Source: UNIV OF WISCONSIN submitted to
CONTROL OF SOFT ROT ERWINIA WITH VIRULENCE PROTEIN INHIBITORS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0208866
Grant No.
(N/A)
Project No.
WIS01072
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2006
Project End Date
Sep 30, 2009
Grant Year
(N/A)
Project Director
Charkowski, A.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
PLANT PATHOLOGY
Non Technical Summary
Bacterial plant diseases cause major yearly economic losses in vegetable and ornamental crops and few disease control options are available. The goal of this project is to identify compounds that inhibit virulence genes of bacterial plant pathogens. These compounds may be developed into new control methods.
Animal Health Component
(N/A)
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21240101160100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1160 - Pathology;
Goals / Objectives
Erwinia carotovora and Erwinia chrysanthemi cause rot and wilt diseases on vegetable and ornamental crops. In the field, these diseases are promoted by environmental conditions such as rain and hot weather or damaging hail. Erwinia diseases are also commonly found in greenhouses on numerous high value ornamental crops and in vegetable storages, where they can lead to the total loss of a crop. In Wisconsin, these pathogens cause significant losses each year, particularly in the potato and ornamental plant industries. As with nearly all bacterial plant pathogens, there are currently no effective chemical control methods for Erwinia diseases in the field, greenhouse or storage. Transgenic plants have been constructed that are resistant to soft rot Erwinia, but they have not been accepted by commercial producers and this is not a realistic control option for the ornamental industry, where multiple varieties of hundreds of species are produced and it is cost prohibitive to make them all transgenic. Recently we discovered that E. chrysanthemi 3937 (Ech 3937) is able to form pellicles, a type of biofilm that forms at the air-liquid interface in cultures, and that several virulence genes, including the type III secretion system (T3SS), motility, bacterial cellulose synthesis, and energy taxis (aerotaxis) all contribute to pellicle formation. The goal of our work is to find new control measures for bacterial plant pathogens and this simple E. chrysanthemi pellicle assay is an important key because it allows us to quickly screen for virulence protein inhibitors, which may be developed into chemical control for soft rot diseases. Homologs of Erwinia virulence genes are widespread in bacterial plant pathogens, thus, control measures based on virulence protein inhibition may also work with other bacteria. Virulence proteins are attractive targets for antimicrobials because many of the targets are widespread in pathogens but not in normal microbiota. Thus, unlike with antibiotics, much of the normal microbiota would not be a source of resistance genes. In addition, although many homologous virulence genes are present in bacterial plant and animal pathogens, there are still significant differences in the virulence proteins of plant and animal pathogens that may allow for plant pathogen-specific antimicrobials to be developed. Therefore, unlike antibiotics, if resistance develops, it will be unlikely to directly compromise human health. Although potential virulence protein targets in Erwinia have been known for years, it was not possible until very recently to identify inhibitors of these targets. Now, due to rapid improvements in high throughput technologies, gene expression reporter systems, the availability of numerous genome sequences, and an improved understanding of virulence gene regulation and protein function, identification of virulence protein inhibitors is feasible. The hypotheses that will be tested in this project include: 1. The E. chrysanthemi 3937 energy taxis proteins regulate virulence genes. 2. Inhibitors of Erwinia virulence proteins can be used to control soft rot
Project Methods
We will determine if aerA or tsr affect the regulation of known E. chrysanthemi virulence proteins. We have experience with multiple quantitative assays that are used to measure E. chrysanthemi virulence proteins, including enzyme assays to monitor secretion of cell-wall degrading enzymes, quantitative real-time PCR (qRT-PCR) to measure gene expression in bacteria grown in culture and in leaves, and reporter bacteria to measure auto-inducer production. These assays will be used to determine if the production of any of these known virulence proteins is altered in the aerA or tsr mutants. .Because both aerA and tsr are energy taxis genes and both are required for pathogenicity but only aerA is required for pellicle formation, aerA and tsr appear to affect the expression of two different, but possibly overlapping, sets of genes. Virulence genes tend to be regulated by multiple positive and negative regulators that are themselves responding to external signals. Thus, the set of genes regulated by aerA and tsr in culture may differ from those regulated during a disease interaction. Therefore, we will use standard gene expression experiments to identify genes regulated by aerA and tsr during the initial stage of the E. chrysanthemi-plant interaction. We will use allelic-exchange to delete the differentially expressed genes to determine if they are required for full virulence. If we identify numerous differentially regulated genes, we will first mutate those that are conserved across species but that have with no known function and will give priority to those regulated by both aerA and tsr. The mutants will be examined for phenotypes known to be affected in aerA and tsr mutants such as reduced virulence and pellicle formation. We will screen compound libraries at the UW Keck Center to identify pellicle inhibitors. We will then use a combination of secondary assays to determine how the candidate compounds are inhibiting pellicle formation. If multiple structurally related inhibitors are found, we will choose one representative compound from this family for our assays. The lowest compound concentration that completely inhibits pellicle formation will be used for the secondary assays. We will also test known biofilm, motility, aerotaxis, and T3SS inhibitors in our pellicle and secondary assays. In addition to inhibiting virulence proteins, the compounds also must not be toxic to eukaryotic cells or be quickly degraded and they need to be available in the plant apoplast. We will use N. benthamiana to determine if the candidate compounds are phytotoxic and if they can inhibit soft rot disease.

