Location: Emerging Pests and Pathogens Research
Project Number: 8062-22410-007-010-I
Project Type: Interagency Reimbursable Agreement
Start Date: Sep 1, 2020
End Date: Aug 31, 2025
Objective:
The project will be divided into six overall objectives:
1) Identification and production of therapeutic molecules to protect trees from (Huanglongbing) HLB.
2) Implementation of a flexible and customizable bench-to-field screening pipeline for molecule evaluation, including broad community access to the pipeline.
3) Data collection to support regulatory approval.
4) Economic analysis of delivery strategies for field deployment.
5) Perform basic research to optimize delivery of molecules to citrus.
6) Train the next generation of scientists in synthetic biology.
Approach:
Already developed bioassays will be combined into a screening pipeline that includes rapid CLas+ extract assays, detached leaf assays for activity in planta against CLas, whole-plant therapeutic molecule infusion with CLas titer and HLB symptom evaluation, psyllid toxicity/transmission assays, and greenhouse and field evaluation of transgenic plants and exogenous molecule delivery methods. Combined evaluation of in-house molecules and molecules submitted by outside researchers will be conducted, with blind sample evaluation and confidential reporting. A minimum of 10 top performing candidates will be advanced to field testing. The pipeline will include in-field evaluation for HLB tolerance and/or psyllid toxicity using one of three delivery strategies: infusion devices, transgenic plants or biotherapy delivery. Many experiments are ready for evaluation now: 4 infusion experiments, numerous events of 5 transgenes expressed, and 2 biotherapy delivery. Data will be collected to support regulatory approval in consultation with agribusiness companies who will financially support regulatory approval. One of these delivery strategies is entirely new to plant biology and we will conduct basic research at the interface of synthetic and plant biology needed to improve its efficacy and maximize field performance. We will train undergraduates in synthetic biology/plant pathology.
b. Outline of methodology to be used to achieve project goals.
1. Therapeutic Molecule Identification. Citrus and ACP bioassays will be used to screen various molecules as a continuation of previously funded projects or on-going research.
2. Bioassay Screening Pipeline. A menu of already developed bioassays will be used and be made available to outside researchers for screening. Assays are already in-use for: 1) Antimicrobial compounds activity against CLas in: CLas+ psyllid homogenates; in planta detached HLB+ citrus leaves; whole potted plants; and in-field tree infusions studies; 2) Detached leaf/potted plant assays for psyllid mortality and CLas transmission studies.
3. In-Field Biomolecule Synthesis and Delivery. Using plant cell culture/synthetic biology, we will optimize a novel non-transgenic biomolecule delivery method for the continuous synthesis and delivery of molecules to protect trees in the field, for which proof-of-concept is complete.
4. Topical Delivery of Therapeutic Molecules. We will develop commercially viable infusion devices based on initial design concepts we have developed and 3D printed in our lab, and tested in the field. Agricultural engineering expertise will be used to evolve this strategy for commercial deployment. At the same time, this system will be used in field plot experiments to evaluate whole plant responses to therapeutic molecule delivery.
5. Transgenic Plant Evaluation. Already developed transgenic plants have demonstrated activity against ACP and/or CLas in initial greenhouse evaluations. Furthermore, we anticipate development of more lines if gene products are identified from the pipeline. Molecular analysis and citrus transformation are routine for the Fort Pierce laboratory and research farm.
