TRANSGENIC SUCCESS-GUIDED REPRODUCTION OF HUANGLONGBING DISEASE RESISTANCE/TOLERANCE IN CITRUS BY GENE EDITING

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1014787
• Grant No.: 2018-70016-27392
• Cumulative Award Amt.: $2,951,933.00
• Proposal No.: 2017-08545
• Project Start Date: Feb 1, 2018
• Project End Date: Jan 31, 2024
• Grant Year: 2018
• Program Code: [CDRE]- Citrus Disease Research and Extension Program

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Microbiology and Cell Science

Non Technical Summary
Huanglongbing (HLB), also called citrus greening, is one of the most devastating diseases of citrus worldwide. In Florida, HLB is a disaster for the citrus industry. HLB is in Texas with spread increasing. In California, there are accelerated finds in residential trees and the insect that spreads the disease continues expanding into commercial areas. Without new solutions, it is possible that these citrus industries may follow a pattern similar to that in Florida. In Florida, HLB has spread to almost all trees, reducing yields and the quality of juice. Since 2005, when HLB was first identified, total citrus production in Florida has shrunk by more than 50%, an unprecedented contraction of the Florida citrus industry that is causing closure of citrus packing and processing plants. Citrus is the major agricultural crop in Florida, valued at $10 billion and provides 76,000 jobs. For many rural communities citrus is the economic base of the local economy. California is only second to Florida in total citrus acreage, but has a larger share of the higher value, fresh market produce. Accelerating pessimism concerning the survival of this major industry is overtaking Florida. There are no identified alternative crops to replace the use of the nearly 435,300 remaining acres of citrus and its infrastructure. Survival of the juice market in Florida requires new plantings to increase overall production. However, without resistant or tolerant citrus trees, controlling the insect that spreads the disease alone is not sufficient to provide productivity in most new plantings. California is being extremely pro-active in its efforts to prevent HLB establishment, and despite that, the insect that spreads the disease is now established. Restoration of the Florida citrus industry requires new trees that better tolerate the infection or reduce the spread of the disease and they are needed immediately. Transgenic citrus trees that are tolerant to HLB and prevent reproduction of the insect that spreads the disease have been produced. However, these trees are genetically modified organisms (GMOs) that that will have to go through the approval process that cannot be accomplished in time to save the Florida citrus industry. Additionally, the industry would have to sell a GMO product. For these reasons, we propose to recreate HLB resistance or tolerance based on the transgenic results using a new technology known as transgene-free gene editing. Citrus trees produced using this new technology will be non-GMOs and without time-consuming regulatory approval can be delivered to the industry more quickly. This will allow development of new groves that will increase the overall production levels to 2005 levels, which will increase the value of this industry and recreate jobs that have been lost in Florida and will provide non-GMO HLB resistant or tolerant trees for the California and Texas industries that may not be ready to use transgenics. Throughout the time of this project, stakeholders will be engaged in the project development and evaluation, and the citrus communities will be informed about the transgene-free gene editing technology. Informative outreach endeavors targeting potentially interested all parties will be organized to raise awareness of the new transgene-free gene editing technology and its application in citrus and to facilitate acceptance of the resulting non-GMO citrus products. It is expected that at the end of the project, citrus growers, industry, consumers, and state/federal regulators will have understood that transgene-free gene editing technology-created non-GMO products are similar to those generated by conventional crop breeding and the market for non-GMO citrus products will be ready.

Animal Health Component

80%

Research Effort Categories

Basic

(N/A)

Applied

80%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	0920	1081	80%
201	0920	1040	20%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0920 - Orange;

Field Of Science
1040 - Molecular biology; 1081 - Breeding;

Keywords

huanglongbing

citrus

disease resistance

disease tolerance

gene editing

transgenic success

Goals / Objectives
The overall goal of this project is to produce citrus trees that are resistant or tolerant to HLB using the CRISPR/Cas9 gene editing technology and to educate the public (including citrus growers, consumers, and regulators) about the CRISPR/Cas9 technology and its benefits to the citrus industry. The central hypothesis of this project is that transgenic approach-generated HLB tolerance and prevention of psyllid reproduction can be reproduced by knocking out negative regulators of citrus defense signaling pathways using the CRISPR/Cas9 technology and the resulting citrus gene knockouts will be exempt from regulations. To accomplish the goal of this application, four specific objectives will be pursued(1) Identify targets for gene editing: Citrus homologous genes encoding negative regulators of a major defense positive regulator will be cloned and the citrus gene that is required for psyllid reproduction will be determined. These genes will be the priority genes and will be simultaneously subjected to CRISPR/Cas9 gene editing and CTV RNAi testing. Citrus homologs of other defense negative regulators will also be identified and tested by CTV RNAi. The most promising candidate genes will be subjected to CRISPR/Cas9 gene editing. Additionally, negative cis-element in the promoter of major defense positive regulators will be identified and tested. If promising, they will be targeted using CRISPR/Cas9.(2) Generate transgene-free citrus varieties by CRISPR/Cas9-mediated gene editing: The transformation protocols for generating citrus transgene-free CRISPR knock-outs will be optimized and transgene-free CRISPR/Cas9 gene editing on the priority genes and other promising genes identified by CTV-based RNAi will be performed.(3) Evaluate CRISPR/Cas9-produced citrus varieties: The CRISPR/Cas9 knock-outs will be tested for HLB responses in the greenhouse/field and will be molecularly characterized.(4) Engage stakeholders in project development/execution process and develop effective outreach programs: Stakeholders will be engaged in the project and outreach activities educating the public will be carried out.

