Source: SOIL CULTURE SOLUTIONS, LLC submitted to
GENERATING ZEBRA CHIP-RESISTANT TOMATOES AND POTATOES USING CRISPR/CAS9
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1022640
Grant No.
2020-33610-31993
Project No.
FLAW-2020-00607
Proposal No.
2020-00607
Multistate No.
(N/A)
Program Code
8.2
Project Start Date
Sep 1, 2020
Project End Date
Apr 30, 2022
Grant Year
2020
Project Director
Boltz, K.
Recipient Organization
SOIL CULTURE SOLUTIONS, LLC
3802 SPECTRUM BLVD STE 142H
TAMPA,FL 336129223
Performing Department
(N/A)
Non Technical Summary
Zebra chip disease is a bacterial disease of potatoes, tomatoes, peppers, and other related crops. The bacteria are transmitted to the plants by an insect, the potato tomato psyllid. Infected plants display a range of symptoms including yellowing leaves, reduced growth and yield, and death. Diseased potatoes used for chips show zebra-like stripes when fried, making them unsellable to the public. Crop loss and increased pesticide application due to zebra chip disease cause significant economic impacts to growers and consumers. Soilcea, in conjunction with Texas A&M, aims to create improved, zebra chip disease-resistant potatoes, tomatoes, and other crops.Soilcea will use a precision breeding technology based on the CRISPRplatform. This system acts like molecular scissors to make small changes to plant genes that make the plant susceptible to zebra chip disease. Unlike GMO crops, CRISPR technology allows us to make changes without introducing foreign DNA. For this Phase I proposal, we will use Moneymaker tomatoes, which, after infection by the psyllid, quickly develop dramatic symptoms of yellowing, curled leaves, stunted growth, and death within eight weeks. Our CRISPR-edited tomato plants will be infected with psyllids and observed for a decrease or absence of disease symptoms.The data collected will be the basis for the development of non-GMO, zebra chip-resistant potato and tomato varieties for farmers in the US and around the world. These varieties will assist growers by increasing yield, preventing crop loss, and decreasing pesticide spending. Decreased need for pesticides will also benefit the environment and slow the development of pesticide-resistant insects.
Animal Health Component
0%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21214691081100%
Goals / Objectives
Goal:Generating Zebra Chip-Resistant Tomatoes and Potatoes using CRISPR/Cas9Soilcea in conjunction with Texas A&M aims to improve potatoes, tomatoes, and other plants by creating varieties resistant to zebra chip disease caused by Candidatus Liberibacter solanacerum.To create improved potato and tomato varieties, Soilcea will use biotechnology, specifically CRISPR precision breeding, to delete proposed target susceptibility genes to create plant varieties with increased resistance to zebra chip disease.Objective 1: Generate Moneymaker tomatoes with CRISPRedits that are predicted to confer resistance to zebra chip disease.CRISPR-Cas9 will be used transgenically to edit the genomes of Moneymaker tomatoes. Plants with the desired edits will be grown to maturity, and seeds or other plant material will then be provided to Texas A&M for resistance testing.Objective 2: Conduct grow room trials to confirm tolerance or resistance to zebra chip disease.Regenerated plants will be tested for zebra chip resistance in grow room trials by Texas A&M. CRISPR-edited plants and control plants will be exposed to potato psyllids with the disease. To determine the impact of the CRISPRedits, tomato plants will be monitored over their life, and if necessary, will measure levels of CLso by qPCR. The technical question that must be addressed is whether the edited tomato varieties are tolerant or resistant to zebra chip disease.
Project Methods
Objective 1: Generate Moneymaker tomatoes with gene edits that are predicted to confer resistance to zebra chip disease.To test for resistance to zebra chip, Soilcea and Texas A&M will work with the Moneymaker tomato variety. Within a month after infection with CLso, these tomato plants begin to exhibit strong visible phenotypes including stunted growth, yellowing and curled leaves, and death. Thus, subtle effects from our gene edits should be more visible in this system compared to plants with a less robust response to CLso. For this study, Soilcea will individually mutate three to five disease susceptibility genes to generate five to ten unique tomato lines for psyllid testing. This will occur at Soilcea's lab at the Tampa Bay Technology Incubator.CRISPR/Cas9 gene editing design and assay: Because the specificity of the CRISPR/Cas9 system comes from the binding of the guide RNA to the target gene, we will use PCR and Sanger sequencing to verify the sequences of our target genes in Moneymaker tomatoes. CRISPR-P 2.0 will be used to identify the best sgRNAs for each gene. We will transcribe several sgRNAs for each target and test the efficiency of these RNAs in in vitro Cas9 assays with DNA substrates produced by PCR. The most efficient sgRNAs will be selected for subsequent steps.Agrobacterium transformation and gene editing: gRNAs will be cloned into a standard T-DNA binary vector containing NPTII selection, a GFP marker, Cas9 driven by a constitutive promoter, and the sgRNA under control of the Arabidopsis U6-1 promoter. For controls, the empty plasmid without the gene-specific region of the sgRNA will be used. Agrobacterium tumefaciens will be used to transform the CRISPR plasmids into tomato cotyledons. We will use established methods shown to be efficient for CRISPR experiments. For each construct, approximately 250 cotyledons will be transformed to ensure we increase the odds of creating a CRISPR-induced mutation.Screening to identify plants with gene edits: To recover stably transformed shoots, cotyledon explants will be grown