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Open Final MS Thesis Updated.Pdf The Pennsylvania State University The Graduate School College of Agricultural Sciences TN5 MUTAGENESIS REVEALS POTENTIAL MECHANISMS OF METABOLIC PARASITISM OF APPLE FRUIT BY ERWINIA AMYLOVORA A Thesis in Plant Pathology by Melissa Finley Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2017 ii The thesis of Melissa Finley was reviewed and approved* by the following: Timothy McNellis Associate Professor of Plant Pathology Thesis Advisor Cristina Rosa Assistant Professor of Plant Pathology Seogchan Kang Professor of Plant Pathology Edward Dudley Associate Professor of Food Science Carolee Bull Head of the Department of Plant Pathology and Environmental Microbiology *Signatures are on file in the Graduate School iii ABSTRACT The gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, a destructive disease of apples, pears, and other Rosaceae species. This study seeks to further elucidate the trophic aspects of the host-pathogen parasitic interaction and which metabolic pathways are required for pathogenicity. Auxotrophic mutants of E. amylovora were generated via Tn5 transposon mutagenesis followed by plating mutagenized bacterial cells on a selective minimal media on which auxotrophs could not grow. Forty-seven confirmed auxotrophic mutants were then inoculated onto immature ‘Gala’ apple fruits in order to evaluate their pathogenicity. The mutated genes were identified by Sanger sequencing of E. amylovora DNA flanking the Tn5 insertion in each auxotrophic mutant. Characterization of transposon insertion sites showed that the following biosynthetic pathways or cellular functions were disrupted: amino acid biosynthesis (19), nucleotide biosynthesis (12), sulfur metabolism (5), nitrogen metabolism (2), survival protein biosynthesis (2), exopolysaccharide biosynthesis (3), and a selection of uncharacterized proteins (4) (the number of mutants of each type is listed in parentheses). We hypothesize that if an auxotrophic mutant is able to cause disease, the mutant must be able to derive the missing metabolites from host tissues. If an auxotrophic mutant is not able to cause disease, this suggests that it cannot derive the missing metabolites from their host. It was determined that the disruption of amino acid biosynthesis, such as for the production of arginine, leucine, and methionine, and nucleotide biosynthesis, such as for the production of purines and pyrimidines, resulted in reduction or elimination of pathogenicity. These two groups of mutants are unable to obtain sufficient amounts of the missing metabolic products from the host tissue in order to complement their metabolism and grow normally. Conversely, mutants with disrupted sulfur metabolism remained pathogenic, indicating that these mutants were able to obtain sufficient amounts of sulfur and sulfur metabolites from the host tissue. In addition, mutants iv defective in several survival proteins and exopolysaccharide biosynthesis were identified during screening as possible auxotrophs, and they displayed reduced or absent disease expression. The question of why these mutants are auxotrophs is still being investigated. In summary, this genetic study revealed new details of the profile of pathogen-accessible metabolites in colonizing the host tissues and furthered understanding of which metabolic pathways are needed for disease development. v TABLE OF CONTENTS List of Figures ......................................................................................................................... vi List of Tables ........................................................................................................................... vii Acknowledgements .................................................................................................................. viii Chapter 1 INTRODUCTION .................................................................................................. 1 1.1 Rationale and Objectives............................................................................................ 1 1.2 Literature Review ....................................................................................................... 3 1.2.1 Fire Blight ....................................................................................................... 3 1.2.2 Erwinia amylovora .......................................................................................... 10 1.2.3 Bacterial Access to Plant Nutrient Niches ...................................................... 11 1.2.4 Iron Acquisition............................................................................................... 14 1.2.5 Tn5 Mutagenesis ............................................................................................. 16 1.2.6 Nutritional Auxotrophy ................................................................................... 18 1.2.7 Erwinia amylovora-Host Interactions ............................................................. 20 1.2.8 Auxotrophy of Erwinia amylovora Illustrates Details of the Host Interactions ....................................................... 23 Chapter 2 METHODOLOGY ................................................................................................. 25 2.1 Experimental Design .................................................................................................. 25 2.2 Data Collection and Analysis Techniques ................................................................. 26 Chapter 3 RESULTS................................................................................................................ 31 3.1 Sequence data ............................................................................................................. 32 3.2 Pathogenicity data ...................................................................................................... 32 Chapter 4 DISCUSSION ........................................................................................................ 37 4.1 Amino Acid Biosynthesis .......................................................................................... 40 4.2 Nucleotide Biosynthesis ............................................................................................. 42 4.3 Sulfur Metabolism ...................................................................................................... 44 4.4 Nitrogen Metabolism ................................................................................................. 45 4.5 Hypothetical Protein Biosynthesis ............................................................................. 47 4.6 Exopolysaccharide Biosynthesis ................................................................................ 48 4.7 Survival Protein Biosynthesis .................................................................................... 49 4.8 In the Context of the Plant Host Tissues .................................................................... 50 4.9 Experimental Pitfalls .................................................................................................. 53 Chapter 5 FUTURE WORK AND CONCLUSIONS ............................................................. 55 CITATIONS ............................................................................................................................ 58 APPENDIX .............................................................................................................................. 71 vi LIST OF FIGURES Figure 1. Work flow diagram .................................................................................................. 25 Figure 2. Fifty Tn5 mutants screened on an LB Kan50 plante with grid; an auxotrophic mutant did not grow on the M9 minimal media ............................................................... 26 Figure 3. Experimental replications of a prototrophic mutant and an auxotrophic mutant from secondary auxotrophic screen ................................................................................ 27 Figure 4. Pathogenicity assay in immature ‘Gala’ apple halves ............................................. 28 Figure 5. Equation for the determination of theoretical genetic saturation ............................. 31 Figure 6. Distribution of pathway types affected by the insertion of the Tn5 transposon ....... 32 Figure 7. Variations in pathogenicity phenotype are divided by the biosynthesis pathway type ......................................................................................... 34 vii LIST OF TABLES Table 1. Nutritional Auxotrophy in Plant-Associated Bacteria .............................................. 19 Table 2. Auxotrophic Tn5 mutant symptom rating metric ..................................................... 29 Table 3. Distribution of Pathogenicity Phenotypes by Affected Pathway ............................... 33 Table 4. Mutants Auxotrophic for Amino Acid Biosynthesis ................................................. 34 Table 5. Mutants Auxotrophic for Nucleotide Biosynthesis .................................................... 35 Table 6. Mutants Auxotrophic for Nitrogen and Sulfur Metabolism ....................................... 36 Table 7. Unexpected Mutants: Mutants Auxotrophic for Exopolysaccharide Biosynthesis, Survival Proteins, and Uncharacterized Proteins ....................................... 36 Table 8. Summary of Tn5 Virulence Screen Results ............................................................... 38 viii ACKNOWLEDGEMENTS
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