Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Citrus industry stakeholders in the USA and abroad, university partners, other industry and government researchers, and US regulatory agencies. Changes/Problems: The PD, Dr. Shatters, accepted a 2-year positionas the Center Director at the USDA ARS European Biological Control Lab in Montpellier, France. The advisory board recommended that Dr. Michelle Heck function as the scientific director of the project in partnership with Dr. Randall Niedz at the USHRL. The advisory board was comfortable with the idea that Dr. Shatters remain the PD of the project and be consulted on all high-level administrative and scientific decisions.Dr. Shatters engageswith this work and participates in project and laboratory meetings via Zoom. A new project coordinator, Lucy Bennett, was hired to replace Kim Wood who accepted another position. The project's scope of work was refinedto focus on field evaluation of molecules based on the feedback from the steering committee. This refocusing was designed to speed commercial delivery of therapeutic treatments and supported by citrus growers' recent willingness to deliver HLB therapies via manual injection. The teamrecently received APHIS BRS and EPA permit approval to begin Symbiont field trials. This has been an ~8 months process. Some data suggest that certain large fusion proteins expressed in S-TIS were proteolytically cleaved during protein secretion from S-TIS in vitro and extracted from on planta Symbionts. To remain focused on delivering solutions to the growers as quickly as possible, current research is focused on S-TIS production of small antimicrobial peptides, that are less susceptible to proteolytic cleavage for direct injection. A new sub-award was developed with Mr. Robert Adair at the Florida Research Center for Agricultural Sustainability for APHIS and EPA approved field evaluations. The sub-awards from two of the project PIs have been terminated and the work of one PI was redirected to focus resources on the most fruitful areas of research. Methods to develop Symbionts using inoculation of Agrobacterium carrying Symbiont-forming plasmids now produce Symbionts on citrus in as little as one month. Thus, we are no longer researching transplantation as a mode of Symbiont formation. Early experiments proved very challenging to get transplantation to work in citrus, although some promising advances in transplantation were made in tomato. What opportunities for training and professional development has the project provided?The projects' labs each train technicians across the project's needs bestowing professionals with a wide breadth of skills. The Heck Lab and IRSC train high school and undergraduate students in standard lab and assay protocol and foster the students' comprehension of fundamental molecular biology and plant pathology. IRSC, the Heck Lab, and USHRL students and staffpresentresearch findings at symposiums and conferences. Conference attendance allows individuals to network, diversify their protocol, and brainstorm new avenues of research.IRSC students presented 8 posters and attended 7 scientific conferences. Developing IRSC curricula in line with the project necessitated staff scientists become informed on instructional strategies and form a view of their work focused on fostering creative innovation. The curricula developed at IRSC convey the industrial applicability of students' education. The Heck Lab and USHRL prepare post-doctorate researchers on diverse protocols to increase their employability. Weekly project meetings encourage staff to engage in the project's wide breadth of undertakings. The CRADA partner hired an IRSC graduate as a technician this summer. The annual stakeholder meeting was in person this year which provided an opportunity for staff to connect with one another and forge connections across disciplines. The meeting also allowed lab personnel to contextualize their work with industry stakeholders and the methods of field trials. How have the results been disseminated to communities of interest?The research groups meet on a weekly basis to maintain effective collaboration. Additionally, in January the groups met for a Stakeholder Advisory Meeting where the various scientists working on the project briefed the committee of growers and industry partners on the progress of the work. The stakeholders provided feedback and shaped the framework for analysis of molecules in the field. Collaborators have given various seminars, research talks, and posters listed in the publications section to agencies, academia, industry, and the wider research community. Dr. Randall Niedz updates industry stakeholders on the research by phone and during in-person meetings at the US Horticultural Research Center and during trips to visit growers in their groves. Dr. Michelle Heck presented Symbiont's potential to treat HLB affected citrus at the 2023 Florida Grower Citrus Show and various other seminars to groups of interest. AgroSource had a booth focused on their injectable oxytetracycline product, Rectify, at the 2023 Citrus Show to interact directly with growers to disseminate product information. Rectify is now being used to treat millions of trees in the state of Florida. They interact directly with growers to disseminateproduct information. Drs. Marco Pitino and Michelle Heck gave talks at the Entomology Society of America meeting. More than ten team members presented their research at a meeting focused on HLB and vascular bacterial pathogens in Clearwater, FL. Drs. Heck and Shatters gave a keynote address at the BioSolutions conference in Reno, NV, which is a conference for agricultural industry professionals working in biological control.Team members developed an informational brochure that has been shared with the heads of the state industry groups and other interested parties. Dr. Shatters has worked to develop international interest in deploying Symbionts for control of insect-transmitted plant vascular diseases. The goal is to advance Symbiont capabilities, effectiveness, and acceptance through engagement with international researchers and creation of international multidisciplinary teams. Dr. Shatters hosted an international workshop with researchers, administrators, policymakers, and stakeholders from the U.S., France, England, Spain, Italy, Greece, and Australia. The results of this workshop included an agreement to develop an International Plant Biosecurity Alliance to integrate interdiction strategies- that include Symbionts- into tools for a biosecurity toolbox. This will increase the resources brought to bear on commercializing Symbionts and improve international acceptance of commodities produced on plants with Symbionts. What do you plan to do during the next reporting period to accomplish the goals?1 Research will focus on optimizing the production of small peptides using S-TIS and providing AMPs for field trials. Psyllid acquisition assays will continue to be part of a lab screen to determine if there is any effect on CLas uptake through feeding on plant material treated with molecules that are working in the field or new classes of antimicrobials that are too expensive to use in the trunk injection screen.ORISE scholar will be trained in this method. Research will investigate the potential of stacking multiple AMPs, ACP neuropeptides, and NCRs in the same plasmid to increase treatment potential using the GAANTRY system. Testing AMPs stacked with an ACP neuropeptide for their potential to interfere with ACP feeding. ACP neuropeptides and NCRs will be evaluated by direct infusion for blocking CLas acquisition and transmission of HLB. Continue development ofS-TIS to ensure enough AMPs are produced for testing and application in the field. Expansion of Vmax technology for nanobody surface display. Possibly also for AMP production if time and funds permit. Construct fully synthetic nanobody VHH libraries using a universal scaffold from domains obtained from animal immunizations. 2 Finalize data collection from Phase I of the direct injection field screen. Develop Phase II of the direct trunk injection screen and incorporate other state industries in the effort (Texas and California). Continue to screen all molecules (and combinations of such) tested in the field through the MIC Assay to identify beneficial interactions for further field evaluation. Update the citrus industry on this transformativeproject. 3A Utilize new expression vectors to produce NCR peptides in S-TIS. If successful, will be an effective method for consistent NCR peptide production. NCR peptide expressing Symbiont vectors are being constructed for in-field Symbiont delivery testing on CLas infected trees. Begin Symbiont field trials at the Florida Research Center to establish baseline parameters for Symbiont inoculation and growth on field trees. Collect environmental monitoring data required by APHIS BRS to remain compliant with the permit. Confirm quantitative methods of detecting therapeutic molecule movement between the Symbiont and host plant. Investigate RNA silencing and plant protein overexpression to control protein export from the Symbiont to the host plant and/ or boost systemic plant immunity. Continue greenhouse trials of Symbionts to identify ideal growing conditions to improve growth and protein export. Validate findings showing a decrease in CLas titer in citrus trees bearing AMP expressing Symbionts with labs in Fort Pierce FL, Ithaca NY, Albany CA, and in field trials. Evaluate attenuating Agrobacterium strains through creation of auxotrophs that cannot survive in the environment but can still deliver DNA to initiate Symbiont formation. 3B Already developed transgenics will be placed in field trials at the USDA, ARS research farm for further evaluation. 3C Replication of field trials to validate previous direct injection findings. AMPs will be tested through direct infusion into CLas-infected potted greenhouse plants using 3D printed infusion devices. Prepare a small library of promising NCR peptides ready for direct injection experiments. Continue evaluating OTC trials on grower farms. A further injection trial will take place in Spring 2024 to observe the effects of injection on the full growing season. Molecules for injection will be determined based on current trial results. 4 Current APHIS/BRS permit will allow Symbiont field trials of 9 constructs &5 AMPsthat will be initiated this fall/winter. Maintain field sites to EPA and APHIS permit expectations. 5 AgroSource will be leading the Economic Analysis, pending identification of lead candidate products from field testing. Licensing negotiations of Symbiont technology are ongoing between AgroSource and USDA ARS. 6A IRSC interns will continue to work with IRSC faculty and grant scientists to complete undergraduate research projects. IRSC faculty will continue to execute a lab module in BSC3465L and BSC 2012, General Biology 1. The curricula will be adjusted based on student performance and feedback. Opportunities will be provided for IRSC students and interns to attend and present at scientific conferences. 6B A 2024 Stakeholder Advisory Committee Meeting is being planned to inform industry partners on research progression and receive feedback on effective means of further engaging the industry. At least three project/grove tours are planned for the coming year that involves taking industry leaders through the project and into the groves to see how the research is developing and to learn about the new research framework. Experiments are conducted in collaboration with citrus growers in their groves and three different citrus industry associated companies that satisfy the extension component of the project. Initiated contract with FUTURUM, Inc. for web-based and publication presentation of grant-associated activities with educational component to describe new technologies to the public. The educationaldescription includesnew screening strategy and Symbionts.

