Source: YALE UNIVERSITY submitted to NRP
SP: ANALYSES OF SUSCEPTIBILITY GENES IN A LIBRARY OF MULTIPLEX GENE EDITED CITRUS PLANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031541
Grant No.
2023-70029-41277
Cumulative Award Amt.
$1,365,000.00
Proposal No.
2023-06870
Multistate No.
(N/A)
Project Start Date
Sep 15, 2023
Project End Date
Sep 14, 2026
Grant Year
2023
Program Code
[ECDRE]- Emergency Citrus Disease Research and Extension Program
Recipient Organization
YALE UNIVERSITY
105 WALL ST
NEW HAVEN,CT 06511-6614
Performing Department
(N/A)
Non Technical Summary
The U.S. citrus industry is facing unprecedented challenges from the spread of huanglongbing disease (HLB) in citrus-growing states like Florida, Texas and California. While traditional breeding approaches using wild relatives are one way of introducing disease tolerance into commercial citrus cultivars, these approaches often result in the loss of the carefully selected attributes of those commercial varieties. Thus, alternative approaches to rapidly generate new cultivars that are resistant to HLB and yet maintain their qualities would be extremely valuable. The proposed project will develop a population of CRISPR/Cas9 gene-edited plants, in which ~1200 HLB susceptibility genes will be targeted for mutation. We will then test these lines for resistance to Xcc (the causative bacteria for citrus canker) and CLas (the causative bacteria for HLB) since broad-spectrum resistance to bacterial diseases can be induced by mutating susceptibility genes. By targeting disease susceptibility genes in this way, we hope to be able to uncover those genetic variants that confer resistance to HLB. Part of this project also aims to develop the genetic resources necessary to carry out this project, including generating a high-quality reference genome for Carrizo citrange, and developing low cost approaches to genotype plants using sequencing. This project will provide a sustainable resource for the citrus community that can be screened for resistance to other diseases and othervalue-added traits in the future, and thus accelerate discoveries that would benefit the U.S. citrus industry for many years. This collection of citrus mutants will also help to identify intervention gene targets most worthy of advanced testing and commercialization. We will also study the societal impact of using CRISPR/Cas9 technologies, with a focus on increasing consumer acceptance of gene-edited citrus.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20109991060100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
The U.S. citrus industry is facing unprecedented challenges from the spread of HLB in citrus-growing states like Florida, Texas and California. While traditional breeding approaches using wild relatives are one way of introducing disease tolerance into commercial citrus cultivars, these approaches often result in the loss of the carefully selected attributes of those commercial varieties. Thus, alternative approaches to rapidly generate new cultivars that are resistant to HLB and yet maintain their qualities would be extremely valuable. The proposed project builds upon previous ECDRE supported work to complete the production of a library of multiplex CRISPR/Cas9 edited susceptibility genes in Carrizo citrange. We will then test these lines (and those previously generated in Valencia) for resistance to Xcc (the causative bacteria for citrus canker) and CLas (the causative bacteria for HLB) since broad-spectrum resistance to bacterial diseases can be induced by mutating susceptibility genes. Through this project, we will generate a high-quality phased reference genome for Carrizo citrange, complete production of the Carrizo citrange mutant library, utilize low-cost genomic sequencing approaches to quantify the mutation rates at each target gene and off-targets, and test Valencia and Carrizo citrange lines for susceptibility to Xcc and CLas using well-established assays. This project will provide a sustainable resource for the citrus community that can be screened for resistance to other diseases and othervalue-added traits in the future, and thus accelerate discoveries that would benefit the U.S. citrus industry for many years. This collection of citrus mutants will contribute to achieving goals of the funding program, including use of CRISPR tools and identification of gene-editing targets, understanding how specific citrus genetic pathways contribute to HLB susceptibility, and consolidation of screening efforts to identify intervention targets most worthy of advanced testing and commercialization. We also propose to study the societal impact of using CRISPR/Cas9 technologies, with a focus on increasing consumer acceptance of gene-edited citrus.
Project Methods
Methods include genome sequencing using PacBio HiFi reads and bioinformatics approaches to generate a high quality phased reference genome for Carrizo citrange; sgRNA synthesis using a commecially available gene synthesis platform; using those sgRNA to generate vectors using molecular biology approaches for plant transformation; and then using a low cost genome sequencing approach to assess and quantify the mutation rates at target and off-target sites. Together these approaches will allow us to select and prioritize edited plants for phenotyping for disease susceptibility/resistance.Methods for phenotyping the resulting plants will include leaf innoculation of Xcc and quantification of the resulting infections using colony counts and PCR. Plants selected for further analyses will be tested for HLB susceptibility/resistance using graft innoculation.Methods for assessing consumer preferences will include surveying 1000 consumers for their perceptions and preferences using a discrete choice experiment, as well as a neuromarketing experiment using 150 subjects using a Frontal Asymmetry Index to assess how consumers' internal motivations are translated into preferences and purchasing decisions.

