Location: Crop Improvement and Genetics Research
Project Number: 2030-21220-003-030-I
Project Type: Interagency Reimbursable Agreement
Start Date: Sep 15, 2025
End Date: Sep 14, 2028
Objective:
The goal of this proposal is the commercial delivery of a sustainable and biologically-based therapeutic delivery platform called “Symbiont technology” to treat citrus trees in production fields to provide protection/symptom alleviation from citrus greening disease (aka Huanglongbing, HLB).
Approach:
a: Develop the best Agrobacterium strain for commercial delivery and regulatory approval. Initial focus will be with the currently identified Agrobacterium strain, 1416, because it forms the highest quality and longest-lived galls (Fig. 4B), we have a high-quality complete genome sequence, and it is already adapted for the GAANTRY delivery system. WETo optimize this strain for commercial use we will 1) Re-introduce the opine genes into 1416 T-DNA; 2) evaluate knock-out mutants that have reduced ability to survive outside the Symbiont (we call these Reduced Free Living (RFL) mutations) and reduced ability to horizontally transfer the Ti-plasmids to other bacteria.
b. Protein Export Optimization. This will be accomplished by evaluating 80 different fusion proteins construction using known plant vascular mobile peptides. Using this fusion strategy we will evaluate HLB-symptomatic citrus response to Symbionts that produce fusions of these AMPs to this export peptide tag.
c. Identify the most effective defensive proteins to express. Students will use an Agrobacterium-based transient leaf infiltration assay to produce desired defensive AMPs and then use filter sterilized extracts from this transient expressing leaf tissue in plate based bacterial growth inhibition assays. They will screen for growth inhibition of Agrobacterium, E. coli and a gram positive Bacillus subtilis strain to identify new AMPs that could be used. Agrobacterium is used as a surrogate for CLas that cannot be cultured. It should be noted that all expression constructs used are designed with an intron to prevent expression when present within Agrobacterium for leaf inoculation thus preventing production of the AMP when the gene is in the Agrobacterium. Any AMPs showing desired activity will then be tested in small potted greenhouse citrus symbiont production for effect on CLas titer and HLB symptoms using 10 trees per construct and comparison to GFP expressing symbiont controls as well as no-symbiont citrus controls.