Impacts
What was accomplished under these goals? 1A Direct infusion trials IDed as the preferred method for initial screening of compounds available in sufficient quantity. A bench-scale assay to quantify inhibition of CLas acquisition by psyllid nymphs from HLB positive leaves is used for compounds not available in sufficient amounts. Several novel AMPs appear to be effective against CLas acquisitionand are being moved to field trial. Dramatic improvement in Symbiont cell culture production in Temporary Immersion Systems (S-TIS) suggests that S-TIS may be a commercially viable method of therapeutic biomolecule production. Plants have been selected for evaluating the use of on-plant Symbionts as a biofactory for desired biomolecules, where the Symbiont is a harvestable commodity. 1B Developed components of a bacterial nanobody surface display system for screening nanobodies targeting CLas effectors. Created nanobody conserved-domain scaffold system for producing synthetic libraries of nanobody variants (variations in the known nanobody variable regions between the three known conserved regions) that can be screened against CLas effector ligands. Developed a control system composed of fluorescent protein ligands and efficient binding cognate nanobodies for optimizing library screening. Binding kinetics demonstrated this control ligand/nanobody system as a superior "display" interaction to benchmark nanobody screening/selection platform. 1C Development of a 3-week assay to screen AMPs using Symbionts allowed ID of AMPs effective against CLas in citrus and "Candidatus Liberbacter solanacearum" in potato. 4 AMPs were discovered that reduce CLas titer and HLB symptoms in a Symbiont screen of 10 AMPs, plus controls, using potted greenhouse citrus plants. 2 Developed system to produce 5,000 CLas inoculated trees for team use. Re-designed the pipeline into a field-to-grower "In Field First" screening program. 3A Research showed that Symbionts are a semi-organized structure comparable to the early stages of lateral root formation instead of the prior belief that they are a mass of undifferentiated cells. 3Ai Genomes of a variety of Agrobacterium isolates, with varying ability to form galls, have been sequenced to identify genes involved in cell activation. Analysis in progress. Continued evaluation of activation gene combinations resulted in discovering certain combinations best for use of Symbiont cell cultures as biofactories while other combinations are best for on-plant Symbiont formation. 3Aii 79 constructs fusing GFP to potential movement signals were screened and 18 candidate molecular export signals were identified for further evaluation. Developed plasmid system for Agrobacterium-mediated engineering of plant cells called GAANTRY; demonstrated to be a suitable platform for producing Symbionts and allowing delivery of up to 50 Kb of DNA (up to ~25 genes) to the plant cells. System allows stacking genes for defensive molecule biosynthesis and the ability for biosynth. pathway manipulation. 3Aiii A method developed for direct Agrobacterium delivery produces Symbionts on citrus within 1 month, has redirected efforts away from plant Symbiont cell transplantation forSymbiont formation. Data shows limited survival of Agrobacterium after inoculation, mitigating some initial regulatory concerns about inoculation as a delivery approach. 3Aiv On-going field studies using wildtype Agrobacterium on three citrus cultivars to evaluate inoculation success- a proxy for Symbionts until permits are approved. Higher success of Symbiont formation in the winter than that observed in summer. 3B Transgenic citrus expressing: Oncocin, an insect AMP from milkweed bug, induced mortality in adult and nymph psyllids, slowed nymph development, and reduced number of eggs. Also increased tolerance to Xanthamonas citri (causing citrus canker). Ready to move to the field. Trypsin Modulating Oostatic Factor (TMOF) from mosquitoes induced psyllid mortality. Ready to move to the field. Jaburetox (an insecticidal peptide derived from Jack bean urease) induced psyllid mortality. Ready to move to the field. Citrus Gene Knockouts: Citrus DMR6 (gene involved in downregulating the salicylic acid defense pathway) downregulation using either dsRNA induced RNAi or CRISPR knockout). Strong tolerance to X. citri observed with associated demonstration of reduced X. citri titer. Cloned plants ready for field. 3C Two ongoing field trials in commercial groves testing direct delivery oxytetracycline (OTC) and delivery devices,showing improved plant performance. A third trial at the USDA, ARS research farm in Fort Pierce, FL is ongoing to screen therapeutic molecules of interest with 80 molecule trials. At least 5 molecule combinations result in vigorous summer flush and warrant further evaluation to optimize management parameters for HLB control. 4 APHIS BRS and EPA permits were written and submitted. Both have recently provided regulatory approval for the field trials that will be initiated in the fall. APHIS BRS advised the team on data needed to relax permit conditions for future Symbiont field trials. 5 Symbiont biology was demonstrated but a field-validated application example has not, yet, been achieved. 13 molecules have been identified in the field injection screening trials that perform as good or better than oxytetracycline for summer flush development, and a process to replicate and validate effects in larger replicated field trials is required (ideally in production groves). 6A A new Cornell University graduate student is working on the project. Two DOE ORISE scholars are training in computational biology and microbiology. High school seniors in Ithaca train in HLB research via the New Visions program. Two Cornell undergraduate interns trained in diverse laboratory practices. Indian River State College (IRSC and HSI) undergraduate students and local high school seniors participate in training modules developed by project staff. AgroSource hired an IRSC graduate as a full-time technician in July 2023. 6B Oral presentations and/or Posters were presented by team members at various international and national conferences (Fl. Entomol..Soc., Int. Congress on Plant Path., Nat. Hort. Soc., Am. Phytopath. Soc., Int. Soc. For Molec. Plant Microbe Interact). PIs presentedseminars to audiences of growers, horticulturalists, and regulatory representatives within the US and internationally on Symbiont technology and delivery of plant-based therapeutics.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Mann, M., Saha, S., Cicero, J.M., Pitino, M., Moulton, K., Cano, L., Hunter, W.B., Mueller, L.A., Heck, M. (2022). Quantification of new and archived Diaphorina citri transcriptome data using a chromosomal length D. citri genome assembly reveals the vectors tissue-specific transcriptional response to citrus greening disease. GigaScience. DOI: 10.1093/gigascience/giac035
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Pitino, M., Fleites, L., Shrum, L., Heck, M., Shatters, R. (2022). Plant production of high affinity nanobodies that block SARS-CoV-2 spike protein receptor binding with human angiotensin converting enzyme. Frontiers in Bioengineering and Biotechnology. DOI: 10.3389/fbioe.2022.1045337
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Kennedy, J.P., Wood, K., Pitino, M., Mandadi, K., Igwe, D.O., Shatters, R., Widmer, T.L., Niedz, R., Heck, M. (2023). A perspective on current therapeutic molecule screening methods against Candidatus Liberibacter asiaticus, the presumed causative agent of citrus Huanglongbing. Accepted by Phytopathology. DOI: 10.1094/PHYTO-12-22-0455-PER
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Steven A. Higgins, David O. Igwe, John S. Ramsey, Stacy L. DeBlasio, Marco Pitino, Randall Niedz, Robert G. Shatters Jr., Laura A. Fleites, Michelle Heck. Plant-derived, nodule-specific cysteine rich peptides inhibit growth and psyllid acquisition of Candidatus Liberibacter asiaticus, the citrus Huanglongbing bacterium. bioRxiv 2023.06.18.545457; doi: https://doi.org/10.1101/2023.06.18.545457
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Saeed Hosseinzadeh and Michelle Heck. Variations on a theme: factors regulating interaction between Diaphorina citri and Candidatus Liberibacter asiaticus vector and pathogen of citrus huanglongbing. Current Opinion in Insect Science. Volume 56, 101025 https://doi.org/10.1016/j.cois.2023.101025.