Progress 10/01/06 to 09/30/09

Outputs
OUTPUTS: The goal of this work was to better understand how bacterial pathogens cause disease and to use this knowledge to develop better control measures for bacterial plant pathogens. The objectives of this project are to determine how energy taxis contributes to virulence and to use the Ech 3937 pellicle assay to identify virulence protein inhibitors. Our first hypothesis is that Dickeya dadantii energy taxis proteins regulate virulence genes. All of the energy taxis (aer) homologs were deleted from D. dadantii 3937. Two aer homolog mutants had no phenotype, one had a severe pleiotropic phenotype, and one appeared to be reduced in biofilm formation and aerotaxis. We chose to focus on this final mutant strain. Unfortunately, we discovered that a phage was adjacent to the aer homolog and it appears that when we attempt to mutate this aer homolog we activate the phage. It is most likely phage-mediated bacterial cell death that was responsible for our biofilm and virulence phenotypes. We re-constructed the mutant using alternate methods and had the same problem with the mutant strain. Thus, this line of work was not possible to continue with available resources and tools. We therefore focused on novel virulence genes that were identified in a related project, with the same overall goal in mind. Essentially, we are looking for novel targets for inhibition of virulence and these novel virulence genes many provide new targets for this goal. Our second hypothesis was that inhibitors of Dickeya virulence proteins could be used to control soft rot. We identified several inhibitors that function well in culture over the course of this project, but none of the small molecules were able to inhibit bacterial virulence in plants. We do not currently have tools available in my lab to determine the fate of these molecules, nor have we tested analogs for many of them. This leaves open many avenues of inquiry. For example, are these small molecules being metabolized by the plant, making them unavailable to the bacterial cells The activities associated with this project include training of one PhD student, Maria del Pilar Marquez. Additional undergraduate students and a high school student also participated in this work. Research results from this project were presented at annual grower meetings, the 2009 IS-MPMI meeting, at three annual APS meetings, and at multiple regional scientific meetings. Our collaboration with a faculty member in the Biology Department at the University of Wisconsin-Milwaukee was assisted by this project. PARTICIPANTS: Amy Charkowski (project director) managed this project, supervised others involved in this project, and completed some of the experiments and the majority of the writing for this project. Maria del Pilar Marquez was the graduate student responsible for the majority of the work associated with this proposal. She mentored Lindsay Hughes and Isaac Yang during this project. Ching-Hong Yang (UW-Milwaukee) and his students collaborated on this project, by reviewing project results and providing assistance with some assays. TARGET AUDIENCES: The target audiences for this project include potato, vegetable, and ornamental producers and processors, and researchers interested in plant-microbe interactions. To deliver information from this project, we presented research results from this project at multiple potato grower meetings, at the 2009 International Society for Molecular Plant-Microbe Interactions meeting, at three annual American Phytopathological Society meetings, and at regional scientific meetings. Results from this work were also presented at invited talks at the University of Colorado and at Hebrew University in Jerusalem. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Changes in knowledge from this project include identification of novel inhibitors of motility and the T3SS for plant pathogens and identification of novel virulence genes that may be targets of additional virulence inhibitor assays. This project was also responsible for training early career scientists and has provided them with increased knowledge of agricultural science.