Project Methods
Citrus varieties that are resistant or tolerant to HLB will be generated using the new gene editing technology known as transgene-free CRISPR/Cas9 technology. Target genes will be identified based on previous transgenic success in which citrus trees that are highly tolerance to HLB have been produced. They will also be identified using a CTV RNAi-mediated screening strategy from a group of candidate genes. Stakeholders will be engaged in the project development and evaluation through direct involvement in the project or the advisory panel that will inform the project including identification and prioritization of research and extension objectives. Teaching materials including Powerpoint presentations, extension articles, and website will be prepared for broad audiences including citrus growers, industry representatives, consumers, and state/federal regulators. Flyers, handouts, and fact sheets in lay language explaining the project will be made available for download on the website and some will be printed for distribution via various means including post offices, grocery stores, and supermarkets. Workshops, seminars, and meetings targeting potentially interested all parties will be organized to raise awareness of the new transgene-free CRISPR/Cas9 technology and its application in citrus and to facilitate acceptance of non-GMO CRISPR products. At the end of the project, citrus growers, industry, consumers, and state/federal regulators will have understood that transgene-free CRISPR/Cas9 technology-created non-GMO CRIPSR products are similar to those generated by conventional crop breeding and the market for non-GMO CRIPSR citrus products will be ready. Surveys will be conducted throughout the project term to collect feedback from the audience. Questions will be designed to test the audience's understanding of the topics presented and their acceptance of the new technologies and their products. Questions will also be designed to understand the public's opinions on their informational needs and the appropriate delivery approaches. Answers to these questions will tell us if we are using effective ways to provide the appropriate information needed by the citrus community. Surveys will also be incorporated into the website to gain feedback from end-users on the outreach information/materials and opinions on future improvement.

Progress 02/01/18 to 01/31/24

Outputs
Target Audience:The extension and outreach activities of the project provide information on HLB and ACP control strategies and the progress of the project to stakeholders including citrus growers, industry, state, and federal regulators, as well as students and the public. These topics were discussed and presented in following events in Florida and elsewhere in 2023: Batuman, O. (2023). Plant pathogens that may exacerbate the HLB effects on citrus trees. Growers Seminar. January 10, 2023; Immokalee, FL. Batuman, O. Emerging viral diseases of citrus and management of their insect vectors. UF-IFAS In-service training to extension Agents. Mar 28, 2023; Immokalee, FL. Batuman, O. (2023). Citrus Pathology and HLB Laboratories' Activities. UF/IFAS Extension Southeast Extension District, SEED Spring Meeting. April 27, 2023; Clewiston, FL. Batuman, O. IPCs' beneficial effects on citrus diseases and challenges they create. UF-IFAS In-service training to extension Agents. May 3, 2023; Immokalee, FL. Batuman, O. (2023). Batuman Lab NIFA projects' overview. Cornell University, Heck Group. May 1, 2023, Immokalee, FL. Batuman O. Citrus Pathology Program for Huanglongbing (HLB) management. Presentation to USDA/Cornell Research Group. June 1, 2023 (Virtual). Batuman, O. HLB and its management in citrus groves. Presentation to citrus Advisory Board. June 9, 2023 (Virtual). Batuman, O. (2023). Citrus Screening: Evaluation of various compounds to prevent infection and spread of CLas in citrus. Stakeholder Advisory Board Meeting, June 9, 2023; Immokalee, FL. Batuman et al. (2023). Collaborative approach between academics, growers and agrochemical industry to discover, develop and commercialize therapies for HLB. Citrus Expo, August 16-17, 2023, Tampa, FL. Batuman et al. (2023). Novel strategies for HLB resistance/tolerance in citrus by gene editing. Citrus Expo, August 16-17, 2023, Tampa, FL. Batuman et al. (2023). Can finger limes help Valencia and Hamlin trees be more tolerant of HLB? Citrus Expo, August 16-17, 2023, Tampa, FL. Batuman et al. (2023). Delivering therapeutic materials through the trunk to treat HLB-infected citrus trees. Citrus Expo, August 16-17, 2023, Tampa, FL. Batuman et al. (2023). Can We Use an Insect Virus to Control ACP in the Groves? Citrus Expo, August 16-17, 2023, Tampa, FL. Mou et al. (2023). Generation of transgenic Huanglongbing tolerant varieties. Citrus Expo, August 16-17, 2023, Tampa, FL. Mou et al. (2023). Approaches toward Huanglongbing tolerance. Citrus Expo, August 16-17, 2023, Tampa, FL. Sanju Kunwar, Ana Redondo, Leigh Archer, Fernando Alferez, Ute Albrecht, Ozgur Batuman (2023). Injecting Oxytetracycline: An Effective Approach for Managing HLB Disease in Citrus. 12th International Congress of Plant Pathology. Lyon, France. August 20-25, 2023. Ozgur Batuman, Sanju Kunwar, Ana Redondo, Denise Manker, Meagan Iott, Thomas Knobloch, Stephane Brunet, and Jeremy Dufour (2023). Novel Plant Defense Inducers for Managing Huanglongbing (Citrus Greening) and Citrus Canker Diseases. 12th International Congress of Plant Pathology. Lyon, France. August 20-25, 2023. Batuman, O. (2023). Huanglongbing (citrus greening) and its management in citrus. University of Cukurova, Adana, Turkey, October 25, 2023. Batuman, O. (2023). Huanglongbing (citrus greening) and its management in citrus. University of Akdeniz, Antalya, Turkey, October 20, 2023. Batuman, O. (2023). Development of an automated delivery system for therapeutic materials to treat HLB-infected citrus. Emergency Citrus Disease Research and Extension Project Directors Annual Meeting. Washington DC, Nov 2-3, 2023. Batuman, O. (2023). Citrus Pathology and HLB Laboratories' Activities. SWFREC Annual Open House Science Demo. November 8, 2023, Immokalee, FL. Batuman, O. (2023). Utilizing HLB tolerant citrus germplasm and understanding their role in mitigating HLB. HLB Discussion Group Meeting. November 20, 2023, Immokalee, FL. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoc was trained in project supervision/management, experimental design, advising graduate students, presentation, and progress report. Two graduate students were trained in experimental design, basic molecular biology techniques including CRISPR/Cas9 vector construction, Agrobacterium-mediated transformation, gene transient expression, CTV inoculation, and collecting and analyzing data. One of the graduate students was also trained in organizing outreach and extension activities. One undergraduate student was trained in general laboratory techniques including preparation of various media and buffers, tissue culture, citrus genetic transformation, and plant maintenance. How have the results been disseminated to communities of interest?Project progresses of the project have been continually communicated with stakeholders including citrus growers, industry, state and federal regulators, as well as students and the public through in-person or virtual meeting presentations, seminars, and discussions. The results have also been disseminated through the project website: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/ What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (1) Identify targets for gene editing: We have finished building all CTV-RNAi constructs and inoculating these constructs into citrus plants. In this year, we continued to maintain both Citrus macrophylla source plants and grafted sweet orange plants that have been infected with CLas. As sweet orange on its own roots is very difficult to maintain, some of the sweet orange plants died. The surviving sweet orange plants were scored twice for HLB symptom development in this year. Most of the CTV-RNAi sweet orange plants showed down-regulation of the target genes and severe HLB symptoms. Among the genes targeted by CTV-RNAi, one gene has been confirmed to reliably provide HLB tolerance when silenced. We propagated the HLB-tolerant sweet orange plants. Although some of the progeny plants are dying at this stage of the experiment, most plants show healthy growth after being confirmed positive for HLB. In summary, we have confirmed at least one gene that can induce HLB tolerance when silenced in commercial scions and several more genes with similar effect on HLB are beingconfirmed. (2) Generate transgene-free citrus varieties by CRISPR/Cas9-mediated gene editing: We have continued testing different new CRISPR/Cas9 vectors for their efficiency in citrus genome editing. A vector with a multi-intron containing Cas9 gene was found to be highly efficient in citrus. The editing efficiency of this vector in 'Carrizo' reached 4.5% (4.5 edited shoots out of 100 shoots in the absence of selection). This efficiency is high considering that the transformation efficiency in 'Carrizo' is often below 10%. This CRISPR/Cas9 vector has been used to generate transgene-free edited plants without using selection marker in both juvenile and mature tissue transformation. We also tested multiplexed gene editing using this vector and found that when three guide RNAs (gRNAs) were used, most of the edited plants have mutations at all three target sites, indicating that this vector is also efficient for multiplexed gene editing. We also made a gene editing vector with the Cas9 gene and the gRNA fused with mobile tRNAs. It has been shown that the mobile tRNA is able to move the Cas9 protein and gRNA to upper parts of the plant. We are producing transgenic rootstocks carrying this vector with a gRNA targeting the promising gene target and will test if the Cas9 protein and gRNA can move into scions to create transgene-free mutations. (3) Evaluate CRISPR/Cas9-produced citrus varieties: The gene-edited plants have been propagated and infected with CLas using ACP in the greenhouse. These plants contain T-DNA and are not transgene-free. Molecular and physiological changes following infection are being observed. (4) Engage stakeholders in project development/execution process and develop effective outreach programs: The project research results were delivered to growers and industrial groups through seminars, local, regional, national, and international meeting presentations, posters, and abstracts. Regular discussions with growers were performed through calls, emails, text messages, and field days on collaborative projects and emerging problems. Other communication methods include attending citrus advisory board meetings, industry meetings, and plant pathology meetings. The project progresses and updated information about HLB are also described on the dedicated webpage for the project. The project webpage has been updated at the beginning of 2024 and can be visited here: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/