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Lombardi, R.; Ramsey, J.; Mahoney, J.; MacCoss, M.; Heck, M.; Slupsky, C.. Longitudinal Transcriptomic, Proteomic, and Metabolomic Response of Citrus sinensis to Diaphorina citri Inoculation of Candidatus Liberibacter asiaticus"

Progress 09/01/21 to 08/31/22

Outputs
Target Audience: Targeted audiences include: 1. APHIS-BRS- regulatory agency responsible for permitting field trials of certain types of therapeutic treatments. 2. USDA, ARS National Program Staff and Administrators 3. Growers and Processors in Florida and California and their commodity support organizations (Florida Citrus Research and Development Foundation and California Citrus Research Board). They are on our advisory board, and we have provided in person meetings that include citrus production grove visits and discussions and seminars. 4. Other research collaborators on this NIFA project: Updates on routine research updates scheduled as Zoom virtual presentations. 5. Private industry that could provide supportive product delivery activities. Under Nondisclosure agreements, we have met with private companies and investors on moving our products into commercialization 6. Students and Staff at collaborating Higher educational institutions- Cornell, Indian River State College (IRSC), University of Florida. Students are both undergraduates and graduates. IRSC serves a community of underrepresented minority students and is designated a Hispanic Serving Institution. Students at all levels of their degree program, from freshman to seniors. This includes both students in the associates and bachelor's degree programs. The approach has been to include both lower-level students (freshman and sophomores) and upper-level students in undergraduate research as means to provide peer mentoring and to encourage persistence in IRSC the biology program. Changes/Problems: - Full interaction with stakeholders and collaborators has been difficult under COVID travel restrictions for the majority of the past two years. Improving conditions should allow for more. - Space limitation has been a major constraint for generating and maintaining the large number of citrus plants needed for this project. However, we have been able to occupy several greenhouse spaces and also to house plants at Picos Farm, which has enabled us to almost reach our objective of generating ~9,000 plants. - The detached leaf assay (DLA) was proposed as an initial step in this project's molecule assay pipeline. This assay would allow us to discard many potential molecules relatively quickly (assay consists of a 7-day incubation period) before investing more time (weeks to months) and resources into whole-plant assays. However, the DLA requires many replicate leaves to detect a potential effect of a molecule and we recently discovered that only a subset of our stock HLB+ Valencia plants (18 of 112) are actually infected with this bacteria. We are working out a trimming system to get these HLB+ plants to produce greater numbers of leaves and plan to graft inoculate uninfected plants, but we are currently left with a limited supply of leaves for this assay. In the meantime, we are discussing potential alternative assays to test molecules, including the psyllid homogenate assay, L. crescens plate assay, or transitioning directly to delivering molecules to whole plants using a 3D-printed direct infusion device developed and produced in this laboratory. - During the period, we have encountered challenges with the reliability of the BioXP. This included failure of the BioXP to complete runs as well as failure to assemble multiple fragment constructs critical for our delivery objective 3A. Working with the supplier, CodexDNA, we have been able to overcome these issues. We have also completed assembly of some constructs on bench. What opportunities for training and professional development has the project provided? - We provide bi-weekly project team meetings to discuss recent work, troubleshoot issues, and plan new work for the project objectives. - We also provide monthly journal club meetings to discuss and analyze recent publications relevant to the project and HLB therapy; covering topics such as new techniques in genome editing, plant immune defense pathways and novel antimicrobial compounds. - Across the project locations, we provided training and professional development for new technicians, graduate and undergraduate students in all aspects of plant and molecular research, such as molecular biology, plant horticulture, tree physiology, proteomics. - A postdoc on this project (Dr. Joseph Krystel) was mentored on submission of an ARSX competition (an internal USDA, ARS Innovations program to support innovative and disruptive technologies) for a method that will support the development of therapeutic molecules to be used for citrus greening. He won this competition and was awarded $100,000 for this supportive work. - We have a postdoctoral researcher, Dr. Brian Rhodes, who is developing new methods for Symbiont delivery. In this work he has learned to use a 3D printer for producing prototypes of direct plant infusion devices and containment vessels for Symbiont transplantation. - Scientists, technicians, postdocs and students attended and presented at professional society meetings in state and out of state. How have the results been disseminated to communities of interest?The research groups continue to have bimonthly meetings to discuss project objectives, data, and roadblocks. Furthermore, a series of Stakeholder Meetings were held in April and May of 2022 where the various scientists working on the project updated members of the citrus industry, scientists in academia and the NIFA Advisory Board, among others, to get feedback on research progress and future plans. Collaborators have given various seminars, research talks and posters listed in the publications section to agencies, academia, industry and the wider research community. What do you plan to do during the next reporting period to accomplish the goals? Obj 1: Molecule discovery and production: Transgenic expression of NCR peptides and psyllid neuropeptides: We will collaborate with researchers at Ft. Peirce to generate new transgenic citrus lines expressing our most promising candidates for direct inhibition of CLas growth and disruption of the psyllid life cycle. We will also evaluate Symbiont expression of these peptides Psyllid Stylet Sheath-Binding RNA Aptamers (SSBRAs): We will test a panel of new constructs to improve RNA aptamer expression and stability in citrus leaves. Psyllid development and survival will be observed on leaves expressing the highest possible dosage of SSBRAs Expand number of AMPs produced in Vmax at scale Surface display technology development in Vmax : Identify a model protein-single-domain antibody interaction and validate affinity/specificity in conventional ELISA/flow cytometry/SPR methods to benchmark expected results for a "display" version of this interaction Optimize and screen display variations for use in "mock selection" experiments to empirically determine display efficiency, enrichment, and utility for discovering tight binders against a selected effector molecule Submit purified effector proteins for animal immunizations to develop pre-primed VHH libraries of nanobodies against high priority CLas effector proteins thought to promote HLB by modulation of the plant immune system and adaptive response Construct fully synthetic VHH libraries using a "universal" scaffold (cAbBCII10) and varying degrees of mutagenesis in the CDR1, CDR2, and CDR3 domains for selection experiments as an orthogonal directed evolution approach to those obtained from animal immunizations Obj 2: Screening Pipeline The remaining ~400 mature citrus plants and >8,000 citrus seedlings will progressively undergo psyllid inoculation over the next year. Subsets of these inoculated plants will be monitored for symptom development and tested for CLas presence with qPCR between 3 and 6 months post-inoculation. Whole-plant testing of potential therapeutic molecules will be initiated using direct plant infusion devices we print using 3D printer located at the USHRL facility. We will identify which biological assay(s) can be used, with our available resources, to begin evaluating the therapeutic potential of internally and externally-provided molecules for this project. Obj 3 Therapeutic Molecule Delivery Obj 3A: Symbiont Delivery: First set of constructs designed to produce secreted antimicrobial peptides shown to work against CLas have been finalized and will be used for symbiont delivery to HLB+ citrus in greenhouse and field testing. We will develop constructs for Symbiont production of therapeutic fusions and test these in greenhouse. We will conduct an evaluation of symbiont delivery strategies