UNDERSTANDING FITNESS COSTS ASSOCIATED WITH FUNGICIDE RESISTANCE USING GENE EDITING

Career development goalsThis project includes professional training goals, as well as research goals which will lead into my long-term career goal, which is to lead a basic/applied plant pathology program at a land-grant university or USDA research station. I intend to tackle the most pressing pathology-related issues faced by my stakeholders, while furthering scientific advancements in my research field. I will build off my expertise in the epidemiology and fungicide sensitivity of highly destructive plant pathogens. Specifically, I am highly interested in the selection and evolution of fungicide resistance in pathogen populations and how this affects the real-world disease control in the field. I also desire to bring cutting edge tools into my applied research program including molecular and machine learning techniques. My previous research has been highly focused on applied field and in vitro research, but I desire to build a research program that brings basic research into the applied world. I will utilize my postdoctoral training and this fellowship to develop my molecular biology skills, primarily in the blooming area of gene editing.Professional training goalsMolecular techniques. My previous research experience has trained me to be proficient in field and laboratory experiments. I have also employed traditional molecular techniques such as PCR, qPCR, and Sanger sequencing. However, this project will require more advanced techniques, technologies, and software, including CRISPR/Cas9, whole genome sequencing, and bioinformatics.In order to become proficient in modern molecular techniques, I will enroll in workshops and trainings on these topics including plant pathology-bioinformatics workshops hosted by the American Phytopathological Society. These trainings will be most useful in the first year of this fellowship and will be primarily sought at that time.Mentoring. As I have been the recipient of wise mentoring throughout my academic career, I desire to reciprocate this to undergraduate research assistants who are interested in plant pathology. I have mentored and managed multiple undergraduate students over the course of my PhD and Master's studies. However, my PhD fellowship training with the Foundation for Food and Agriculture Research instructed me that mentoring can be more effective if it includes more than just teaching of laboratory techniques. This proposal's budget contains funding for undergraduate research assistants, and I want to support them with professional development opportunities in addition to laboratory training.Mentor and manage one to two undergraduate assistants for the duration of the project.Mentorship will include monthly meetings and discussion of UMD Career Center materials, research goals, and an Individual Development Plan (IDP).Extension. A core motivation of my research is for it to be as applicable and helpful to the stakeholder as possible; addressing critical needs in the agricultural industry. My previous extension experience has primarily taken the form of presentations at grower meetings.I will continue in these efforts by giving two extension presentations per year, including the Maryland Grape Growers Association Annual Meeting, and the Mid-Atlantic Fruit and Vegetable Convention.To explore and develop further, I will foster writing extension materials by writing one extension publication per year. These publications will incorporate the field-applicable findings of my research including fungicide resistance management and fruit rotting disease control in small fruit.Research goalsThis proposal seeks to clarify the significance of fitness costs to methyl benzimidazole carbamates (MBCs), quinone outside inhibitors (QoIs), and phenylpyrroles using gene editing techniques and B. cinerea as a model pathogen. Conclusions from these transformations and fitness cost tests will inform fungicide resistance management decisions. This may enable the longer effective lifespan of the limited number of effective fungicides. Specifically, gene editing techniques will be conducted on fungicide-sensitive parental strains, transforming their genotypes with mutations that have been associated with fungicide resistance. These mutations will be the replacement of glutamine with alanine at position 198 (E198A) in ß-tubulin for MBC resistance, replacement of glycine with alanine at position 143 (G143A) in cytochrome b for QoI resistance, and the deletion of leucine at position 497 in the transcription factor Mrr1 for phenylpyrrole resistance. This will also be conducted sequentially to produce multi-fungicide resistant strains. Following transformation, the new genotype and accompanying fungicide resistance will be confirmed through sequencing and in vitro testing. Third, the fitness will be compared between the mutant and parental isolates. These simple objectives will lead to powerful conclusions of the significance of fitness costs associated with these fungicide groups.I will investigate the fitness cost associated with genetic mutations leading to fungicide resistance via three specific objectives:Edit the genome of fungicide sensitive parental B. cinerea isolates in a stepwise manner to cause three fungicide resistance associated mutations.Confirm that editing was successful with no off-target alterations and confirm the resistance phenotypes.Assess the fitness of parental and mutant isolates with different combinations of fungicide resistance associated mutations.