Publications

  • No publications reported this period


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Activities from this project include the training of a PhD student, Courtney Jahn, who defended her thesis in July 2008. Experiments addressing both objectives of this project were completed. Products include numerous bacterial mutants and optimization of methods for bacterial mRNA purification. PARTICIPANTS: The individuals who worked on this project include the project director, Amy Charkowski, and a graduate student, Courtney Jahn. An undergraduate, Josiah Howley, also assisted with this project. TARGET AUDIENCES: The target audience of this work includes other plant pathologists and growers of vegetables and ornamentals. Information from this project was reported in peer reviewed articles and at two potato and vegetable grower meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our impacts were described in the abstracts of our publications from this project: The genome sequence of the Enterobacteriaceae phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) revealed homologs of genes required for a complete flagellar secretion system and one flagellin gene. We found that D. dadantii was able to swim and swarm but that ability to swarm was dependent upon both growth media and temperature. Mutation of the D. dadantii fliA gene was pleiotropic, with the alternate sigma factor required for flagella production and development of disease symptoms but not bacterial growth in Nicotiana benthamiana leaves. The flagellar sigma factor was also required for multiple bacterial phenotypes, including biofilm formation in culture, bacterial adherence to plant tissue, and full expression of pectate lyase activity (but not cellulase or protease activity). Surprisingly, mutation of fliA resulted in the increased expression of avrL (a gene of unknown function in D. dadantii) and two pectate lyase gene homologs, pelX and ABF-0019391. Because FliA is a key contributor to virulence in D. dadantii, it is a new target for disease control. RNA integrity is critical for successful RNA quantitation for mammalian tissues, but the level of integrity required differs among tissues. The level of integrity required for quantitation has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from Dickeya dadantii, a bacterium refractory to RNA isolation. Bacterial lysis with Trizol using standard protocols consistently gave low RNA yields with this organism. Higher yields due to improved bacterial cells lysis was achieved with an added hot SDS incubation step, but RNA quality was low as determined by the RNA Integrity Number (RIN). Contaminating DNA remained a problem with the hot SDS-Trizol method; RNA samples required repeated, rigorous DNase treatments to reduce DNA contamination to levels sufficient for successful real-time qRT-PCR. A hot SDS-hot phenol RNA method gave the highest RNA quality and required only two DNase treatments to remove DNA. The assessment of RNA integrity using the Agilent 2100 BioAnalyzer was critical for obtaining meaningful gene expression data. RIN values below 7.0 resulted in high variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. We found that RNA preparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels. This work provides guidelines for RNA isolation and quality assessment that will be valuable for gene expression studies in a wide range of bacteria.

Publications

  • Jahn, C. E., A. O. Charkowski, and D. K. Willis. 2008. Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation. J. Microbiol. Meth. 75:318-324.
  • Jahn, C. E., D. K. Willis, and A. O. Charkowski. 2008. The flagellar sigma factor FliA is required for Dickeya dadantii virulence. Mol. Plant-Microbe Interact. 21:1431-1442.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Activities: Quantitative real-time PCR assays were developed and genetic analysis of one of the virulence genes being examined was conducted. These are required for confirmation of potential virulence protein inhibitors. One one graduate student was mentored on this project. Events: Research related to this work was presented at the American Phytopathological Society meeting (one poster, one talk). New fundamental knowledge: The sigma factor controlling flagella production, also regulates other virulence genes and is required for full virulence. The hrpX/Y two component system, which is required for biofilm formation and full virulence, also regulates other potential virulence genes not involved in biofilm formation. Collaborations fostered by the project: A collaborative manuscript with Dr. Ching-Hong Yang was submitted and another is in preparation. PARTICIPANTS: Project Director = Amy Charkowski Graduate student = Courtney Jahn Ms. Jahn presented the following abstract at the American Phytopathological Society meeting and is currently working on two manuscripts describing work from this project. The flagellar sigma factor FliA is required for full virulence and biofilm formation of Dickeya dadantii C. E. JAHN (1), A. O. Charkowski (1) (1) University of Wisconsin-Madison, Madison, WI, USA Phytopathology 97:S50 The ability to move in a directed manner affords certain advantages to host-adapted bacteria including the ability to move toward a preferred host, access infection sites, and escape back into the environment. The best understood flagella regulatory pathways are the transcriptional hierarchies of Escherichia coli and Salmonella Typhimurium. However data from other bacterial species shows that the regulatory cascades and environmental effects on flagella gene expression cannot be generalized. The enteric phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi 3937) produce peritrichous flagella that are maintained in planta. The recently sequenced D. dadantii genome revealed homologs of all flagella genes, but arrangement of the operons differs from other enterobacteria. Recently, we found that FliA, the alternate sigma factor required for flagella production, is also required for full virulence and biofilm formation. In related species, FliA regulates virulence genes in addition to flagella genes. In D. dadantii, FliA is required for full expression of pectate lyase activity, which may account, in part, for the reduced virulence of the fliA mutant. Because FliA contributes to virulence and not solely to the regulation of motility it is a new target for disease control. TARGET AUDIENCES: Students and Researchers interested in bacterial genetics and plant disease control owners of small and large farms, as well as greenhouses. Ornamental and Vegetable Growers.

Impacts
The outcome of this project is an increase in the fundamental knowledge of regulation of virulence and biofilm formation genes in an important plant pathogen. This increase in knowledge will help us interpret results from our high throughput assay used to find virulence protein inhibitors.

Publications

  • Yap, M.-N., Yang, C.-H., Charkowski, A. O. 2008. The response regulator HrpY of Dickeya dadantii 3937 regulates virulence genes not linked to the hrp cluster. Mol. Plant-Microbe Interact. (accepted)