to improve the efficiency of Agrobacterium-free symbiont delivery to citrus. We will evaluate the on-plant symbiont biofactory production strategy in plant systems shown to rapidly produce multiple symbionts. We will test Symbiont cell culture pilot scale systems developed by Agricultural Engineers for producing therapeutic biomolecules. We will begin testing the experimental symbiont vectors for efficacy against Liberibacter solanacearum. The optimal symbiont protein export configuration for systemic protein delivery to the plant will be identified using various cell penetrating peptides and/or expression enhancement through viral amplification. We are also working on a BRS permit to enable field inoculations of citrus with one of the novel plasmids. The primary focus of the planned field trials is to study the survival of Agrobacterium in symbionts, whether the bacteria form symbionts on uninoculated, nearby plants, and to test one or more candidate antimicrobials for CLas growth inhibition either in local symbiont tissue or systemically. This will be the first field trial using Agrobacterium tumefaciens carrying the PGR genes necessary to form a symbiont and will inform future studies and the regulations that may surround this novel technology.? Nanobody sequences will be used to generate constructs to deploy nanobody using Symbiont technology in citrus trees.? Obj 3B: Transgenic Delivery: Field plantings of transgenics expressing Onyx and Topaz AMPs will be initiated. Greenhouse testing of transgenics expressing Onyx and Topaz against CLas transmission/acquisition by psyllids and HLB disease symptom development will be evaluated. ACP colonization, greenhouse and field trials for Topaz transgenics. Canker screening to identify transgenics with broad spectrum disease resistance, including 37 new transgenic constructs and additional lines/varieties of previously tested constructs. Detached leaf assay testing of Jade transgenics (insecticidal AMP) for anti-CLas and anti-ACP activity. Renewal of BRS permit for CBI containing transgenics (current period ends 12/2023) Secure BRS release permits for symbiont field trials and conduct data collection for future SOP adjustments to a non-transgenic status. Obj 3C: Direct Plant Infusion(DPI): Continue the field study and advance the investigation on molecules of interests with greenhouse studies using 4-year-old citrus trees grown in pots. Four treatments testing two therapeutical molecules are going to be established using 16 trees. The molecules will be delivered at the same concentration using a direct tree infusion device. After 2 and 4 weeks the trees will be assessed for CLas titer and tree size. Upon completion of the greenhouse study the same molecules will be evaluated under field conditions. Obj 4: Regulatory Strategy: - Secure regulatory permit for field testing at our research farm and initiate discussions on data needed for commercial use approval. Obj 5. Economic Assessment: - Develop economic assessment of plant produced biomolecule biofactories versus in vitro growing of symbionts for therapeutic biomolecule production: Use this information to develop roadmap for best system to produce therapeutics that could be used in direct delivery to citrus to control HLB symptoms. Obj 6: Outreach: Obj 6A. Education During the next reporting period, IRSC faculty (D'Elia, Carroll) will implement a newly designed lab module in BSC 2010, General Biology 1. The curriculum will be adjusted based on students' performance and feedback. Opportunities will be provided for IRSC students and interns to attend and present at scientific conferences. IRSC students will be placed with scientists at USDA and Agrosource for internship opportunities. Obj 6B: Extension - Provide field evaluation of Symbiont system to advisory board consisting of growers and citrus industry representatives. - Initiate field trials to be used in field-day for citrus industry to demonstrate delivery strategies and field studies. - Continue website development and population for public presentation of advances.?

Impacts
What was accomplished under these goals? Note: Symbiont technology was referred to by the technical acronym 'PHACT' in our original proposal. Obj 1: 50% Complete Several previously identified NCR peptides active against CLas induced significant decreases in CLas persistence and transmission to psyllid nymphs in detached treated citrus leaves. Screened a panel of native psyllid neuropeptides for their ability to interfere with psyllid fecundity. Obj 1A. Vmax bacterial system was successfully used for production of antimicrobial peptides (AMPs) with scaled-up production of the AMP, Jade. Identified two host plant systems that support rapid symbiont development and will demonstrate their utility as biofactories for biomolecule production. Optimized Symbiont culture methods to produce therapeutic molecules. A continuous production system was developed and run for several months. We are currently working with agricultural engineers on pilot scale up devices. Obj 1B.Developing a model single-domain antibody molecule using a "mock selection" (GFP targeting enhancer and minimizer single-domain antibodies) that will provide a framework for screening enriched Single variable domain on a heavy chain (VHH) libraries from animal immunizations. Obj 1C.IRSC and ARS-USHRL developed an undergraduate screening protocol based on symbiont expression of bacteriocins. Obj 2: 25% Complete Approximately 1,050 healthy, >2 years old citrus plants have been obtained for greenhouse testing and 714 have been psyllid inoculated. More than 8,000 citrus seedling plants were generated seed and will be inoculated. Bioassay was optimized for citrus varieties, control solutions, and tissue sampling locations to reduce variability in the detached leaf assay. Twenty-nine molecules from outside collaborators have been submitted for testing. Initial tests with an oak extract have been performed working with organic chemists to purify active compounds. Obj 3A: 50% Complete Subobj 3Ai. Demonstrated species-specific interactions between Agrobacterium and plant species. Three Agrobacterium (AP2 (rhizogenes), JGT105 (tumefaciens; aka EHA105) and 1416 (tumefaciens)) were tested on tomato, citrus, Arabidopsis and grape. Genes necessary for gall formation were evaluated. Production of a biofactory/cell penetration system was developed to ID the best system for symbiont export. Initiated computational analysis for Agrobacterium genomic sequences that vary in their gall production phenotype Analyzed Mandarin RNAseq data to ID citrus genes to manipulate with Symbiont technology. Used proteomics to survey global protein expression in symbionts A. tumefaciens galls and host plant tissues and confirmed similarity between wild-type A. tumefaciens transformation and symbionts generated from engineered plasmids. Found that effects on gene expression in the host plant outside of the symbiont were minimal, suggesting a low burden on the host. This was explored in a field trial to quantify yield of potatoes bearing wild-type A. tumefaciens symbionts for a growing season. Subobj 3Aii. 79 constructs with candidate movement proteins fused to GFP were generated and 47 have been screened in tomato. Four improved export of GFP outside of the symbiont. Evaluations are on-going in citrus. The remaining 32 constructs are under evaluation.? Six plasmids designed to be small and modular to facilitate manipulation were constructed carrying the RUBY cassette and different iterations of the plant growth regulator (PGR) cassette and tested in tomato, tobacco, and citrus. The varied PGR cassettes were evaluated for effect on symbiont morphology and homogeneity on plants and in tissue culture. We identified versions that are best suited for different applications of this project. Several modes of biomolecule delivery from symbionts to host plants were evaluated: mobile protein fusions, RNA export with viral vectors, and small peptide export via the xylem. Subobj 3Aiii. Symbionts generated using transgenic citrus as the plant donor. This produces symbionts with 100% therapeutic expressing cells without the need for cell culture selection, reducing the time needed before grafting and RITA procedures. Subobj 3Aiv. A two-year evaluation of our Symbiont technology on citrus plants in the field is on-going with native Agrobacterium infections. No negative effects on plant evaluation parameters were observed. Regulatory approval for production of engineered symbionts is on-going. Obj 3B: 60% Complete Continued data and sample collection for current transgenic field trials of therapeutic plantibodies submitted to the testing pipeline. Submitted for reauthorization of the current BRS release permit covering transgenic field trials. Replicated plants for new field trials of Onyx AMP and TMOF constructs that will be initiated in 2023. Received 232 transgenic citrus lines representing 37 additional antimicrobial and resistance gene constructs for in vitro pipeline testing. Extensive testing of AMP expressing transgenics on ACP. Onyx AMP transgenics reduce natural ACP endosymbiont populations, delayed nymphal development and reduced adult eclosing. Whole plant infestations with Topaz have also shown ACP mortality comparable to that seen in DLA. DMR6 transgenics increased defense gene expression and Canker resistance. Lines are now replicated for greenhouse and field trials. Obj 3C: 25% Complete The engineering team investigated scientific literature and commercial products to understand state-of-the-art devices and systems for direct delivery of treatments into trees and plants. Various greenhouse trials have been initiated. Obj 4: 15% Complete We have completed consultations with APHIS Biological Regulatory Service to determine the appropriate permit needs for field trials to test Symbiont delivery. We are now actively working with them to develop a submission packet. Full submission is anticipated in November 2022 and anticipate approval by February 2023. Obj 5. 