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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Insects and Horticulture Research » Research » Research Project #428857

Research Project: Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease

Location: Subtropical Insects and Horticulture Research

Project Number: 6034-22320-004-018-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Mar 1, 2015
End Date: Feb 29, 2020

Objective:
Research is designed to provide short-term citrus treatment solutions and long-term development of resistant transgenic citrus by completion of three main objectives. 1. Develop a comprehensive capability in assembling, integrating, and analyzing existing and emerging ‘omics and biology data on HLB to create an efficient research environment that speeds solution discovery. Initial focus: CLas, the ACP gut and salivary gland secreted and transmembrane proteins, ACP immune system and citrus phloem. 2. Design and deliver small molecule therapeutics for blocking HLB: RNA interference (RNAi)-inducing double stranded RNAs (dsRNAs), RNA aptamers, and peptide inhibitors for blocking transmission of CLas, and compounds from nontoxic small molecule libraries that stop CLas growth in the tree. Provide a grove-deployable delivery strategy optimized for therapeutic delivery of inhibitor molecules or combinations that allows economically viable citrus production in the face of HLB. 3. Engage research community, growers, regulators, and consumers to understand the best technology to advance, the best pathway for advancement and to education the stakeholders about the science foundation and application strategies of this CAPS grant.

Approach:
1. Protein-protein interaction blockers-peptides and RNA aptamers We propose to couple co-immunoprecipitation (coIP) and targeted proteomics to identify the receptors within the psyllid that interact with the gut binding peptides characterized by the Shatters Lab. The peptides used in this work were hexameric peptides with C-terminus ending in Gly-Gly-biotin linker-ligand. Using quantitative mass spectrometry, differences in protein stoichiometry can be determined in addition to the reliable identification of specific binding partners. Next, we will use a targeted proteomic platform, which includes selected reaction monitoring (SRM) and co-IP to specifically quantify the level of the gut surface receptors during CLas infection of the psyllid, in different psyllid tissues, during psyllid development, in different psyllid populations, and during RNAi experiments to silence the receptors. 2. RNAi- Oral delivery of dsRNAs as RNAi inducers. Drs. Shatters, Hunter and Stover conduct research on the use of orally delivered dsRNAs as RNAi-inducers that produce phenotypes in the psyllid that could be adapted as psyllid control strategies (mortality, development malformations, blocking CLas transmission, etc. This project will focus only on topical application strategies for therapeutic delivery of dsRNA exogenously applied to citrus and subsequent systemic movement of the dsRNA in the plant followed by psyllid uptake during feeding. Research will be conducted to identify the best targets or combination of targets and most effective candidates will be tested in field trees using several application strategies that will be optimized to support dsRNA stability and efficient dsRNA uptake by the plant. 3. Delivery Strategies Therapeutic Molecule Delivery. Drs. Shatters and Stover have obtained successes in optimizing therapeutic delivery of “antibiotic”-type bactericides using several strategies including a novel approach for metered delivery. This research focuses on adapting this strategy to delivery of interdiction molecules discovered in the proposed research. Transgenics. The Stover lab will work on developing transgenic citrus expressing genes to deliver therapeutic peptides and or dsRNA species identified in this research. Working with the Thomson lab (USDA Albany, CA), we have developed transgenic tobacco and citrus lines which contain an exchange /recombination site permitting comparison of numerous transgene variants were all plants have a single transgene copy inserted into the same genomic position. In the first year of this project, these founder lines will be used to optimize GUS expression exploring several promoters (AtSS, D35S, Citrus ScAMPs promoter-Belknap lab) and the presence/absence of a 5’ ubiquitin monomer leader, introns and 3’ untranslated sequences. The Citrus ScAMPs and At SS have high levels of phloem-specific expression. HLB-resistance screening will be conducted using methodology which is routine at USHRL: no-choice CLas-infected-psyllid feeding for one week followed by two months of exposure in a free-flying CLas-infected-psyllid house.