Publications

• Type: Websites Status: Published Year Published: 2023 Citation: Transgene-free citrus using CRISPR CAS9 (project website; Last Modified: Tue, 11 Jan 2024): https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Sarkar, P., Vazquez, J.S., Zhou, M. Levy, A., Mou, Z., Prbovic, V. (2023) Efficient multiplexed gene editing with a multi-intron containing Cas9 gene in citrus. (Under review)

Progress 02/01/22 to 01/31/23

Outputs
Target Audience:The outreach and extension activities of the project provide information on progress of the project and on HLB and ACP control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. These topics were discussed and presented in following events in Florida and elsewhere in 2022: Batuman O., Britt-Ugartemendia, K., Kunwar, S., Redondo, A. and Alferez, F. (2022). Potential management tools for Huanglongbing (HLB) in Florida citrus groves. 16th International Citrus Congress. Mersin, Turkey. November 6-11, 2022 (abstract). Kunwar, S., Redondo, A., Manker, D., Iott, M., Knobloch, T., Brunet, S., Dufour, J. and Batuman, O. (2022). Novel systemic acquired resistance (SAR) inducers for managing Huanglongbing (citrus greening) and citrus canker diseases. 31st International Horticultural Congress. Angers, France. August 14-20, 2022(abstract). Batuman O., Britt, K., Kunwar, S., Redondo, A. and Alferez, F. (2022). Florida citrus growers' potential 'toolbox' for Huanglongbing (HLB) management: an alphabet soup (ISVs, PDIs, IPCs, NATI, etc.). 15th International Symposium of Plant Virus Epidemiology. Madrid, Spain. June 5-8, 2022(abstract). Mou et al. Approaches towards Huanglongbing tolerance. Ag Tech Expo. November 2, 2022 (Abstract). Batuman O., Britt-Ugartemendia, K., Kunwar, S., Redondo, A. and Alferez, F. (2022). Potential management tools for Huanglongbing (HLB) in Florida citrus groves. 16th International Citrus Congress. Mersin, Turkey. November 6-11, 2022. Kunwar, S., Redondo, A., Manker, D., Iott, M., Knobloch, T., Brunet, S., Dufour, J. and Batuman, O. (2022). Novel systemic acquired resistance (SAR) inducers for managing Huanglongbing (citrus greening) and citrus canker diseases. 31st International Horticultural Congress. Angers, France. August 14-20, 2022. Batuman O., Britt, K., Kunwar, S., Redondo, A. and Alferez, F. (2022). Florida citrus growers' potential 'toolbox' for Huanglongbing (HLB) management: an alphabet soup (ISVs, PDIs, IPCs, NATI, etc.). 15th International Symposium of Plant Virus Epidemiology. Madrid, Spain. June 5-8, 2022. Batuman, O., Nov 10, 2022: Potential management tools for Huanglongbing (HLB) in Florida citrus groves. 16th Int. Citrus Congress 2022, Mersin, Turkey. Batuman, O., Nov 8, 2022: HLB and management options in Florida. Workshop 3 at the Int. Citrus Congress 2022, Mersin, Turkey. Batuman, O., Aug 16, 2022. Novel systemic acquired resistance (SAR) inducers for managing Huanglongbing (citrus greening) and citrus canker diseases. 31st International Horticultural Congress. Angers, France. Batuman O., Jun 8, 2022. Florida citrus growers' potential 'toolbox' for Huanglongbing (HLB) management: an alphabet soup (ISVs, PDIs, IPCs, NATI, etc.). 15th International Symposium of Plant Virus Epidemiology. Madrid, Spain. June 5-8, 2022. Batuman, O. HLB and its management in citrus groves. Nov 16, 2022, Adana, Turkey. Batuman O. Florida citrus growers' potential 'toolbox' for Huanglongbing (HLB) management. Sep 22, 2022; Department of Plant Pathology, Chinese Academy of Sciences. Batuman, O. Citrus Pathology Program. Sep 15, 2022; Ft. Myers, FL. Batuman, O. Precision trunk injection method for treating HLB. Aug 16-17, 2022; Ft. Myers, FL. Batuman, O. CRB Antimicrobial Meeting: HLB Projects. May 23-25, 2022; San Francisco, CA. Batuman, O. Huanglongbing (aka citrus greening) Disease of Citrus. Apr 6, 2022; Woodland, CA. Batuman, O. Citrus disease trends we should heed: Phytophthora, HLB, and Leprosis. Jan 10, 2022. Immokalee, FL. Websites: Transgene-free citrus using CRISPR CAS9 (project website; Updated September 27, 2022): https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/ Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three postdocs were trained in project management, experimental design, advising graduate students, presentation, and progress report. Three graduate students were trained in experimental design, basic molecular biology techniques including CRISPR/Cas9 vector construction, protoplast manipulation and transfection, Agrobacterium-mediated transformation, gene transient expression, and CTV inoculation, as well as collecting and analyzing data. One of the graduate students was also trained in organizing outreach and extension activities. One undergraduate student was trained in general laboratory techniques including preparation of various media and buffers, tissue culture, citrus genetic transformation and plant maintenance. How have the results been disseminated to communities of interest?Progresses of the project have been continually disseminated to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public through in-person meeting presentations, seminars, and communications. The results have also been disseminated through the project website: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/ What do you plan to do during the next reporting period to accomplish the goals?The project is in the second NCE year. We have identified candidate targets for gene editing and have generated edited plants. However, these plants carry the transgene. We are testing different CRISPR/Cas9 vectors for producing transgene-free mutant plants. In this year, we will the best vector to produce transgene-free gene edited plants.