10% Complete Developed cost analysis of in vitro therapeutic biomolecule production in engineered symbionts comparison and IDed ways to reduce production costs through media modifications. Obj 6: 30% Complete Obj 6A. We provided students at Indian River State College (IRSC) and Cornell University training and research experience in synthetic biology, microbiology, molecular biology and to explore how these disciplines are used in agriculture. Thirteen students (29 total to date) completed the updated BSC4434L lab exploring protein expression in plants. Continuation of the development of a GFP demonstration curriculum: IRSC faculty and staff have worked with scientists to develop curriculum for instructions labs for BSC3465 Biotechnology II. The curriculum changes were implemented in the fall of 2021 in BSC3465L (13 students enrolled) at IRSC. We developed a new two-week lab module for our introduction to biology (BSC2010L: General Biology I). The module will introduce students to agricultural biotechnology and expand their knowledge of degree programs and career options. IRSC students attended four scientific conferences, five scientific poster presentations and several won awards for their presentations. Obj 6B. Our team has presented a talk and two posters about the work done as part of this project at grower meetings. Marco Pitino introduced Symbiont technology at the annual Citrus Show in Fort Pierce, and Guilherme Locatelli presented two posters at horticultural meetings. Project Summary: - Peer-reviewed publications: 6 - Non-peer-reviewed publications: 7 - Presentations: 7 - People reached: 500+

Publications

• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Borovsky, D., Roug�, P. and Shatters Jr, R.G., 2022. The ribosome is the ultimate receptor for trypsin modulating oostatic factor (TMOF). Biomolecules, 12(4), p.577.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Higgins, S. A., Mann, M., and Heck, M. 2022. Strain tracking of Candidatus Liberibacter asiaticus, citrus greening disease pathogen, enabled by high-resolution microbiome analysis of the Asian citrus psyllid. Phytopathology. https://doi.org/10.1094/PHYTO-02-22-0067-R
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Igwe DO, Higgins SA, Heck M. An excised leaf assay to measure acquisition of Candidatus Liberibacter asiaticus by psyllids associated with citrus Huanglongbing disease. 2022. Phytopathology. 112(1):69-75.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Krystel, J., Liu, H., Hartung, J., & Stover, E. 2021. Novel Plantibodies Show Promise to Protect Citrus from Greening Disease,?Journal of the American Society for Horticultural Science,?146(6), 377-386. Retrieved Sep 30, 2022, DOI: 10.21273/JASHS05078-21
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Mann, M., Saha, S., Cicero, J.M., Pitino, M., Moulton, K., Hunter, W.B., Cano, L. M., Mueller, L. A., and Heck, M. 2022. Lessons learned about the biology and genomics of Diaphorina citri infection with Candidatus Liberibacter asiaticus by integrating new and archived organ-specific transcriptome data. GigaScience. https://doi.org/10.1093/gigascience/giac035
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ramsey JS, Ammar ED, Mahoney JE, Rivera K, Johnson R, Igwe DO, Thannhauser TW, MacCoss MJ, Hall DG, Heck M. Host plant adaptation drives changes in Diaphorina citri proteome regulation, proteoform expression, and transmission of Candidatus Liberibacter asiaticus, the citrus greening pathogen. 2022. Phytopathology. 112(1):101-15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Fitzgerald J, Massimino C, et al. Isolation of four Sinorhizobium meliloti bacteriophages from Florida soil. Florida Undergraduate Research Conference (FURC). UCF, Orlando, FL. Feb 18-19, 2022. (Poster)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Heck M, Fleites L. Plant Symbionts: a new method to express therapeutic molecules leveraging the Agrobacterium genetic engineering tool kit. American Phytopathological Society Annual Meeting in Pittsburgh, PA (August 6-10, 2022)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Locatelli G, Shatters RG, Rhodes B, Rossi L, Shrum L, Fleites L, Zagorski P, Heck M, Pitino M. Using a Symbiont Strategy to produce and deliver therapeutic molecules to fight candidatus Liberibacter asiaticus. Florida State Horticultural Society Annual Meeting, Sarasota, FL. June 5-7, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Massimino C, Fitzgerald J, Nielander M, Maybank M, Blossom H, Carroll M, Holland C, & DElia T. Characterization of a novel jumbo phage for potential agricultural applications. First South Florida Translational Research Symposium. FIU, Center for Translational Science, Port St. Lucie, FL. Apr 13-14, 2022 Awarded First Place in Division
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Massimino C, Fitzgerald J, Nielander M, Maybank M, Blossom H, Carroll M, Holland C, & DElia T. A Novel Sinorhizobium meliloti Jumbo Phage: Isolation and Genome Sequencing. Florida Undergraduate Research Conference (FURC). UCF, Orlando, FL. Feb 18-19, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Massimino C, Fitzgerald J, Nielander M, Maybank M, Blossom H, Carroll M, Holland C, & DElia T. Characterization of a novel jumbo phage for potential agricultural applications. 9th Annual Life Sciences South Florida (LSSF) STEM Undergraduate Research Symposium. Virtual. Apr 23, 2022. Awarded Second Place
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Nielander M, Fitzgerald J, Massimino C, Maybank M, Seibenick T, Blossom H, Carroll M, Holland C, D'Elia T. Complete Genome Sequence of a Mu-Like Sinorhizobium meliloti Phage Isolated from Soil. Florida Undergraduate Research Conference (FURC). UCF, Orlando, FL. Feb 18-19, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Fleites L and Sullivan S. Connecting Synthetic Biology with Plant Disease Control. Seminar for Indian River State College students enrolled in Biotechnology 2. September 26, 2022.
• Type: Other Status: Published Year Published: 2021 Citation: Heck M. Modifying plant traits without modifying plant genes: a novel solution for citrus greening disease with the potential to change the future of agriculture. Seminar for PPPMB. September 29, 2021.
• Type: Other Status: Published Year Published: 2022 Citation: Heck M and Shatters RG. New strategy for controlling citrus greening disease: a model for combating AMR concerns in crop disease management. ARS Antimicrobial Resistance Workshop, August 16, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Pitino M. Advances in Therapeutics Delivery in the Battle Against Citrus Greening Disease. Florida Citrus Show, Fort Pierce, FL. January 26-27, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Shatters R, Heck M. Modifying plant traits without modifying plant genes: a novel solution for citrus greening disease with the potential to change the future of agriculture. Plant Breeding Symposium. Virtual. September 14, 2021
• Type: Other Status: Published Year Published: 2021 Citation: Shatters R, Heck M., Pitino M. Modifying plant traits without modifying plant genes: a novel solution for citrus greening disease with the potential to change the future of agriculture. Virtual Seminar to Office of National Programs, Crop Production and protection, Weeds and Invasive Pests. December 17, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Following is the citation: Locatelli, G., Rossi, L., Pitino, M., Zagorski, P., Heck, M. L., & Shatters, R. (2022, July). Symbiont: A Novel System to Deliver Therapeutic Compounds to HLB-Affected Citrus Trees. In 2022 ASHS Annual Conference. ASHS.

