Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
Citrus HLB is the most devastating citrus disease. We aim to expedite the release of non-transgenic HLB-resistant/tolerant citrus varieties through a multi-tiered decision-making approach including 1) assessment of genome-edited citrus plants previously generated by a transgenic approach for HLB resistance/tolerance, fruit quality and yield and other horticultural traits. 2) Accelerate screening and evaluating of target genes for non-transgenic genome editing usingCandidatusLiberibacter psyllaurous (synonymCa.L. solanacearum)-tomato as a surrogate by evaluating approximately 100 tomato mutants related to immune response and cell death that were previously generated.3)Generation of non-transgenic genome-edited citrus varieties for HLB susceptibility genes using our newly developed co-editing technology via mature tissue transformation to bypass juvenility.4)Evaluation of non-transgenic genome-edited citrus varieties for HLB resistance/tolerance, fruit quality and yield and other horticultural traits.5)Delivery of non-transgenic genome-edited citrus varieties via extension and outreach approaches, regulatory approvals, registration, and release.
Animal Health Component
90%
Research Effort Categories
Basic
10%
Applied
90%
Developmental
0%
Goals / Objectives
The overall goal of the project is to expedite the release of non-transgenic HLB-resistant/tolerant citrus varieties through a multi-tiered decision-making approach.Thecentral hypothesis is that citrus resistance/tolerance against HLB can be engineered throughgenome editing of genes critical for the CLas-triggered dysfunctional immune responses and celldeath. Five objectives are proposed: 1) Assessment of genome-edited citrus plants previously generated by a transgenic approach for HLB resistance/tolerance, fruit quality and yield and other horticultural traits. We will evaluatepreviously generated transgenic genome-edited C. sinensis lines. 2) Accelerate screening and evaluating of target genes for non-transgenic genome editing using Candidatus Liberibacter psyllaurous (synonym Ca. L. solanacearum)-tomato as a surrogate by evaluating approximately 100tomato mutants that were previously generated. 3) Generation of non-transgenic genome-edited citrus varieties for HLB susceptibility genes. This will be conducted using our newly developed co-editing technology via mature tissue transformation to bypass juvenility. 4) Evaluation of non-transgenic genome-edited citrus varieties for HLB resistance/tolerance, fruit quality and yield and other horticultural traits. 5) Extension and outreach.
Project Methods
Objective 1:Greenhouse assays. Twelve-month-old propagated genome-edited lines and wild-type will be inoculated with CLas via grafting. CLas-negative genome-edited lines and wild-type plants will serve as controls. We will quantify CLas in the inoculated plants with qPCR. Measurements of ROS production, starch accumulation, callose deposition around sieve pores, and phloem tissue cell death will be conducted every 3 months within the 12-month duration. We will monitor HLB symptoms monthly and investigate tree growth (trunk diameter, height, and canopy) annually. Since many edited lines were manipulated in plant immunity, we will investigate whether their resistance to other common citrus pathogens are affected in greenhouse studies using one bacterial pathogen Xcc (canker) and one fungal pathogen Mycosphaerella citri (greasy spot) for the testing.Field trials. The genome-edited lines and wild-type of 1.5-year-old will be planted in fenced sites that are approved by USDA APHIS Biotechnology Regulatory Service (BRS) with Permit Under 7 CFR 340 for testing transgenic citrus in FL, TX and CA. For each edited line at each site, we will plant 8 propagated plants. Eight wild-type plants will be used as controls. Randomized block design will be used. We will quantify CLas titers every 3 months, monitor the disease severity and incidence of HLB of the edited lines and control plants. We will investigate ROS, callose deposition, starch accumulation and phloem cellAdditionally, we will monitor other diseases, such as greasy spot, for disease severity and incidence every 3 months in FL, CA, and TX. To test whether the genome edited plants are affected in normal citrus growth, we will evaluate the size of the seedlings (height and trunk diameter), leaf morphology, leaf fresh weight and area, rate of photosynthesis, gas exchange, leaf chlorophyll content, root density, carbohydrate metabolism, and phytohormone analysis of the modified and wild-type seedlings. We will evaluate fruit yield and quality when ready. Sensory and consumer preference and flavor analyses will be evaluated.Objective 2: Testing tomato mutants against CLps infection. To inoculate tomato plants with CLps, we will utilize the tomato psyllid B. cockerelli western biotype. This genetic line of B. cockerelli is infected with CLps haplotype B. For each mutant, five plants will be inoculated with one plant as a biological replicate. Tomato plants used for inoculation will be 6-week-old and psyllids will be allowed to feed for one week inside of the cage. After one week of feeding psyllids will be subsequently removed to ensure all plant biological replicates are inoculated for the same duration of time. For each batch, five wild-type tomato plants will be inoculated with CLps as controls. To confirm that inoculations are successful the top-tier leaves will be tested for CLps infection using real-time quantitative PCR (qPCR). Once CLps infection is confirmed, we will compare disease symptoms,. For the tomato mutants showing resistance/tolerance against CLps, we will evaluate how mutations affect ROS levels, callose deposition around sieve pores, and cell death of phloem tissues, tomato plant growth, morphology, flowering, fruit yield and quality. Objective 3: Co-editing vectors for the target genes will be constructed. Non-transgenic genome-edited citrus varieties will be generated via mature tissue transformation. To verify the transformants to be non-transgenic, PCR amplification of Cas12a or GFP will be conducted. To eliminate the possibility of transgenes of small fragments from transformed plasmids, all putative non-transgenic genome-edited lines will be subject to whole-genome sequencing.Objective 4: Greenhouse and field trials will be conducted as described in Objective 1 except that non-transgenic genome-edited citrus plants do not need fenced sites and do not need to apply permit Under 7 CFR 340 from USDA APHIS BRS.Objective 5: We will organize workshops, grower meetings, field day events to highlight the progress in generation of non-transgenic genome-edited citrus varieties via mature tissue transmission, and showcase the performance of the genome-edited lines (both non-transgenic and transgenic) in HLB resistance and other traits. Information will be published in industry magazines.