zinc finger protein-VP16
fusion (or Gal4 DBD-based effector) to amplify the signal via a transcriptional
auto-feedback loop.
1D. Determine if the Arabidopsis constructs will function in lettuce leaves using PEG
transformation.
2. Construct a two-component system to increase the specificity of Arabidopsis
detection of E. coli.
2A. Combine the plant two-hybrid system with the amplification scheme developed
in Aim 1 to achieve both specificity and sensitivity of detection.
2B. Test the two-hybrid and combined systems in lettuce leaves by protoplast
transformation.
___________________________________________________
Milestones addressed during the 9th quarter:
Continuation of Milestone 1C (Transcription auto-feedback loop). Due to budget
constraints (no salary during this period) a very limited amount of work was conducted.
However, a number of the autofeedback constructs were cloned into pCAMBIA vectors
suitable for transformation of Arabidopsis plants. As time permits, these will be
introduced into Arabidopsis in the remaining time for evaluation of the autofeedback loop
system.
Item c. Results Obtained Related to Previously-Identified Problem Areas
Item d. Any Significant Changes to Contractor’s Organization or Method of Operation, to
Project Management Network, or to Milestone Chart
None
Item e. Problem Areas Affecting Technical or Scheduling Elements
None
Item f. Problem Areas Affecting Cost Elements
None
Item g. Cost Curves Showing Actual and Projected Conditions Throughout the Contract
7
Item h. Any Cost Incurred for the Reporting Period and Total Contractual Expenditures as of
Reporting Date
Total Amount Available $ 193,125.00
Total Expenditures $ 193,026.23
Encumbrances $ 111.38
Expenditures for the 7th quarter $ (163.17)
Amount Remaining $ (12.61)
Item i. Person-hours Expended for the Reporting Period and Cumulatively for the Contract
During this reporter period:
Prof. Eva Czarnecka-Verner (Co-PI) 0 hr; 01OCT09-31DEC09
Lance Verner (Biological Scientist) 0 hr; 01OCT09-31DEC09
0 total person-hours were expended for the reporting period.
Note: Lance Verner was taken off the contract April 2, 2009, and Dr. Czarnecka was
taken off the grant September 17th, 2009.
Cumulative for contract:
4,048 person-hours were expended cumulatively for the contract.
(No change from last report)
Item j. Any Trips and Significant Results
None
8
Item k. Record of all Significant Telephone Calls
None
Item l. Summary of Engineering Change Proposal (ECP) Status
None
Item m. Contract Schedule Status
Item n. Plans for Activities During the Next Reporting Period
Milestones to be addressed during the 10th quarter:
Continuation of Milestone 1C (Transcription auto-feedback loop). We plan to
transform selected auto-feedback- loop constructs into Arabidopsis in order to evaluate
the system in whole plants.
Item o. Name and telephone Number of Preparer of the Report
William B. Gurley and Eva Czarnecka-Verner; Telephone: (352) 392-1568
Item p. Appendices for Tables, References, Photographs, Illustrations and Charts
None
9
Conclusions
Transient assays using Arabidopsis protoplasts have been used to determine the
effectiveness of the auto-feedback loop on elicitor induction. We successfully lowered basal
activity of investigated promoters by strategic removal of pathogen-responsive distal elements,
thus significantly improving flg22 inducibility. An additional approach to lower basal activity by
the use of a repressor was also successful. Additional cloning and transient assays were
conducted during the last quarter (no salary support) to prepare select constructs for
transformation into Arabidopsis for evaluation.
10
Appendix A-1
Literature Cited
Asai, T., Tena, G., Plotnikova, J., Willmann, M.R., Chiu, W.L., Gomez-Gomez, L., Boller, T.,
Ausubel, F.M. and Sheen, J. (2002). MAP kinase signalling cascade in Arabidopsis
innate immunity. Nature 415: 977-83.
Bergey, D.R., Howe, G.A. and Ryan, C.A. (1996). Polypeptide signaling for plant defensive
genes exhibits analogies to defense signaling in animals. Proc Natl Acad Sci U S A 93:
12053-8.
Ciolkowski, I., Wanke, D., Birkenbihl, R.P. and Somssich, I.E. (2008). Studies on DNA-binding
selectivity of WRKY transcription factors lend structural clues into WRKY-domain
function. Plant Mol Biol 68: 81-92.
De Sutter, V., Vanderhaeghen, R., Tilleman, S., Lammertyn, F., Vanhoutte, I., Karimi, M., Inze,
D., Goossens, A. and Hilson, P. (2005). Exploration of jasmonate signalling via
automated and standardized transient expression assays in tobacco cells. Plant J 44: 1065-
76.
Huffaker, A., Pearce, G. and Ryan, C.A. (2006). An endogenous peptide signal in Arabidopsis
activates components of the innate immune response. Proc Natl Acad Sci U S A 103:
10098-103.
Huffaker, A. and Ryan, C.A. (2007). Endogenous peptide defense signals in Arabidopsis
differentially amplify signaling for the innate immune response. Proc Natl Acad Sci U S
A 104: 10732-6.
Ryan, C.A. and Pearce, G. (2003). Systemins: a functionally defined family of peptide signals
that regulate defensive genes in Solanaceae species. Proc Natl Acad Sci U S A 100 Suppl
2: 14577-80.
Yoo, S.D., Cho, Y.H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell
system for transient gene expression analysis. Nat Protoc 2: 1565-72.
Zipfel, C., Robatzek, S., Navarro, L., Oakeley, E.J., Jones, J.D., Felix, G. and Boller, T. (2004).
Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428: 764-
7.
Czarnecka-Verner, E. , Pan, S., Salem, T. and Gurley, W.B. (2004). Plant class B HSFs inhibit
transcription and exhibitaffinity for TFIIB and TBP. Plant Mol. Biol. 56, 57-75.
11
12