Impacts
What was accomplished under these goals? (1) Identify targets for gene editing:We have a set of all 22 constructs being screened for HLB tolerance phenotype. However, some of these constructs just have one plant being screened for the HLB phenotype. From the earlier screen, one of the plants with a target gene silenced is HLB positive but still stands well and the other two silenced plants were found to be HLB negative. The propagated progeny plants from the original HLB positive plant that shows no symptoms segregated with 50% of the progenies showing no HLB symptoms and 50% showing clear HLB symptoms. They were all confirmed HLB positive by qPCR. This result indicates that the targeted gene is a good candidate gene for editing and that CTV-mediated silencing is not stable after propagation. We have a second set of plants that went through the HLB screening and are waiting for symptom development. (2) Generate transgene-free citrus varieties by CRISPR/Cas9-mediated gene editing: We have tested multiple CRISPR/Cas9 vectors and continue testing new ones for their efficiency in citrus genome editing. The vector named pZM18 was used in the experiments that involved both Agrobacterium-mediated transformation of and biolistic introduction of DNA into juvenile citrus explants. The experiments with the "gene gun" were not successful as there was no regeneration of shoots from explants. Multiple citrus cultivars (Carrizo, Duncan, and Hamlin) were used for Agrobacterium-mediated transformation with this vector, but the transformation rate was low and all the shoots that were white also expressed GFP, suggesting they had T-DNA inserted in their genome. A new vector called pZM-PDS was used for Agrobacterium-mediated transformation of Carrizo explants. In experiments with this vector, we recorded higher transformation rate. We have selected multiple shoots that exhibited an albino (white) phenotype. Interestingly, two of these shoots seem to not have T-DNA present in their genome. These shoots are therefore potential transgene-free edited shoots. We plan to continue improving the protocol to increase the portion of such plants. In addition, smaller Cas proteins such as SpCas12f1 and Cas12j (CasΦ) are being investigated for their ability to efficiently edit the citrus genome using protoplasts. (3) Evaluate CRISPR/Cas9-produced citrus varieties: Sweet orange plants with one candidate gene edited have been clonally propagated and infected with CLas using ACP. A graduate student is investigating the molecular and physiological changes following infection. (4) Engage stakeholders in project development/execution process and develop effective outreach programs:?Project research results were disseminated to growers and other industry personnel through seminars, local, regional, national, and international meeting presentations, posters, and abstracts. Routine communications with growers were carried out through calls, emails, text messages, and field days on collaborative projects and new problems. Other communication means include attending citrus advisory board meetings, industry meetings, and plant pathology meetings. Information about the project and project progresses are also described on the dedicated webpage for the project. The project webpage has been updated and can be visited here: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/

Publications

• Type: Other Status: Published Year Published: 2022 Citation: HLB Reduces Phytophthora Management Efficacy. Citrus Industry News  February 2022. https://citrusindustry.net/2022/02/21/hlb-reduces-phytophthora-management-efficacy/
• Type: Other Status: Published Year Published: 2022 Citation: HLB Strategies From Zoomokalee. Citrus Industry News  January 2022. https://citrusindustry.net/2022/01/14/hlb-strategies-from-zoomokalee/