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:The target audience for this project can be split into several groups: Other scientists, agencies, citrus industry groups and growers, faculty, undergraduate and graduate students at collaborating universities. Other scientists and agencies include researchers in and adjacent to horticultural sciences, predominantly at USDA; as well as scientists in other agencies, universities and private research institutes. Specifically, our researchers have given multiple presentations and seminars about this project including those listed in the "publication" section of this report. Audiences for these seminars include the USDA Office of National Programs, Staff at the Boyce Thompson Institute, Staff at the USDA-ARS Ithaca Location, a attendees to the USDA AMR Workshop and the Joint-Agency Microbiome Symposium. Citrus industry audience members include growers and related interest groups, mainly from the major growing regions of Florida, California, and Texas. Students and faculty reached by our efforts are mainly at Indian River State College (IRSC), Cornell University and the University of Florida. Students are both undergraduate and graduate level. IRSC serves a community of underrepresented minority students and is designated a Hispanic Serving Institution. At IRSC, the approach has been to include both lower-level students (freshman and sophomores) and upper-level students in the undergraduate research as means to provide peer mentoring and to encourage persistence in the biology program. At Cornell and Univ. Of Florida, students have the opportunity to follow a more traditional approach, working in a research lab, conducting individual research related to the project. Changes/Problems:Throughout the reporting period, 11 of the 18 lead project PIs had COVID-19 restrictions on lab access and hiring that have significantly hindered project progress. Even those labs that didn't have severe restrictions have reported additional hurdles and suboptimal working efficiencies compared to pre-pandemic times. The labs that had the most trouble were the USDA locations in Fort Pierce, Dawson, Albany, and Wapato. Lab access restrictions are ongoing, however almost all open positions have now been filled and are awaiting start dates. What opportunities for training and professional development has the project provided?Initially weekly, and later biweekly grant meetings allowed for project members to learn the broader aims of the project and to understand the methods used in the research. These meetings also provide training in a broad range of topics: synthetic biology, molecular biology, genomics, proteomics, plant breeding, plant pathology, transgenics, plant cell biology, experimental design, commercialization, economics, agriculture engineering, among others. Project members and students also participated in biweekly journal clubs to develop skills in critically reading and assessing scientific publications and practicing presentation skills. The critical assessment skills provide a foundation for their own future scientific writing. Lab technicians, interns and students supporting this project have expanded their skillsets substantially in the areas of proteomics, bioinformatics, text mining, tissue culture, plasmid design, and molecular biology including electroporation of bacteria, PCR, RT-qPCR, protein purification, Western blots, and sequencing analysis. The project team held specific training meetings for IRSC students and staff on the protocols for development of symbionts, protein extraction, SDS-PAGE and transient gene expression in Nicotiana benthamiana. Training was provided by USDA and AgroSource scientists and research assistants. Training was completed through meetings in person in the lab, and supplemented with videoconference meetings as needed to support the process. During the 2020-2021 IRSC faculty began the process to offer the Biotechnician Assistant Credentialing Exam (BACE) at IRSC in order to provide professional development to students. This certification is an industry-recognized exam that was developed at the University of Florida and vetted by BioFlorida, which represents over 3,000 companies. One of the interns from this grant completed the certification, and additional members will be eligible next year. Work related to this grant, including discussion with scientist at the USDA and Agrosource, and attending the grant meetings, were very valuable when designing the study material related to biotech industrial processes and applications of research methods. Project team members and students were able to gain professional development through attendance and presentations at the following professional conferences and meetings: 2021 Florida Phytopathological Society meeting 2021 Florida Academy of Sciences meeting 24th Biennial Evergreen Phage Meeting in 2021 How have the results been disseminated to communities of interest?To engage the scientific community, our researchers have given multiple presentations and seminars about this project including those listed in the "other products" section of this report. Audiences for these seminars include the USDA Office of National Programs, Staff at the Boyce Thompson Institute, Staff at the USDA-ARS Ithaca Location and attendees to the USDA AMR Workshop and the Joint-Agency Microbiome Symposium, both in August of 2021. Outreach efforts for the Citrus Industry and Growers: Our collaborators on the Science for Citrus Health Team have generated a webinar and podcast. This team comprises outreach specialists, postdocs and graduate students at UC Riverside. The webinar was conducted on February 24th, 2021 and was entitled "Emerging technologies to manage Asian citrus psyllid and Huanglongbing". The format was 20 minute talks by expert presenters with a panel discussion at the end. The invited speakers were Dr. Michelle Heck (USDA), Prof. Dr. Bryce Falk (UC Davis), and Prof. Dr. Bryony Bonning (University of Florida). Dr. Heck covered the objectives of the NIFA Delivery Systems CAP Grant and the potential application strategies. The webinar was advertised in Texas and Florida as well as California, where 1.5 CEU (pesticide license) credits were offered. The webinar was well-attended with 163 participants and a 78% response to the required poll questions contained in the presentations. The webinar lasted for 103 minutes and included 16 international participants. Based on poll responses overall, attendees found the webinar to be informative, educational, and relevant to their pest management work. On June 14th, a podcast with Dr. Michelle Heck was released on Apple Podcasts and Spotify. The podcast has had 55 listens and in it, we discussed the podcast as well as Dr. Heck's career trajectory. In this approach, we drew an audience for those curious about the guest and then delved into details about the grant project. Dr Robert Shatters gave a seminar discussing the project to the Indian River Citrus League, a Florida-based citrus grower association. Dr. Shatters gave a presentation "Advances in therapeutics for citrus HLB control: Discovery and Delivery" at the 2021 Florida Citrus Show in Fort Pierce, FL on May 13th, 2021. Our advisory board is made up of industry leaders and scientists from across the US. We engaged the advisory board in our first meeting on January 21, 2021. This was a virtual meeting in which the research team presented a summary of our efforts to the advisory board, our first point of contact with the broader citrus growers' community. Student and Faculty Outreach Efforts: Development of a GFP demonstration curriculum: IRSC faculty and staff have worked with scientists on our team to develop curriculum for instructions labs for BSC3465 Biotechnology II. The methods have been developed to work within the semester timeline and provide training to students in expressing genes (GFP and RUBY) in plants (symbiont and transiently), protein purification, protein quantitation and analysis. IRSC students, faculty met with the AgroSource scientists multiple times for in-lab instruction on implementing their technology. General curriculum has been created to instruct students in the fall of 2021 in BSC3465L (13 students enrolled) at IRSC on using Agrobacterium to introduce and express genes in plants and lab protocols have been made to supplement lab instructions. A total of 5 interns worked through year one on learning the skills for analysis of symbionts on plants and to begin assays to test expression of potential antimicrobial genes in plants and their effect on bacterial growth. This work is to pilot an inquiry-based approach that could be implemented in a teaching lab. Students in the IRSC biotechnology course would screen proteins of interest and report back to the grant partners their findings. This experience would increase student interest and ownership of their project and time in the lab. Students would also learn how to complete research and record notes as if they were working at a biotechnology company. The interns also isolated three bacteriophage from a Sinorhizobim meliloti and sequenced the genomes to screen for potential gene candidates to screen in their research system. Students have presented a poster of their work at the Evergreen Phage conference and prepared a video overview. Students have also participated in journal clubs hosted by members of the grant. During the 2020-2021 IRSC faculty (D'Elia) began the process to offer the Biotechnician Assistant Credentialing Exam (BACE) at IRSC. This certification is an industry-recognized exam that was developed at the University of Florida and vetted by BioFlorida, which represents over 3,000 companies. Review materials were developed for students and an online support system was developed for the first cohort of students to take the exam. One of the interns from this grant completed the certification, and additional members will be eligible next year. Work related to this grant, including discussion with scientist at the USDA and Agrosource, and attending the grant meetings, were very valuable when designing the study material related to biotech industrial processes and applications of research methods. Several Cornell students are currently involved in citrus greening research associated with the project. A rising Cornell freshman worked as a technician performing analysis on the proteins produced in plant tissues. Two graduate students in the Heck group are pursing projects related to citrus greening not directly funded by the NIFA award but informed generally by the science of citrus greening disease discussed at NIFA project meetings. IRSC students attended webinar by Dr. Wayne Hunter from USDA-ARS on RNA interference technology. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Molecule Discovery and Production Complete our screen of transgenic citrus lines in detached leaf assays. Initiate further screens of these transgenic lines (e.g. grafting healthy transgenic material on infected rootstocks and monitoring CLas infection) before making final recommendations for field trials of any transgenic lines. Some candidate genes may be tried in the context of symbiont delivery prior to field trials with transgenics. Test if surface application or infiltration of leaves with aptamers can reduce transmission of CLas to healthy leaves by infected adult psyllids, or if the presence of the aptamer inhibits feeding by either adult psyllids or nymphs in a detached leaf assay format. Test if transient expression of these aptamers in detached leaves or plant cuttings effects CLas transmission or psyllid feeding. Ideas to implement tree delivery of aptamers will be developed. Testing symbionts for protections against fungal or viral infection by plants hosting symbionts and/or treated with symbiont exudates. Optimize scaled production of AMPs in Vmax Support sequencing and characterization of Agrobacterium strains Surface-display technology enablement in Vmax Developing assays to assess surface anchoring of nanobodies Developing constructs for surface display of nanobodies Generating pre-primed libraries of nanobodies against select effectors that are hypothesized to be important for CLas survival and propagation. Determine if genomic background influences effect symbiont forming efficiency. Double and triple stack the individual plant growth regulator genes to study of dosage effects on symbiont formation. Determine which of the eight known Cell Penetrating Peptides (CPP) we have cloned are best suited for protein secretion from plant cells. Objective 2: Bench-to-Field Assay Pipeline We will continue optimization and deployment of the detached leaf assay as an early screening approach for new therapeutic molecules. Initiate testing of outside party provided molecules. Expand field study and start data collection in experimental and commercial groves. Objective 3: Therapeutic Molecule Delivery Objective 3A: Symbiont Delivery. Explore multiple routes to improve peptide delivery from symbionts to tissues throughout the host plant. These approaches will include the use of known movement proteins (proteins that naturally traverse large distance of the plant's vascular system to accumulate in specific tissues), modification of symbiont metabolism to manipulate bulk flow of sap out of the symbiont thus carrying therapeutic molecules to the rest of the plant, expressing peptides using virus sequences that have evolved to efficiently move from cell to cell in the host plant, and the exploration of RNA sequences and structures that encourage RNA export from the symbiont Candidate sequences demonstrating promising increase in protein translocation will be used to generate new constructs with molecules that strongly inhibit CLas multiplication. These will be screened in the greenhouse prior to selection of best sequences to advance to field trial testing. The tissue selected for 3D bioprinting will be combined with different bio-inks to generate a matrix/scaffold to improve transplantation success of symbiont tissue on the host plant. Objective 3B: Transgenic and Genome Editing Move plants that are at different levels of evaluation through out the transgenic pipeline analysis. Different transgenic citrus varieties expressing antimicrobial and insecticidal peptide will be evaluated for gene expression and DLA, selected lines will be propagated in GH by rooting cuts. Already propagated plants are being assessed for HLB tolerance at GH level, by grafting with CLas+ Rough lemon or cage inoculation with CLas+ ACP. RNAi and CRISPR DMR6 downregulated Carrizo grafted into Carrizo WT will be double bud inoculated with Clas+ Rough Lemon and evaluate for HLB tolerance. Extra CRISPR/Cas DMR6 downregulated Carrizo clones will be establish in the field for HLB analysis. dsRNA-DMR6 Hamlin clones will be evaluated for DMR6 gene expression and will be challenged with Xcc using detached leaf inoculation. As proof of concept for Symbiont delivered genome editing, the most current construct version will be augmented with gRNA sequences targeting phytoene desaturase as a visual reporter of editing. This construction will be used to generate Symbionts on in vitro citrus seedling and assess CRISPR-Cas activity in adjacent tissue. Objective 3C: Direct Plant Infusion (DPI) Complete a research and development plan to produce equipment to allow direct plant infusion in commercial citrus groves. Plans and prototypes of infusion devices will be prepared and tested. A Postdoc engineer is being recruited to assist in device design, construction, and testing. Replicated field evaluation of uptake chemistries will be conducted to identify penetrants that best support replicated fluid uptake from the same attached delivery device. Large scale production of AMP in in vitro symbiont system will be conducted and AMP extract will be harvested and tested in plants. Plant systems for in planta production of AMPs in symbionts used as biofactories will continue to be evaluated. Objective 4&5: Data Collection to Support Regulatory Approval & Economic Assessment - Information on current mitigation practices used commercially, their success rates, and cost of implementation will be gathered to support assessment of commercial viability of therapeutic candidates identified in screening. Objective 6: Outreach The project website will be completed and launched IRSC faculty will implement the gene expression and analysis lab using GFP and RUBY in symbionts and transient expression. The curriculum will be adjusted based on students' performance and feedback. Students and faculty at IRSC will continue to work with partners at USDA and AgroSource on the development of the inquiry based lab with goals to implement this late in year two, or early year three. IRSC students will be placed with scientists at USDA and AgroSource for internship opportunities. IRSC students will be provided the opportunity to complete the Biotechnician Assistant Credentialing Exam (BACE)

Impacts
What was accomplished under these goals? NOTE: PHACT has been renamed symbiont since the award has been made and this terminology will be used in all award documentation moving forward. Obj 1: Molecule discovery and production: 25% Complete We investigated novel candidate therapeutics: We analyzed 166 transgenic plants that had been encoded to express four novel peptides. Confirmed that at least 40 of those plants were successfully genetic engineered. We identified seven additional candidate aptamer sequences and fed citrus psyllids an artificial diet containing these aptamers. The results indicate the aptamers may disrupt psyllid feeding and/or transfer of CLas bacteria. We investigated the effects of several peptide candidates on the growth of a culturable bacterial relative of CLas and the effect of these peptides on CLas growing in infected citrus leaves. Obj 1A. Determine the optimum production system for each molecule type We identified and optimized constructs, protocols, strains, conditions, and media to maximize the expression of antimicrobial peptides (AMP) in Vmax. Obj 1B. Can the Vmax system be developed into a nanobody screening and selection platform? We completed the construction and testing of preliminary designs for surface display in Vmax. We demonstrated that nanobody constructs were retained within the cellular fraction and not secreted anymore due to presence of anchoring tags. Obj 1C. Using Symbiont as a direct screen for therapeutics Developed and implemented a training module at IRSC for using Symbiont as a direct AMP screen: Students screened 30 different symbiont expression constructs encoding different putative AMPs. Obj 2: Screening Pipeline: 10% Complete