Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 999 - Citrus, general/other | 1160 - Pathology | 100% |
In this project, we will develop and deliver a kind of 'therapy' that relies on small dsRNA molecules that mimic psyllid gene sequences. When the dsRNAs are applied foliarly to citrus trees (phloem) and ingested by psyllids during feeding, the dsRNA initiates a response that knocks down, or silences psyllid gene expression. RNA-interference (RNAi) can result in reduced expression of genes (proteins), which in turn can impair important molecular and cellular functions in the target organism. Previously, we demonstrated that RNAi effectively silences gut gene expression in psyllids, some resulting in psyllid mortality, and others that interfere with Liberibacter invasion and establishment in the psyllid gut, resulting in reduced Liberibacter transmission. We also have shown that by using multiple dsRNAs together, or 'stacking' them, the effects can be multiplied. Stacking has the added benefit that psyllids will be unlikely to develop resistance to 'therapeutic' dsRNA treatment. DsRNA therapy offers a highly target-specific, non-toxic (humans, off-target organisms, and environmental) that does not require GMO technology for effective delivery of an effective dose to the plant, and will control CLas and ACP simultaneously, leading to reduced psyllid population size and lowered CLas levels in citrus trees. Further, highly promising light laser delivery technology has been used to create tiny wounds in the epidermis of citrus leaves, and when dsRNA is applied foliarly with effective delivery amendments (surfactants, penetrants, and protectants), the dsRNA molecules are directed into the phloem. Effective lasers are already available for laboratory and field applications, including models designed for use in citrus groves. Together RNAi and laser-enhanced foliar delivery of dsRNAs promise to provide a significant new tool to HLB management. These breakthroughs address timely needs of the citrus industry by creating new ways forward that promise to return production to near pre-HLB levels, permit re-establishment of groves in HLB hard-hit locales, aid in reclaiming HLB infected groves, and make it economical to expand new tree plantings to meet market demands. This project focuses on the design and delivery of effective, target-specific dsRNAs already proven to cause psyllid mortality or interfere with psyllid-mediated CLas transmission, following dsRNA ingestion. Herein, we couple translational research and extension pillars of discovery, design, efficacy testing, validation, and characterization, with a promising delivery system for HLB control.
The goal of this project is to develop 'therapy' that uses small dsRNA molecules delivered to the plant phloem, that when ingested by psyllids, induces RNA-interference (RNAi). In a previous project, we (and others) have demonstrated that gene silencing of psyllid genes can cause high mortality, and/or interfere with Liberibacter invasion of the psyllid gut, and potentially reduce rates of transmission. The project will define optimal delivery parameters for 'best dsRNA performers' based on dsRNA dose, efficacy, and persistence criteria. The long-term goal is to implement RNAi in citrus IPM programs to reduce psyllid vector numbers and lower CLas inoculum levels. The project objectives are:
Objective 1. Optimize laser-foliar uptake of dsRNA into phloem of tomato seedlings. Initially, a number of different solutions will be tested as carriers of dsRNA using a range of laser settings to optimize dsRNA delivery to the phloem. Laser assisted delivery in foliar applications will be enhanced with compounds e.g. lipophilic/ hydrophilic surfactants and penetrants. Nanoparticle spheres of known sizes that enter the phloem post-laser treatment will serve as quantitative markers to evaluate the success of the treatment. Next, we will optimize the conditions for laser-leaf uptake of fluorescent-dsRNA, a tracer molecule that mimics the size of dsRNA. Once parameters are established for dsRNA phloem delivery, 20-30 dsRNA candidates will be tested for knock down using the PoP-CLso system.Objective 2. Use fl-dsRNA to track delivery and localization, through direct uptake in sucrose feeding chambers, and indirect uptake, through psyllid feeding on plants to ingest fl-dsRNA.Objective 3. Determine the minimal effective dose by sequentially reducing the amounts of delivered dsRNA to the psyllid, previously quantified by spectrophotometric analysis, in a feeding chamber (Obj. 2) until changes in knockdown and phenotype are not observed. 