Progress 02/01/21 to 01/31/22

Outputs
Target Audience:The outreach and extension activities of the project provide information on progress of the project and on HLB and ACP control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. These topics were discussed and presented in following events in Florida in 2021:The outreach and extension activities of the project provide information on progress of the project and on HLB and ACP control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. These topics were discussed and presented in following events in Florida in 2021: Batuman, O. Development of a citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International industry collaborators meeting (France and Germany). January 5, 2021. Virtual Presentation. Batuman, O. Needle assisted trunk infusion (NATI) and Automated Delivery System (ADS). The California Subtropical Work Group Seminar. January 15, 2021. Virtual presentation. Batuman, O. Development of a novel automated delivery system for citrus trees. Ag Tech Expo. May 11, 2021. Virtual presentation. Batuman, O. Citrus disease trends we should heed: Phytophthora, HLB and Leprosis. Growers Meeting. January 20, 2021. Virtual Presentation. Batuman, O. What is the main cause of fruit drop in the groves in HLB era? SWFREC Advisory Board Meeting. January 26, 2021. Virtual Presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. Industry collaborators meeting. January 26, 2021. Virtual presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. Industry collaborators meeting. February 2, 2021. Field Demonstration. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing in Florida Citrus groves. Rotary Club Invited Seminar. February 9, 2021. Virtual presentation. Britt, K., Gebben, S., Stevens, K., Levy, A., Al Rwahnih, M., Batuman, O. A new Diaphorina citri-associated reo-like virus in Asian citrus psyllid populations in Florida citrus groves. Florida Phytopathological Society Meeting. May 13-14, 2021. Abstract. Britt, K. A new Diaphorina citri-associated reo-like virus in Asian citrus psyllid populations in Florida citrus groves. Florida Phytopathological Society Meeting. May 13-14, 2021. Virtual Presentation. Britt, K., Gebben, S., Stevens, K., Levy, A., Al Rwahnih, M., Batuman, O. A new Diaphorina citri-associated reo-like virus in Asian citrus psyllid populations in Florida citrus groves. American Phytopathological Society Meeting. August 6-10, 2021. Abstract. Batuman, O. Citrus HLB and Blight. Growers Meeting. March 14, 2021. Field Demonstration. Batuman, O. SWFREC Citrus Pathology program overview. The Immokalee Foundation's middle school Career Day Program. March 16, 2021. Virtual Presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing in Florida Citrus groves. Growers Meeting. April 7, 2021. Virtual presentation. Batuman, O. Novel management methods of Huanglongbing in Florida Citrus groves. Quarterly SWFREC Foundation Board of Directors meeting. April 13, 2021. In person presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International academic collaborators meeting (Germany). April 21, 2021. Virtual Presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. SWFREC Citrus Advisory Board Meeting. April 27, 2021. In person presentation. Batuman, O. Impact of HLB and Hurricane Irma on citrus groves. Growers Meeting. May 1 and 3, 2021. Virtual presentation and Field Demonstration. Batuman, O. Citrus tree decline. Growers Discussion Meeting. May 18, 2021 Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. Industry collaborators meeting. May 27, 2021. Field Demonstration. Batuman, O. Needle-assisted trunk infusion (NATI) of therapeutic material for controlling HLB and its psyllid vector. Meeting with Division of Agricultural Environmental Services, Florida Department of Agriculture and Consumer Services. June 2, 2021. Virtual presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. Texas Academic Collaborators Meeting. June 3, 2021. Virtual presentation. Batuman, O. Development of a citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International industry collaborators meeting (France). June 7, 2021. Virtual Presentation. Batuman, O. Needle-assisted trunk infusion (NATI) of therapeutic material for controlling HLB and its psyllid vector. Industry collaborators meeting. June 30, 2021. Virtual Presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. Texas Academic collaborators meeting. July 2, 2021. Virtual presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International academic collaborators meeting (Germany). July 6, 2021. Virtual Presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. SWFREC Foundation Board Meeting. July 13, 2021. In person presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing. SWFREC Citrus Advisory Board Meeting. July 27, 2021. In person presentation. Mou, Z., Dawson, W., Grosser, J., Orbovic, V., Dutt, M., Levy, A., El Mohtar, C., Batuman, O., Irey, M. Seeking long-term solutions to the Huanglongbing disease. Ag Tech Expo. May 11, 2021. Abstract. Websites: Transgene-free citrus using CRISPR CAS9 (project website; Updated April 7, 2021): https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/ Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two postdocs were trained in overall project management, experimental design, advising graduate students, presentation, and progress report. Two graduate students were trained in experimental design, basic molecular biology techniques including vector construction, protoplast manipulation and transfection, Agrobacterium-mediated transformation, gene transient expression, and CTV inoculation, as well as collection and analysis of data. One of the graduates was also trained in organizing outreach and extension activities. One undergraduate student was trained in general laboratory techniques including preparation of various media and buffers, tissue culture, bacterial cell culture, citrus genetic transformation and plant maintenance. How have the results been disseminated to communities of interest?Progresses of the project have continually been disseminated to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public through virtual meeting presentations, seminars, and in-person communications. The results have also been disseminated through the project website: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/. What do you plan to do during the next reporting period to accomplish the goals?Currently, the project is making excellent progresses. We will follow the proposed experimental design in the proposal with a focus on generation of transgene-free CRISPR mutant plants to accomplish the project goals.

