Progress 12/01/14 to 11/30/16
Outputs
Target Audience:Through the reporting period of 12/2015 - 11/2016, I have reached the following Target Audiences: A contributed talk at the International Symposium on Plant-Microbe Interactions in Portland, OR (July, 2016) A poster presentation at the International Symposium on Microbial Ecology in Montreal, Quebec (August, 2016) Instruction and laboratory hands-on engagement with predominantly Hispanic-speaking high school students from Tucson High Magnet School, an Hispanic Serving Institution (HSI) in Tucson, Arizona. Instruction in laboratory research technique for 2 undergraduate researchers, including one from underrepresented scientific demographics, at the University of Arizona in Tucson, Arizona. After the conclusion of the reporting period, work related to these funds were shared at: An invited seminar at Cornell University, Plant Pathology and Plant-Microbe Biology Section. Ithaca, NY (February, 2017). A poster presented at the American Society for Microbiology Conference on Mechanisms of Interbacterial Cooperation and Competition. Washington D.C. (March, 2017). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In addition to stimulating interactions at international conferences, this fellowship provided the basis for a USDA NIFA AFRI Foundational Grant that was submitted and funded in collaboration with my postdoctoral advisor. Additionally, I was able to secure a tenure track assistant professorship at The Pennsylvania State University as a direct result of this fellowship. How have the results been disseminated to communities of interest?Results of this project have been disseminated in a peer-reviewed publication to the scientific community. Both I and an undergraduate student who worked on this project, have presented our research at national conferences reaching scientific professionals, as well as graduate, undergraduate, and high school students. I gave an invited lecture at the USDA ARS station in Salinas, California, reaching agricultural researchers. I have used my results as the basis for outreach to high school students from a Hispanic Serving Institution, Tucson High Magnet School. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In addition to the accomplishments noted in the previous report, I have expanded on the genetic understanding of tailocin resistance and sensitivity within Pseudomonas syringae. First, I have sequenced and identified the causative mutations for tailocin resistance in two pathovars of P. syringae, pv. glycinea (Pgy) and pv. phaseolicola (Pph). All mutations map to lipopolysaccharide (LPS) biosynthetic genes. I confirmed by LPS isolation and SDS-PAGE that all Pph mutants were affected in O-antigen biosynthesis. All predicted mutations were confirmed to be causative by allele-swap mutagenesis between the parent wild type strain and the derivative tailocin-resistant mutants. In all cases, the mutant allele confered tailocin resistance and loss of O-antigen. Additionally, I confirmed that the LPS mutations resulted in loss of virulence toward their host plant compared to the wild type strain. This loss of virulence (roughly 1 log less growth at 24 hours post infiltration) was complementable by inclusion of the wild type allele into the mutant background. Second, I discovered that mutations in P. syringae pv. actinidiae that result in tailocin resistance (transposon insertions that disrupt LPS O-antigen biosynthesis) results in gained sensitivity to an alterantive bacteriocin produced by P. syringae pv. syringae. This result is unique and will form the basis for future research endeavors. Additionally, this points to the possibility of incorporating multiple bacteriocins into a control strategy that may help slow the emergence of bacteriocin resistance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Hockett, K.L., Baltrus, D.A. Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds. J. Vis. Exp. (119), e55064, doi:10.3791/55064 (2017).
- Type:
Journal Articles
Status:
Other
Year Published:
2017
Citation:
Hockett, K.L., Baltrus, D.A. Conditionally Redundant Bacteriocin Targeting by Pseudomonas syringae. In preparation.
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Progress 12/01/14 to 11/30/15
Outputs
Target Audience:Through the reporting period of 12/2014 - 12/2015, I have reached the following Target Audiences: An invited seminar to agricultural researchers at the USDA ARS in Salinas, California. A talk and poster presentation to scientific researchers (professors, post-docs, graduate students) at the 2015 Gordon Research Conference in Microbial Population Biology in Andover, New Hampshire. A talk at the centennial bacteriophage meeting to scientific researchers (professors, post-docs, graduate students) at San Diego State University, in San Diego, California. A talk for the School of Plant Sciences seminar at the University of Arizona, in Tucson, Arizona. Instruction and laboratory hands-on engagement with predominantly Hispanic-speaking highschool students from Tucson High Magnet School, an Hispanic Serving Institution (HSI) in Tucson, Arizona. Instruction in laboratory research technique for 4 undergraduate researchers, including two from underrepresented scientific demographics,at the University of Arizona in Tucson, Arizona. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?To date, this project has provided training and professional development both for myself as well as undergraduate students whom I have mentored. For myself this project has provided multiple professional development opportunities. First, I was able to attend the Gordon Research Conference in Microbial Population Biology, an international conference focused on evolutionary and ecological aspects of microbiology. I was also able to visit a collaborator (Dr. Carolee Bull) at the USDA Agricultural Research Service in Salinas, California. In addition to collaborating on research projects, I was able to meet with Dr. Bull regarding student mentorship, an area in which Dr. Bull has received national awards. I have applied the mentorship guidance provided by Dr. Bull, while working with 5 undergraduate student researchers. The students I have mentored under this project have received training both in laboratory research as well as scientific writing and presentations. One student received summer funding from the Undergraduate Biology Research Program to work on analyzing bacteriocin resistant mutants. Another student presented this research at two national meetings, the American Phytopathological Society's Annual Meeting and the Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) Annual Meeting. Thus this student has received both laboratory training and professional development as a result of this project. How have the results been disseminated to communities of interest?Results of this project have been disseminated in a peer-reviewed publication to the scientific community. Both I and an undergraduate student who worked on this project, have presented our research at national conferences reaching scientific professionals, as well as graduate, undergraduate, and highschool students. I gave an invited lecture at the USDA ARS station in Salinas, California, reaching agricultural researchers. I have used my results as the basis for outreach to highschool students from a Hispanic Serving Institution, Tucson High Magnet School. What do you plan to do during the next reporting period to accomplish the goals?I will continue to pursue the research plan as described in my fellowship application. Specifically, I will: Identify the mutations leading to bacteriocin resistance. Identify the cause of the non-heritable resistance phenotype. Evaluate the loss of virulence in the heritable resistant mutants. Characterize the genetics underlying bacteriocin production in the noted strains. Evaluate modified methods for applying bacteriocins to prevent plant disease.