Progress 09/15/23 to 09/14/24

Outputs
Target Audience:The target audience includes research scientists and other interested professionals in academia and industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three undergraduates have been trained in plant molecular biology and plant tissue culture as a result of this award. Two postdoctorals have had one on one mentoring and have accessed on campus professional training activities. How have the results been disseminated to communities of interest?1. Through publication (Sagawa et al., Plant Methods 2024). 2. Through seminars/talks: U.C. Riverside seminar (V. Irish) Basile Memorial Lecture, New York NY (V. Irish) Minor Use Foundation HLB symposium, Sao Paolo, Brazil (V. Irish) CAES Symposium, New Haven, CT (V. Irish) IRC-HLB VII Conference Riverside CA 2024 (Y. Jacob) Fudan Univ., Shanghai, China (Y. Jacob) Zhejiang Univ., Hangzhou, China (Y. Jacob) Huazhong Agricultural Univ., Wuhan, China (Y. Jacob) 3. Through poster presentations: Plant and Animal Genome conference, 2024 (G. Thomson) IRC-HLB VII Conference Riverside CA 2024 (B. Mermaz) YPA symposium 2024, New Haven CT (B. Mermaz) YPGA symposium 2024, New Haven, CT (M. Tachev) 4. Through social media: X (formerly Twitter) and LinkedIn, biweekly communications to share updates on the project (B. Mermaz) What do you plan to do during the next reporting period to accomplish the goals?We will continue to generate and characterize the CRISPR/Cas9 multiplex mutant lines. Of the lines already generated, we will embark on Xcc (Xanthomonas citri citri) susceptibility assays, to determine if any lines exhibit potential broad spectrum resistance. We have already identified one line which shows reduced suscseptibility to Xcc. Of the lines already generated, a subset will be sent to Prof. D. Seymour, U.C. Riverside, who will initiate testing of these lines for HLB susceptibility or resistance using grafting assays. In addition, approximately 750 gene edited and control plants will be sent to Prof. M. Dutt, University of Florida, for eventual field trials to determine what lines are mutant for HLB susceptibility genes.

Impacts
What was accomplished under these goals? 1. Significant progress was made in generating CRISPR/Cas9 multiplex knockout lines for candidate susceptibility genes. This progress was due in part to improvements in vector design, as reported in Sagawa et al, Plant Methods, 2024. 2. In addition, an affordable and convenient whole genome sequencing protocol was established to rapidly and comprehensively genotype these lines. A publication summarizing this sequencing approach is in preparation. 3. Progress was made in completing a high-quality phased reference genome for Carrizo citrange; a publication summarizing these data is in preparation. 4. Plans have been put in place and permits obtained to ship CRISPR/Cas9 knockout lines to Florida for future field trials and analyses.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: An efficient multiplex approach to CRISPR/Cas9 gene editing in citrus. Sagawa CHD, Thomson G, Mermaz B, Vernon C, Liu S, Jacob Y, Irish VF. Plant Methods. 2024 Sep 28;20(1):148. doi: 10.1186/s13007-024-01274-4.