Impacts
What was accomplished under these goals? (1) Identify targets for gene editing: Citrus macrophylla trees carrying CTV RNAi constructs for all the proposed genes have been generated. At least one C. macrophylla plant for each construct has been used to test the stability of the construct. Eight CTV RNAi vector constructs have been confirmed to be stable by RT-PCR using primers within CTV upstream and downstream of the insertion site as compared to the parent binary plasmids. We are working to confirm the stability of other CTV RNAi vectors in C. macrophylla. Sweet orange seedlings expressing one of the prioritized constructs have gone through one round of HLB test. Results showed that several CTV RNAi plants of this construct are HLB tolerant. We are propagating these plants to test the stability and inheritance of the HLB tolerance in these plants. Sweet orange seedlings expressing two other prioritized constructs have been inoculated with Candidatus liberibacter asiaticus (Clas) using its Asian citrus psyllid vector. The plants are growing in the greenhouse for symptom development. Sweet orange seedlings expressing other stable CTV RNAi constructs are being inoculated with Clas-infected psyllids in the greenhouse. (2) Generation of transgene-free citrus varieties by CRISPR/Cas9-mediated gene editing: The CRISPR mutant citrus plants that have been produced carry T-DNA, but have nevertheless been inoculated with Clas-infected psyllids in the greenhouse. They are kept in a clean greenhouse for symptom development. We focused on generation of transgene-free CRISPR mutant plants and have tested different approaches. First, we determined the best cationic lipid nanoparticles to deliver donor DNA and optimized a protocol using Lipofectamine™ LTX Reagent with PLUS reagent to mediate DNA delivery into protoplasts. A Cas9 construct containing a gRNA targeting a citrus gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine recorded a high cell viability percentage (45%) compared with the other transfection agents. Nine mutant plants were identified based on the endonuclease mismatch detection assay and Sanger sequencing. Our results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus. Second, ribonucleoprotein particle transfection using cationic lipid nanoparticles as a DNA carrier has been carried out on Sweet orange protoplasts. We are optimizing the methods for gRNA synthesis and transfection. Some mutations have been detected by the mismatch detection assay, but no edited plants have been recovered by this method. Third, two CRISPR/Cas9 vectors with high gene-editing efficiency in citrus have been identified and are being used for Agrobacterium-mediated transient expression for production of transgene-free CRISPR mutant plants. (3) Evaluate CRISPR/Cas9-produced citrus varieties: We have produced CRISPR mutant citrus plants, but they carry T-DNA insertions. These plants have nevertheless been inoculated with Clas-infected psyllids in the greenhouse. They are kept in a clean greenhouse for symptom development. (4) Engage stakeholders in project development/execution process and develop effective outreach programs: Although in-person interactions with growers and industry in 2021 were still limited by the Covid-19 pandemic, routine communications with growers were carried out via phone calls, emails, and text messages on collaborative projects and new arising problems. Project research results were disseminated to growers and other industry personnel via virtual seminars and presentations locally, regionally as well as nationally, and through email discussions. Other means of communicating research progress and discussing grower's needs included attending virtual meetings including citrus advisory committee meetings, industry meetings, and plant pathology meetings. Information about the project and project progress was also described on the dedicated webpage for the project. The project webpage has been updated and can be visited via this link: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Merritt, B.A., Zhang, X., Triplett, E.W., Mou, Z., Orbovic, V. (2021). Selection of transgenic citrus plants based on glyphosate tolerance conferred by a citrus 5-enolpyruvylshikimate-3-phosphate synthase variant. Plant Cell Reports 40:1947-1956.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Lovatt, c., Narvaez, J., Levy, A., Acanda, Y., Meritt, B., Ravanfar, R., Orbovic, V. (2021). Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp. Transgenic Research 30:687-699.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Acanda, Y., Welker, S., Orbovi?, V., Levy, A. (2021). A simple and efficient agroinfiltration method for transient gene expression in Citrus. Plant Cell Reports 40:1171-1179.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Li, Q., Canton, M., Wu, H., Zhang, X., Zale, J., Mou, Z. (2021). Efficient artificial microRNA vectors for gene silencing in citrus. Plant Cell Reports 40:2449-2452.

Progress 02/01/20 to 01/31/21

Outputs
Target Audience:The project's outreach and extension activities provide information on progress of the project and on HLB and ACP control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. These topics were discussed and presented in following events in Florida in 2020: Batuman, O. (2020). Development of a citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. CRDF Advisory Board Meeting. December 2020. Virtual presentation. Batuman, O. (2020). Needle assisted trunk infusion (NATI) and Automated Delivery System (ADS). 2nd NIFA Project's Advisory Board Meeting. October 2020. Virtual presentation. Torres, J. (2020). Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. UC Davis-UC Berkley collaborators meeting. August 2020. Virtual presentation. Batuman, O. (2020). Needle-assisted trunk infusion (NATI) of therapeutic material for controlling HLB and its psyllid vector. Inviao scientific board invited seminar. July 2020. Virtual presentation. Batuman, O. (2020). Needle-assisted trunk infusion (NATI) of therapeutic material for controlling HLB and its psyllid vector. Department of Plant Pathology invited seminar. January 2020. Gainesville, FL. Batuman, O. (2020). Huanglongbing (a.k.a. Citrus Greening) Disease of Citrus. Department of Plant Pathology Bacteriology Lecture. January 2020. Gainesville, FL. Batuman, O. (2020). Citrus Pathology Program. UF Citrus Agents Meeting. January 2020. Immokalee, FL. Batuman, O. (2020). Automated delivery system: can therapeutics be delivered through citrus trunk? Growers Meeting. September 2020. Virtual presentation. Yosvanis Acanda and Amit Levy. (2020). Poster. Nucleic acid and nucleoprotein delivery Into citrus cells using the Helios gene gun system: its potential for gene editing in citrus. American Society for Horticultural Science annual conference. Aug 9, 2020. Yosvanis Acanda. (2020). lecture. Citrus transient expression. Citrus Research and Education Centere seminar series. Nov. 2020 The project information is also described in the project website: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/ Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three postdocs were trained in project design, experimental design, advising graduate students, presentation, and progress report. Three graduate students were trained in experimental design, basic molecular biology techniques including vector construction, Agrobacterium-mediated transformation, gene gun bombardment, and CTV inoculation, as well as collection and analysis of data. One of the graduates was also trained in arranging outreach and extension activities. One undergraduate student was trained in general laboratory techniques including preparation of various media and buffers, tissue culture, bacterial cell culture, citrus genetic transformation and plant maintenance. How have the results been disseminated to communities of interest?Results of the project have been disseminated to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public through virtual meeting presentations and seminars. The resuts have also been disseminated through the project website: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/. What do you plan to do during the next reporting period to accomplish the goals?The project is moving forward smoothly. We will follow the detailed experimental design in the proposal to accomplish the project goals.

