Source: FORTIPHYTE, INC. submitted to NRP
TABA SUPPLEMENT FOR USDA PHASE I SBIR: DEVELOPMENT OF TOMATO WITH RESISTANCE TO BACTERIAL SPOT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1019393
Grant No.
2019-33610-29805
Cumulative Award Amt.
$106,500.00
Proposal No.
2019-01007
Multistate No.
(N/A)
Project Start Date
Jul 1, 2019
Project End Date
Sep 21, 2020
Grant Year
2020
Program Code
[8.2]- Plant Production and Protection-Biology
Recipient Organization
FORTIPHYTE, INC.
663 COLUSA AVE
BERKELEY,CA 947071517
Performing Department
(N/A)
Non Technical Summary
The disease Bacterial Spot is a significant issue for commercial tomato growers, particularly in warm and humid environments such as the Southeastern United States. This disease can cause large losses in yield and quality and is often controlled by the application of chemicals such as copper, which is expensive and has broad toxicity. As a result of this disease, consumers often have access to lower quality tomatoes at a higher price while farmers make less profit. Attempts to breed new tomato varieties with resistance to this disease have been largely unsuccessful.We recently identified two genes from a wild tomato species which are involved in disease resistance to Bacterial Spot. These genes are immune receptor genes, which enable the plant to recognize when it's under attack by the invading pathogen and activate the plant's natural defense system. For this project we will test whether these two genes enable cultivated tomato to be immune to Bacterial Spot. Although possible, it would be very difficult and time consuming to use traditional breeding to move these genes into cultivated tomato. Instead, we will use genome editing technology to generate a tomato variety which is equivalent to that which would be obtained through traditional breeding. This variety will enable farmers to grow fresh market tomatoes without relying on expensive and potentially hazardous chemicals to control Bacterial Spot. This will increase farmer profitability, improve the environmental sustainability of tomato production, and help to provide consumers with high quality tomatoes at an affordable price.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21214601060100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
The goal of this project is to support a larger effort to develop and commercialize a tomato variety with resistance to the disease Bacterial Spot. This project focuses on testing the efficacy of two resistance genes, Zar1 and Jim2, for their ability to confer resistance to this pathogen and to generate homozygous cisgenic tomato seed with these traits.The objectives of this project are:1. Determine if Zar1 and Jim2 confer resistance to Bacterial Spot in tomato2. Test the efficiency of a cisgenic DNA delivery system3. Obtain homozygous cisgenic Zar1 and Jim2 tomato lines4. Bulk Zar1 and Jim2 tomato seeds for a future field trial
Project Methods
The efficacy of the Zar1 and Jim2 genes will be tested by generating tomato lines expressing these genes and then measuring their resistance to Bacterial Spot. Disease resistance will be measured both quantitatively (using a bacterial growth assay) and qualitatively (by looking at visual disease symptoms).The cisgenic gene delivery system will be tested by using high-throughput DNA sequencingto identify the nature of individual DNA integration events. We plan to analyze more than 200 integration events to measure the efficiency of the delivery system as well as to obtain cisgenic tomato lines.The results of these experiments will be presented atconferences and in scientific publications. We will also engage directly with seed companies, large tomato buyers, and individual tomato farmers to both understand their needs and disseminate our results.

Progress 07/01/19 to 02/28/21

Outputs
Target Audience:The target audience for our work was members of the tomato seed industry and academic researchers working on tomato disease resistance. We presented some of our work at the 2019 Tomato Disease Workshop and Tomato Breeders Roundtable in Clearwater, Florida. We also presented at the 2020 Seed Central startup showcase meeting and held one on one discussions with representatives with several tomato seed companies. Changes/Problems:We made some modifications to the cisgenic gene delivery system to improve efficiency and make the system easier to use. These changes were made after obtaining some preliminary results. The modified system enabled us to succesfully obtain the desiredcisgenic insertion events in tomato. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We generated tomato lines expressing ZAR1 and JIM2 and tested them for resistance to Xanthomonas perforans, the causative agent of tomato bacterial spot. We observed that the line are indeed resistant to this pathogen by qualitative and quantitative disease assays. We tested a cisgenic delivery system for the generation of tomato lines expressing ZAR1 and JIM2 but containing no non-plant DNA elements. The efficiency of the initial delivery system was low, but after making some modifications we obtained the desired cisgenic insertion events. We selectedhomozygous individualsin the next generation and selfed these plants to generate seed for a field trial.