100 ng dsRNA total fed to a psyllid in a sucrose feeding chamber will affect significant gene expression knockdown with a resulting altered phenotype in the psyllid. Preliminary results thus far indicate that this amount of dsRNA may be well below the detectable levels by the above-described methods. The minimal effective dose will be estimated by quantifying dsRNA prior to delivery, followed by gene knockdown via qPCR and bioassay to test effects of dsRNA on development, mortality, or transmission.Objective 4. Characterize the persistence of a measurable dose of dsRNA first in the plant (both tomato and citrus) over time for various delivered doses. Measure relative quantities of dsRNA by fluorescence or absolute quantities by chemiluminescence of plant tissue subjected to foliar application of fl-dsRNA, over time. Using the altered development phenotype in nymphs, conduct a time-course study to determine the duration of 'effective RNAi' in psyllids. A serial bioassay will be carried out to quantify persistence of RNAi in psyllids, measured by qPCR.Objective 5. Optimal candidate ACP-specific dsRNAs will be validated for ACP gene knockdown based on results in Obj. 4, first in CLas-infected citrus leaves/seedlings, then for young trees (greenhouse), and finally in field trials. Methods for delivery, efficacy, validation, quantification in citrus trees and ACP will be according to PoP methods. Our commercial partner will be involved in field-testing in Yr-3.Objective 6. We will use Illumina NextSeq to conduct dual host-pathogen RNA sequencing to characterize large and small RNAs, in relation to dsRNAs, and determine the extent of RNAi fidelity to our chosen targets in psyllids (ACP and PoP) and a non-target phloem-feeder, the whitefly. The mRNA of associated bacterial species will also be sequenced. Together, both data sets will produce global profiles reflecting host and pathogen gene expression.Objective 7. University of Florida, Research and Education Centers in Fort Pierce and Lake Alfred, and University of Arizona will partner with Texas A & M University and University of California, Riverside to provide outreach and extension for the project to HLB affected states. We will deliver practical, research-based solutions to combat HLB that improve the sustainability of the citrus industry, and are effective for area-wide management of HLB. The proposed outreach plan is multi-tiered to ensure constant stakeholder engagement and effective dissemination of results.
Target Audience
The target audience focus was directed toward citrus producers and other stakeholders associated with the citrus industry, field/crop managers, extension specialists/agents, departments of agriculture, researchers at universities and in industry, post-docs and graduate students attending conferences and workshops in the fields of entomology, plant pathology, virus-vector biology, plant protection, insect ecology, agronomy, biopesticide use or development, and/or molecular diagnostics use or development. Results were disseminated through presentations at professional meetings, abstracts and proceedings, and publications.
Changes / Problems
The ACP biological system is difficult to work with and fraught with problems associated with the biology of the vector and of the pathogen-citrus host pathosystems. Much more time than anticipated was required to establish effective ACP feeding systems for nymphs and adults. The large number of PoP genes screened (~200 targets) required enormous coordination within and between the collaborating labs, and the teams rose to the occasion despite hardships and the inability to purchase molecular and disposable reagents and supplies during and even after the pandemic. Hurricane damage to greenhouses/colonies in Florida during the project resulted in major set-backs and lost experiments. The CoVID pandemic disrupted work in both labs and after post-docs left the AZ lab to take jobs, it was difficult to recruit new young scientists to complete the work left underway. One additional no-cost extension was requested but denied, despite the extraordinary circumstances. The PI and co-PI and authors of the publications, accepted/in press (with publications fees to pay), and those to be submitted are committed to completing, submitting, and publishing during 2024.
Training & Professional Development
At least ten post-doctoral associates and two graduate students were trained during this project in Arizona and Florida. Technicians and technical staff in the Brown and Qureshi labs learned new techniques, methodologies, and skills. Our collaborators lab staff were also exposed to dsRNA technology for biopesticide development. All of the post-docs and graduate students were placed in positions at universities, in industry, and/or went on for a Ph.D. after completing a M.S. degree. One technician was hired by industry.
Dissemination Streams
The results have been reported at conferences involving the citrus industry and other industry stakeholders and at professional meetings including profession plant pathology and entomology societies.
Next Reporting Steps
Nothing Reported