Impacts
What was accomplished under these goals? (1) Identify targets for gene editing: Citrus macrophylla trees carrying CTV RNAi constructs for a total of 23 genes encoding negative regulators of the citrus immune system have been generated. These constructs have been prioritized based on protein levels of a major citrus positive immune regulator in the C. macrophylla plants. Sweet orange seedlings graft-inocuated with one of the prioritized constructs have been tested for HLB tolerance in the greenhouse. Preliminary result showed that this construct is promising. Other CTV RNAi constructs have been graft-inoculated into sweet orange seedlings and establishment of CTV systemic infection has been analyzed. These plants will be Inoculated with Candidatus liberibacter asiaticus using its Asian citrus psyllid vector. Stability of the CTV RNAi constructs in citrus plants has also been tested and will be monitored for longer time. (2) Generation of transgene-free citrus varieties by CRISPR/Cas9-mediated gene editing: CRISPR mutant citrus plants have been generated, but these plants contain the T-DNA and are not transgene free. Nevertheless, these plants were propagated and the progenies will be tested for HLB responses. Efforts were focused on generation of transgene-free CRISPR mutant plants using CRISPR/Cas9 constructs that have been shown to be efficient for producing gene editing events through Agrobacterium-mediated transient expression. CRISPR/Cas9 editing via DNA delivery in citrus protoplasts was also experimented and an in-house protocol has been established and will be optimized. Furthermore, genome editing by direct delivery of CRISPR/Cas9 ribonucleoproteins in citrus protoplasts was tested and the protocol will be established. In addition, a cheap and easy Agroinfiltration method was developed to qucikly test CRISPR/Cas9 constructs before entering the transformation pipeline. Finally, the CTV replicon was studied for potential usage in gene editing and results showed that the replicon replicates and accumulates in callus cells in the explants. (3) Evaluate CRISPR/Cas9-produced citrus varieties: Nothing to report. (4) Engage stakeholders in project development/execution process and develop effective outreach programs: In-person interactions with growers and industry this year were limited by the Covid-19 pandemic and research shutdown. However, regular communications with growers were conducted via phone calls, emails, and text messages on collaborative projects and new arising problems. Project research results were disseminated to growers and other industry personnel via virtual seminars and presentations locally as well as regionally, and through email discussions. Other means of communicating research progress and discussing grower's needs included attending virtual meetings including citrus advisory committee meetings, industry meetings, and plant pathology meetings. Information about the project and project progress was also described on the dedicated webpage for the project. The project webpage can be visited via this link: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Dutt, M., Mou, Z., Zhang, X., Tanwir, S.E., Grosser, J.W. (2020). Efficient CRISPR/Cas9 genome editing with citrus embryogenic cell cultures. BMC Biotech. 20:58. https://doi.org/10.1186/s12896-020-00652-9

Progress 02/01/19 to 01/31/20

Outputs
Target Audience:The project's outreach and extension activities provide information on progress of the project and on HLB and ACP control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. These topics were discussed, presented and provided in handouts to attendees in following events in Florida in 2019: Batuman, O. (2019). Citrus Pathology Program (Volunteered presenter). Presentation for UF Plant Science Council Students. SWFREC, Immokalee, FL, December 5, 2019. Batuman, O. (2019). 1st Annual Advisory Board Meeting (Organizer and presenter). Hands-on demonstrations and grower/industry presentation and handout. SWFREC, Immokalee, FL, October 2, 2019. (Volunteered) Batuman, O. (2019). Greenhouse demonstration (Volunteered presenter) - Guided greenhouse tour and demonstrations. SWFREC, Immokalee, FL, June 11, 2019. Batuman, O. (2019). Citrus Farm Field Day (Co-Organizer and Volunteered presenter) - Guided field tour and demonstrations and grower/industry presentation and handout. SWFREC, Immokalee, FL, May 16, 2019. Batuman, O. (2019). Citrus Field Day (Co-Organizer and Volunteered presenter) - Guided field tour and demonstrations and grower/industry presentation and handout. SWFREC, Immokalee, FL, April 18, 2019. Batuman, O. (2019). SWFREC Foundation Board Meeting (Invited presenter). Grower/industry presentation, SWFREC, Immokalee, January 22, 2019. Batuman, O. (2019). Citrus Advisory Committee Meeting (Invited presenter)- Grower/industry presentation and handout. SWFREC, Immokalee, FL, January 18, 2019. Batuman, O. (2019). Workshop (Invited presenter)- Scouting and managing citrus diseases. Grower/Industry presentation and handout. SWFREC, Immokalee, FL, January 16, 2019. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three postdocs were trained in project design, experimental design, advising graduate students, presentation, and progress report. Three graduate students were trained in experimental design, basic molecular biology techniques including vector construction, Agrobacterium-mediated transformation, gene gun bombardment, and CTV inoculation, as well as collection and analysis of data. Four undergraduate students were trained in general laboratory techniques including preparation of various media and buffers, tissue culture, bacterial cell culture, and citrus plant maintenance. How have the results been disseminated to communities of interest?Results of the project have been disseminated to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public through guided field and laboratory tours and demonstrations, poster displays and educational handouts, meeting presentations and short blurbs. What do you plan to do during the next reporting period to accomplish the goals?We will follow the detailed experimental design in the proposal to accomplish the project goals.

Impacts
What was accomplished under these goals? (1) Identify targets for gene editing: A total of 23 genes encoding negative regulators of the citrus immune system have been silenced with CTV RNAi constructs. In order to quickly evaluate the effects of these constructs on the citrus immune system, an antibody against a major citrus positive immune regulator was successfully developed. The antibody has high specificity that allowed quantification of the protein levels of the citrus immune regulator in the CTV RNAi infected Citrus macrophylla seedlings. Several constructs were found to significantly increase the protein levels of the citrus immune regulator. As C. macrophylla seedlings do not produce a pronounced phenotype upon infection with HLB, sweet orange seedlings were graft-inoculated with the different CTV RNAi constructs using budwood from the "source" C. macrophylla plants. The constructs that significantly increased the citrus immune regulator levels in C. macrophylla seedlings have been prioritized in screening for HLB phenotypes. Inoculation of the sweet orange seedlings with Candidatus liberibacter asiaticus using its Asian citrus psyllid vector is ongoing. (2) Generation of transgene-free citrus varieties by CRISPR/Cas9-mediated gene editing: Continuous efforts were focused on optimization of Agrobacterium-mediated transient expression of CRISPR/Cas9 constructs. The 18 CRISPR/Cas9 constructs with various combinations of promoters and gRNA cassettes were used for the optimization experiments. A large number of explants were transformed with Agrobacteria carrying CRISPR/Cas9 constructs and the resulting shoots were screened. Several CRISPR/Cas9 constructs were found to be more efficient than others and produced gene editing events. These constructs are being used for generating transgene-free gene editing events. the extension strategies and outreach included visiting citrus growers on site to assess their specific needs and to gain an overview of their production and management practices and acquire knowledge about specific issues of concern. In addition, regular communications with growers were conducted via phone calls, emails, text messaging, and personal visits to follow up on collaborative projects and inquire about new arising problems. Project research results were also disseminated to growers and other industry personnel via seminars and presentations locally as well as regionally, and through personal discussions and email messages. Other means of communicating research progress and discussing grower's needs included attendance of citrus advisory committee meetings and other industry meetings that were regularly held at the University of Florida Southwest Florida Research and Education Center (SWFREC) or other locations statewide (eight times throughout the year). Seminars held at SWFREC and other centers were conducted in collaboration with extension agents and continuing education units. A dedicated webpage for the project has been set up and running. The project webpage can be visited via this link: https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Erpen-Dalla Corte L, M Mahmoud L, S Moraes T, Mou Z, W Grosser J, Dutt M. Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique. Plants (Basel). 2019 Dec 13;8(12). pii: E601. doi: 10.3390/plants8120601.