Publications


    Progress 07/01/19 to 09/21/20

    Outputs
    Target Audience:The target audience for our work was members of the tomato seed industry and academic researchers working on tomato disease resistance. We presented some of our work at the 2019 Tomato Disease Workshop and Tomato Breeders Roundtable in Clearwater, Florida. We also presented at the 2020 Seed Central startup showcase meeting and held one on one discussions with representatives with several tomato seed companies. Changes/Problems:We made some modifications to the cisgenic gene delivery system to improve efficiency and make the system easier to use. These changes were made after obtaining some preliminary results. The modified system enabled us to succesfully obtain the desiredcisgenic insertion events in tomato. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? We generated tomato lines expressing ZAR1 and JIM2 and tested them for resistance to Xanthomonas perforans, the causative agent of tomato bacterial spot. We observed that the line are indeed resistant to this pathogen by qualitative and quantitative disease assays. We tested a cisgenic delivery system for the generation of tomato lines expressing ZAR1 and JIM2 but containing no non-plant DNA elements. The efficiency of the initial delivery system was low, but after making some modifications we obtained the desired cisgenic insertion events. We selectedhomozygous individualsin the next generation and selfed these plants to generate seed for a field trial.

    Publications


      Progress 07/01/19 to 06/30/20

      Outputs
      Target Audience:Some of the work supported by this project was presented at the 2019 Tomato Breeders Workshop and Disease Roundtable in Clearwater, Florida in November 2019. The talk was titled "Disocvery of bacterial resistance traits and generation of a tomato variety with immunity to Xanthomonas, Pseudomonas and Ralstonia." The audience consistented of faculty, postdocs, and students from academic institutions as well as many individuals from tomato seed companies and the tomato industry. In addition to this presentation, we have discussed the work with several seed companies as well as academic collaborators over the past year. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The Project Director attended and presented a talk at the 2019 Tomato Breeders Roundtable and Disease Workshop in November 2019 to present some of the results of this work. This provided an opportunity for professional development. How have the results been disseminated to communities of interest?A portion of the work was presented at the 2019 Tomato Breeders Roundtable and Disease Workshop in Clearwater, Florida to academic researchers and representives from the tomato industry. The work has also been discussed with academic collaborators and an academic paper is planned for the future. What do you plan to do during the next reporting period to accomplish the goals?The project is mostly completed, but we are continuing to generate and test some new tomato lines. We've seen that some of the tomato lines have stronger resitsance than others and we are investigating the cause of this. We are also interested in testing the resistance against additional Xanthomonas strains, as well as testing the genes in different tomato backgrounds and in combination with other disease-resistance traits.

      Impacts
      What was accomplished under these goals? Objective 1: Determine if Zar1 and Jim2 confer resistance to bacterial spot in tomato. We obtained tomato lines transformed with Zar1 and Jim2. We found that they were resistance to Xanthomonas perforans containing the effector protein XopJ4 and that the resistance was lost if XopJ4 was deleted (as expected). We observed the resistance phenotype through qualitative suppression of disease symptom development as well as quantitative reduction of bacterial growth. Objective 2: Test the efficiency of a cisgenic DNA delivery system. Based on data from our first attempt to make cisgenic tomato plants we modified the delivery system. Using this modified system we were able to generate clean cisgenic insertion events in tomato. Approximately 5 to 10% of tomato lines transformed using our cisgenic delivery system have the desired clean insertion. Objective 3: Obtain homozygous cisgenic Zar1 and Jim2 tomato lines. We selfed primary tomato transformants with clean cisgenic insertion events to obtain plants with homozygous cisgenic insertions. We used high-throughput sequencing, PCR and Sanger sequencing to confirm that nature of the insertions and test for the zygousity. Objective 4: Bulk Zar1 and Jim2 tomato seeds for a future field trial. We selfed homozygous tomato plants expressing Zar1 and Jim2 to obtain several thousand seeds to enable a future field trial. ?

      Publications