on selective media containing kanamycin. Further, shoots will be screened for GFP using fluorescent microscopy. When shoots are large enough, leaf DNA will be used for PCR with primers that flank the Cas9 cut site. To identify mutations, T7 endonuclease assays will be used to detect heteroduplexes in the PCR products. Because homozygous mutations will not be detected using this method, an equal amount of wild type DNA will be included with each sample. Homozygous mutants should show approximately 50% uncut DNA. Any PCR products with T7 cleavage will then be Sanger sequenced to identify insertions, deletions, or point mutations in the target gene. The sequencing traces will be analyzed using ICE (https://ice.synthego.com/#/).Generation of plant material for psyllid testing: Ideally, we would grow and genotype T1 plants then use T2 homozygous mutants for psyllid infection. However, that approach is precluded by the time constraints of this grant. If we identify a T0 homozygous mutant, we will genotype tissue from several areas of the plant to verify it is not a chimera, and then collect the T1 seeds for testing. A second approach is to plant T1 seeds from parents that have a mutation. We will then genotype the seedlings and use the homozygous mutants for further experiments. A final possibility is to take cuttings from homozygous T0 plants and propagate them to use for psyllid inoculation.Expected results: We expect to have one or more independent mutant plant lines for at least three of the zebra chip-susceptibility genes. One of the concerns with using transgenic CRISPR systems is that mutations can be made throughout the life of the plant, producing chimeras with cells containing different mutation events. Because we will not have time to outcross the transgene and segregate independent mutations, our analysis could be hindered by having multiple alleles present in the plant. An alternative approach that eliminates chimerism is to use purified Cas9 and gRNA RNPs and transform them into protoplasts. Since the resulting plants are regenerated from single cells, the mutation should be present throughout the plant. This is not our primary approach because it takes more time to regenerate plants from protoplasts.Objective 2: Conduct grow room trials to confirm tolerance or resistance to zebra chip disease.Plant growth and inoculation: Dr. Tamborindeguy's lab will grow the tomato plants from Moneymaker tomato seeds with edits provided by Soilcea as well as unedited Moneymaker tomato controls. They will then grow tomatoes for six weeks and will assign those plants to one of the following two treatments: Lso-infection or control. Moneymaker tomatoes will be treated as in the Lso-infected treatment. Plants in the Lso-infected treatment will be infested with 3 to 5 potato psyllid (Bactericera cockerelli) adults from the Lso- infected colony while plants in the control treatment will be infested with 3 to 5 potato psyllid adults from the Lso-uninfected colony. There will be at least ten plants in each control treatment and at least 15 Soilcea provided plants from each line in the Lso-infected treatment. After one week the psyllids will be removed and the plants will be maintained as regularly done in our laboratory. During the trial, all growth conditions will be recorded, including pot size, growing medium, fertilizer, and environmental conditions.Collect plant phenotypic data: The plants will be observed and scored weekly for disease development. During and at the end of the trial, plant height and stem diameters will be measured, and, at the end, total plant biomass will be recorded. Additionally, assessments of zebra chip symptoms will be conducted and accompanied by photographs and detailed descriptions of symptoms. CLso presence has strong disease symptoms on Moneymaker tomato plants, and thus we expect to observe strong disease pressure from control plants. Dr. Tamborindeguy's lab has observed that within 3 to 5 weeks, Moneymaker tomato plants infected by CLsoB exhibited smaller new leaves and some discoloration. After week 5, those infected plants showed strong differences from uninfected controls, including stunting, curling, yellowing, and some necrotic areas. After week 6, infected plants were in the early stages of death with dying leaves and no growth. And after week 8, the infected plants either had already died or were dying.Compile data and qPCR for CLso: If the edited tomato plants do not show the typical disease symptoms, while the control plants do show the expected disease symptoms, further analysis will be conducted. Specifically, edited plants that do not display symptoms in the infected treatment will be tested for CLso presence using qPCR and leaf samples from those plants will be stored at -80°C in case further information about the CLso strain is needed. DNA concentrations and Ct values will be recorded each time. We will use t-tests and a p-value approach to statistically calculate which edited, inoculated plants are different from unedited controls.Expected results: If the experiment delivers plants with reduced disease symptoms, the edited plants may have tolerance to zebra chip disease, which still could have commercial potential. If the experiment delivers plants completely without disease symptoms and no to significantly reduced CLso titers, the edited plants will be considered resistant to zebra chip disease. The biggest limitation of our approach is that mutations in a single gene may not confer zebra chip resistance. Our long-term plan is to combine mutations within a single plant, but finding the ideal combination may prove time consuming. Additionally, finding the best mutation in a gene may require either extensive trial and error or additional experiments to determine the region of interaction between the SDE and target protein.