Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
(N/A)
Non Technical Summary
Plant diseases are a critical threat to agriculture worldwide, and fungicides are a valuable tool for disease prevention. Due to widespread use of fungicides, plant pathogen populations, including the highly destructive pathogen Botrytis cinerea, are becoming resistant to the available fungicides. When pathogens become resistant to a fungicide, they may be less competitive and aggressive than they would be if they were sensitive to the fungicide, which is called a fitness cost. The fitness costs associated with resistance to the most important fungicides are not fully understood, leading to challenges in providing accurate guidance on managing fungicide resistance.This project aims to clarify the significance of fitness costs associated with resistance to three frequently relied upon fungicide groups. Gene editing tools such as CRISPR/CAS9 and mitochondria-targeting nucleases will be utilized to induce targeted genetic mutations that are known to cause fungicide resistance into B. cinerea. Then, laboratory evaluations of the fitness of these fungicide-sensitive and fungicide-resistant strains will be conducted. This project is investigating fitness costs in a new way using these gene editing tools, and may provide a clearer picture of fitness costs. Results from this project will help to inform grower fungicide choices and fungicide resistance management strategies. In the long-term, this could result in prolonged or renewed effective use of these valuable disease control tools.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Goals / Objectives
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.
Project Methods
Objective 1: Edit the genome of fungicide sensitive parental B. cinerea isolates in a stepwise manner to cause three fungicide resistance associated mutations.Methods. Three fungicide sensitive strains of B. cinerea from different hosts and locations will be selected for downstream transformations. Three mutations are the target of this proposal, with 1) replacement of glutamic acid with alanine at position 198 (E198A) in ß-tubulin for MBC resistance, 2) replacement of glycine with alanine at position 143 (G143A) in cytochrome b for QoI resistance, and 3) the deletion of leucine at position 497in the transcription factor Mrr1 for phenylpyrrole resistance. Specifically, for mutations 1, 2 and 3, the DNA sequences 5'-GAG-3' (glutamic acid) will be changed to 5'-GCG-3' (alanine), the sequence 5'-GGT-3' (glycine) will be changed to 5'-GCT-3' (alanine), and the sequence 5'-GCG-3' (leucine) will be deleted, respectively. For mutations 1 and 3, CRISPR/Cas9 mediated transformation will be utilized using the polyethylene glycol transformation of protoplasts (Redman and Rodriguez 1994), and a guide RNA specific to each target site will be designed. During the transformation, the Cas9 enzyme will cause a double stranded break at the target site, and homology directed repair in the presence of donor DNA containing the desired mutated sequences will occur. Then, single transformant protoplasts will be regenerated in vitro and stored as fungal cultures. Mutation 2 will involve different methodology, since the CRISPR/Cas9 system has not been reliably used for altering mtDNA, like the target of this mutation 2, cytochrome b. The G143A mutation would require a G to C mutation. One method that will be suitable for producing this mutation is "mitoRE", a nuclease-based approach as described in the background above (Bacman et al. 2009; Barrera-Paez and Moraes 2022). The nucleotides that make up the codon at position 143 also match restriction sites, which can be targeted in this method. After transformation, isolates will be stored and prepared for screening for successful mutants. These mutations will be conducted on isolates in a stepwise fashion to produce multi-fungicide resistant isolates. The proposed order of mutation selection will also follow the order of step-wise accumulation of mutations that has been observed in field populations of B. cinerea (Li et al. 2014).Objective 2: Confirm that editing was successful with no off-target alterations and confirm the resistance phenotypes.Methods. Individual transformant isolates will be revived in vitro and will be screened for successful transformation by extracting DNA and conducting an allele-specific PCR that either amplifies the wild-type or mutant genotypes. This will be conducted for 10 to 20 transformants in order to evaluate the efficiency of the transformation. Then, isolates with confirmed mutant genotypes alongside parental, fungicide sensitive strains will be plated on fungicide amended media at a dosage at which only resistant isolates can grow. These dosages have been previously established for each chemical class that will be investigated (Fernández-Ortuño et al. 2014). Upon confirming the resistance phenotype, the respective target gene (ß-tubulin, cytochrome b, or Mrr1) of a small selection of mutants will be Sanger sequenced to absolutely confirm the presence of the desired mutation. One concern with gene editing is the potential for off-target alterations. Even with a highly specific guide RNA, off-target mutations could occur in other parts of the genome in regions that completely or mostly match the guide RNA. During the planning process, the RNA Design Checker tool (Integrated DNA Technologies Inc., IDT, Coralville, IA) will be used to design the guide RNA and will be checked for other matching sequences in the B. cinerea genome using BLAST. To check for potential off-target alterations after the transformation, the program Cas-OFFinder (http://www.rgenome.net/cas-offinder) will be utilized to check for regions of the genome that closely match the guide RNA. Primers will be designed to amplify and sequence these regions and they will be screened for off-target mutations. Furthermore, MinION and Illumina Whole Genome Sequencing will be utilized to sequence the entire genome to ensure no off-target mutations occurred.Objective 3: Assess the fitness of parental and mutant isolates with different combinations of fungicide resistance associated mutations.Methods. First, the stability of each mutation will be evaluated by successively re-plating mutant isolates and then confirming the mutation with the allele-specific PCR mentioned above. Then, four fitness metrics will be compared between the isolates, the mycelial growth rate, the sporulation rate, the spore germination rate, and sclerotia production. These will be tested at low (11 °C), medium (22 °C), and high (30 °C) temperatures. Furthermore, isolates with the deletion of L497will be evaluated for their ability to endure osmotic (salt) stress, as this has been previously linked with phenylpyrrole resistance (Fernández-Ortuño et al. 2015). After in vitro experiments, mutant and wild type isolates will be inoculated onto detached fruit of different B. cinerea hosts including strawberry, grape, and apple. The disease growth rate (lesion diameter) will be measured following inoculation.I plan to publish two papers from this work, with both submitted to well respected peer reviewed journals by the end of this funding period. One paper will cover the editing of the mtDNA gene cytochrome b, and the second paper will cover the other aspects of the three aims of this project. The quality and impact of the work will be evaluated by the impact factor of the journal in which it is published.I will present preliminary work at Reinhardsbrunn International Symposium in 2023 and complete results at Plant Health 2024, where peer response will indicate the value of the work. I will also present applicable results and fungicide resistance management strategies at two regional stakeholder meetings per year including the Mid-Atlantic Fruit and Vegetable Convention and the Maryland Grape Growers Association annual meeting.