Impacts
What was accomplished under these goals?
Crop loss resulting from microbial plant pathogens is a major obstacle to efficiently producing food and fiber for the world's growing population. Microbially-caused plant diseases result in losses well in excess of 1 billion dollars annually, within the US. There are two major approaches for preventing plant diseases caused by bacteria: use of a resistant crop variety or treatment with a chemical biocide. Resistant crop varieties can be effective preventing disease, but have certain drawbacks. Such resistant varieties are time and resource intensive to develop, and they can have a lower market value than non-resistant varieties. Additionally, in many cases microbial pathogens can overcome or "break" resistance by acquiring mutations that allow the pathogen to infect the resistant variety. Pathogen control that relies on chemical biocides can likewise become ineffective if the target pathogen acquires mutations conferring resistance to the chemical. Additionally, chemical biocides potentially exhibit deleterious environmental an health consequences for producers and consumers. Thus, there is a need to develop new methods for controlling plant diseases that will beeffective and environmentally benign. Bacteriocins are protein-based chemicals produced by bacteria that are toxic toward other bacteria. They exhibit many qualities that make them ideal candidates for preventing plant diseases incited by bacteria. Bacteriocins are highly specific in their targeting as opposed to traditional chemical biocides. Bacteriocins are effective at concentrations that are substantially lower than traditional biocides. Finally, as bacteriocins are protein-based, they are not predicted to persist or disseminate within the environment as chemical biocides can. Before bacteriocins can be implemented as pathogen-control compounds, however, there a a number of aspects that need to be addressed, including the specificity of bacteriocin targeting, dynamics of bacteriocin resistance, and how effective will bacteriocins be in controlling plant diseases. While this project is ongoing, I have made progress toward the research objectives described above as detailed. In this work, I have discovered that a bacteriophage-derived bacteriocin (termed tailocin), is the major determinant of killing activity within Pseudomonas syringae, and that this tailocin is broadly conserved across P. syringae strains. This finding served as the basis for a peer-reviewed publication. Additionally, I have identified two genes that are responsible for specific targeting spectra. Using molecular genetics, I have exchanged these two genes among strains, yielding an expected shift in targeting spectra. These findings have been leveraged to engineer a mock biocontrol P. syringae strain that has been used in biocontrol assays (see below). I have tested the effectiveness of P. syringae biocontrol strain, which has been engineered to target a radish pathogen, for in planta control of said pathogen. The results from these experiments have been encouraging, but have also shown that further research is needed to produce an effective biocontrol agent. Importantly, my results have shown that the engineered biocontrol strain is maintained at roughly 100-fold greater population level than control strains. However, this strain was not able to significantly affect the pathogen population or disease incidence/severity. This lack of control likely stems from the engineered biocontrol strain has a noticeable growth defect relative to control strains as well as the relative timing of applications of the pathogen and biocontrol. Another round of experiments is being conducted aimed at improving control, which will include engineering a new strain with a chromosomally located targeting cassette and application of the biocontrol prior to the pathogen. In examining the dynamics of bacteriocin resistance, I have discovered three forms of resistance: heritable, partially-heritable, and non-heritable resistance. This finding represents a significant change in knowledge, as it was unexpected to observe partially- and non-heritable resistance, which will have implications for the application bacteriocins as pathogen control compounds. These results a wholly novel. Importantly, I have also discovered that those strains with heritable resistance exhibit a loss of virulence in that they have a significant population deficit, achieving just 1/10 the population level when compared to the parent, non-resistant strain. The strains that exhibit non-heritable resistance do not suffer a similar loss of virulence. These finding not only are significant from a biocontrol standpoint, but are also significant (and thus represent a change in knowledge) with regard to the influence of microbe-microbe interactions on plant-microbe interactions. Strains that exhibit heritable resistance are currently being sequenced to identify causative mutations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Hockett KL, Renner T, Baltrus DA. 2015. Independent co-option of a tailed bacteriophage into a killing complex in Pseudomonas. mBio 6(4):e00452-15. doi:10.1128/ mBio.00452-15.
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