Progress 02/01/18 to 01/31/19

Outputs
Target Audience:Outreach activities in Florida are designed to provide information on progress of our project and on HLB and ACP control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. These topics were discussed, presented and provided in handouts to attendees in following events held in Florida in 2018: Guided field and laboratory tours and demonstrations at the Open House 2018, the Southwest Florida Research and Education Center (SWFREC), Immokalee, FL (November 15, 2018) Poster display at the 3rd Materials Innovation for Sustainable Agriculture (MISA) annual symposium, University of Central Florida, Orlando (October 31-November 2, 2018) Presentation at the Citrus Advisory Board meeting, at SWFREC, Immokalee FL (October 12, 2018) Poster display and educational handout at the Citrus Expo, Ft. Myers (August 15-16, 2018) Presentation at the Southwest Florida Research and Education Foundation Board meeting, at SWFREC, Immokalee FL (August 24, 2018). Poster display at 6th Annual Graduate Student Poster Symposium, SWFREC, Immokalee FL (June 2018) Guided field tour and demonstrations and grower/industry presentation and handout at Citrus Field Day at SWFREC, Immokalee, FL (2017-2018) Short blurbs during number of lab and field demonstrations (industry, local schools, master gardeners, the Leadership Lee County group, the Florida Master Naturalists, Young Professionals Organization from Minnesota, homeowners, etc.) (2017-2018). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three postdocs were trained in project design, experimental design, advising graduate students, presentation, and progress report. Two graduate students were trained in experimental design, basic molecular biology techniques including vector construction, Agrobacterium-mediated transformation, gene gun bombardment, and CTV inoculation, as well as collection and analysis of data. Two undergraduate students were trained in general laboratory techniques including preparation of various media and buffers, tissue culture, bacterial cell culture, and citrus plant maintenance. How have the results been disseminated to communities of interest?Results of the project have been disseminated to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public through guided field and laboratory tours and demonstrations, poster displays and educational handouts, meeting presentations and short blurbs. What do you plan to do during the next reporting period to accomplish the goals?We will follow the detailed experimental design in the proposal to accomplish the project goals.

Impacts
What was accomplished under these goals? (1) Identify targets for gene editing: A total of 23 negative regulators of the citrus immune system were selected. Truncated open reading frames (ORFs) (350-600nts) in the antisense orientation were individually cloned into the position between p23 and 3'NTR under the control of the duplicated coat protein controller element (CP-CE) of CTV. The constructs were determined by restriction digestion and confirmed by sequencing. All CTV RNAi constructs were agro-inoculated into Nicotiana benthamiana. Virions were semi purified from systemically infected tissues and inoculated onto Citrus macrophylla (the most amenable citrus host for mechanical inoculation with CTV virion preps). The citrus plants were confirmed positive by ELISA (with antisera against the CTV coat protein) two months post inoculation. In addition, stability of the truncated ORFs was confirmed by RT-PCR using CTV primers upstream and downstream of the insertion position. As C. macrophylla does not reveal a pronounced phenotype upon HLB infection, the different CTV RNAi constructs are being grafted to sweet orange. (2) Generation of transgene-free citrus varieties by CRISPR/Cas9-mediated gene editing: Efforts were focused on a few negative regulators of the citrus immune system. A total of 18 CRISPR/Cas9 constructs with various combinations of promoters and gRNA cassettes have been constructed. More than 46,000 explants were transformed with Agrobacteria carrying CRISPR/Cas9 constructs and more than 100,000 shoots were screened. Gene editing events were identified by restriction enzyme digestion and sequencing, but the efficiency of mutation was low. No transgene-free gene editing event has been identified. Gene gun bombardment of plasmid DNA, messenger RNA (mRNA), and viral RNA/replicons have been tested. In vitro transcribed and capped mCherry mRNA was precipitated into gold microparticles and bombarded into citrus leaf epidermis, stem bark and epicotyl segments. Its translation was confirmed by visualization under the fluorescent microscope 24 hours after bombardment. This experiment confirmed that capped mRNA could be efficiently delivered into citrus cells and successfully translated to protein. Furthermore, the genome of the turnip vein clearing virus (TVCV) was transcribed in vitro and bombarded into N. benthamiana leaves using the Helios gene gun. Results showed that the in vitro transcribed virus genome was expressed and systemic infection of N. benthamiana was observed 8 days after bombardment. Citrus cell suspension cultures were transformed with CRISPR/Cas9 constructs and plants have been regenerated for molecular analyses. Conventional protoplast transformation utilizing PEG has resulted in several putative edited lines. A new transfection mediated protoplast transformation technique using commercially available cationic-lipid nanoparticles with or without the addition of cell penetrating peptides (CPP) was explored. Protoplast transformation efficiency was greatly improved using cationic-lipid nanoparticle encapsulated DNA and transient EGFP expression could be observed every time. Addition of CPP further improved transient DNA uptake. This protocol was further optimized to develop a marker gene free genome editing system. A fusion EGFP-Cas9 recombinant protein construct has been produced and purified protein was used for protoplast transfection. However, yield is very low at the moment. (3) Evaluate CRISPR/Cas9-produced varieties: No results. (4) Engage stakeholder in project development/execution process and develop effective outreach programs: Outreach and extension efforts in Florida are designed to provide information on progress of the project and on HLB and ACP control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. During this period, efforts were focused on introducing the strategies that are being developed for controlling HLB and ACP in citrus groves in Florida. Among the strategies that are being communicated across, CRISPR gene editing, the use of CTV vector to express/silence various genes that may confer control to HLB and/or ACP in citrus groves and using ACP-infecting viruses to develop a novel biological control strategy were the most intriguing topics for a wide range of audience. These topics were discussed, presented and provided in handouts to attendees in eight events held in Florida in 2018.

Publications