We began optimizing rapid bench scale experiments to test candidate compounds. We extended the use of the detached leaf assay (DLA) to investigate the ability of psyllid nymphs to acquire CLas from infected citrus leaves. Developed a method for end-point grower evaluations and initiated grower field trials at 26 different sites for an external party molecule. We met with citrus growers and scientists and collected therapeutic molecules that can be tested in the greenhouse and in the field. Obj 3 Therapeutic Molecule Delivery Obj 3A: Symbiont Delivery: 20% Complete Subobj 3Ai. Optimize cell activation genes Genes required for symbiont formation were reintroduced to three disarmed agrobacterium strains to help us determine if genomic background influences symbiont forming efficiency. Initial tests in tomato and tobacco began 07/13/21. Performed leadup work to study the effects of genes effects on symbiont forming efficiency and dosage effects. Wild type agrobacterium strains were tested in field grown citrus trees to monitor symbiont formation, longevity, and impact on overall plant health. Four wild type agrobacterium strains were selected and prepared for genome sequencing to identify potential factors beneficial to symbiont development. Subobj 3Aii. Optimizing molecule export and systemic movement in plant. We designed and generated a minimal symbiont vector to allow for easy modification and generation of future constructs. Designed 90+ constructs with sequences fused to fluorescent proteins to visualize expression and transport in planta. Candidate sequences were selected for expression in tomato and used to generate new plasmids with BioXP and GAANTRY cloning systems. We conducted microscopic and proteomic analysis of symbionts expressing fluorescent proteins to learn about the physiology of symbionts and host plant vascular system. Using proteomics we identified and quantified highly expressed proteins secreted by the symbiont. This information helps us understand the symbiont/host plant interaction and design constructs to optimize production and secretion of therapeutic molecules to the host plant. Subobj 3Aiii. Optimizing symbiont cell culture for transplantation Tissue culture techniques have been further optimized to facilitate culturing of symbiont tissue and selection of high expressing cells. A new plasmid was designed and generated using marker that is visible to the naked eye to facilitate in vitro selection of tissue. Tissue has been selected with characteristics that can be adapted to a new transplantation system using 3D bioprinting. Subobj 3Aiv. Can Symbionts function as an HLB control strategy? We have initiated a replicated field trial. Working on field trial design with statisticians from Ithaca. Also hired a post-doc to lead this work. Obj 3B: Transgenic Delivery: 20% Complete Three transgenic citrus varieties expressing AMPs were produced this year. A set of these plants were selected for cloning and HLB greenhouse evaluation. Another set has been prepared for gene expression analysis and DLA. RNAi and CRISPR/Cas DMR6 downregulated Carrizo clones were evaluated for Xanthomonas citri (Xcc) tolerance and the best clones were prepared for further HLB tolerance evaluation. Extra clones were prepared to be transferred to the field. Four RNAi DMR6 Hamlin lines were cloned for future bacteria response analysis. We designed a construct for expressing the CRISPR-Cas machinery and symbiont promoting PGR genes and a protocol was developed to produce symbionts on juvenile tissue from in vitro germinated citrus seedlings. This is a necessary step for the regeneration of whole plants from edited shoots. We identified multiple plantibody molecules for inclusion in the pipeline. Constructs expressing the plantibodies have been stably transformed into citrus. The most advanced of these lines have completed DLA and greenhouse experiments where they showed significant inhibition of CLas growth. Field trials are now underway at the Picos farm location. Obj 3C: Direct Plant Infusion(DPI): 15% Complete Preliminary greenhouse trials are in progress to test therapeutics via DPI to the citrus vascular tissue. We compiled protocols to assess plant physiology in the field, performed initial evaluation of research farm citrus trees and we are finalizing the experimental design. Our engineer collaborators visited the lab in Fort Pierce, FL to be familiarized with citrus grove management practices and equipment to inform DPI equipment development. We developed liquid in vitro symbiont cell culture system and demonstrated purification of transgenic proteins of interest. Obj 4: Regulatory Strategy: 5% Complete We conducted planning meetings to ensure the proper data is collected during Objective 1, 2 and 3 trials to sufficiently initiate privately (non-grant) funded regulatory studies. Obj 5. Economic Assessment: 5% Complete We gathered preliminary data on the US citrus market and status of CLas threat in different citrus producing regions. We finalized agreements with two contract research organizations to assist with data collection. Obj 6: Outreach: 20% Complete Obj 6A. Education We developed an instructional lab for BSC3465 (biotechnology II). Curriculum was developed to accompany the teaching lab experiments and include handouts, protocols, and slides. Students attended journal club meetings hosted through the grant. Students attended webinar by Dr. Wayne Hunter from USDA-ARS on RNA interference technology. Students were trained to evaluate a subset of BioXP generated constructs. IRSC began offering the Biotechnician Assistant Credentialing Exam (BACE) to provide professional development to students involved in the grant. The exam was first offered in summer 2021. Obj 6B: Extension We presented several seminars to the scientific community, our advisory board and the industry as outlined in the "Other products" section of this report. We created a webinar and podcast discussing the project. We built a project website that will be published in coming weeks. We created a twitter account for public engagement. Peer-reviewed publications: 5 Non-peer-reviewed publications: 1 Presentations: 10 People reached: 500+

Publications

• Type: Other Status: Published Year Published: 2021 Citation: Fitzgerald J., Massimino C, Maybank M, Nielander M, Blossom H, Carroll M, Holland C and DElia T. Isolation of four Ensifer meliloti bacteriophages from Florida soil. Poster presentation. 2021. 24th Biennial Evergreen Phage Meeting. Video summary of poster: https://youtu.be/wGnZOYafgUI
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Hosseinzadeh, S., S.A. Higgins, J. Ramsey, K. Howe, M. Griggs, L. Castrillo, and M. Heck, Proteomic Polyphenism in Color Morphotypes of Diaphorina citri, Insect Vector of Citrus Greening Disease. J Proteome Res, 2021. 20(5): p. 2851-2866.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Igwe, D.O., S.A. Higgins, and M. Heck, An excised leaf assay to measure acquisition of "Candidatus Liberibacter asiaticus" by psyllids associated with citrus Huanglongbing disease. Phytopathology, 2021. In press.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ramsey, J.S., E.L. Chin, J.D. Chavez, S. Saha, D. Mischuk, J. Mahoney, J. Mohr, F.M. Robison, E. Mitrovic, Y. Xu, S.R. Strickler, N. Fernandez, X. Zhong, M. Polek, K.E. Godfrey, J.J. Giovannoni, L.A. Mueller, C.M. Slupsky, J.E. Bruce, and M. Heck, Longitudinal Transcriptomic, Proteomic, and Metabolomic Analysis of Citrus limon Response to Graft Inoculation by Candidatus Liberibacter asiaticus. J Proteome Res, 2020. 19(6): p. 2247-2263
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Stuehler, D. S., Hunter, W. B., Carrillo-Tarazona, Y., Espitia, H., Bell, T., Mann, H. R., & Cano, L. M. (2021). Picorna-like Virus Discovered in Wild Lime Psyllid Leuronota fagarae Burckhardt (Hemiptera: Psylloidea). Florida Entomol, 97(4), 1481-1492
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Grando MF, Krystel J, and Stover E. Antioxidants Enhance Agrobacterium Transformation and Shoot Production from Sweet Orange (Valencia) and Grapefruit (Ray Ruby) Epicotyls. American Society of Horticultural Sciences Annual Meeting, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Krystel J, Grando MF and Stover E. Adaptation of a Detached Leaf Assay for Screening Transgenic Citrus for HLB Resistance. American Society of Horticultural Sciences Annual Meeting, 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Shatters RS and Heck M. Modifying plant traits without modifying plant genes: A novel solution for citrus greening disease with the potential to change the future of agriculture. Office of National Programs Seminar July 1, 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Shatters RS and Heck M. Modifying plant traits without modifying plant genes: A novel solution for citrus greening disease with the potential to change the future of agriculture. ARS Research Solutions for AMR Workshop, August 25, 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Shatters RS. Advances in Therapeutics for Citrus HLB Control: Discovery and Delivery. Presentation at the 2021 Florida Citrus Show, May 13th, 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Shatters RS. Update on therapeutics for treating HLB infected citrus. Presentation to the Indian River Citrus League Board, November 11, 2020.