Source: KANSAS STATE UNIV submitted to
DEVELOPING AN INFRASTRUCTURE AND PRODUCT TEST PIPELINE TO DELIVER NOVEL THERAPIES FOR CITRUS GREENING DISEASE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1005600
Grant No.
2015-70016-23028
Project No.
KS603372
Proposal No.
2014-10154
Multistate No.
(N/A)
Program Code
CDRE
Project Start Date
Mar 1, 2015
Project End Date
Feb 28, 2017
Grant Year
2015
Project Director
Brown, S. J.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Biology
Non Technical Summary
U.S. citrus growers have a critical need for grove-deployable management practices that keep healthy citrus from becoming infected and infected trees from becoming symptomatic. We present a systems-based pipeline approach delivering commercial, grove-deployable solutions using a novel therapeutic delivery strategy and citrus transgenics. A data integration and analysis platform combining existing complex -omics/biological data with molecular/cellular research will steer hypothesis-driven testing of inhibitors of multiple molecular pathways to provide solutions that can be optimized by combinatorial delivery to citrus. Research areas will include: 1) molecular interaction inhibitor discovery (gut membrane binding peptides, RNA aptamers and non-toxic chemical library screening) to block psyllid acquisition/ transmission of HLB and/or growth in the plant; 2) dsRNA delivery to induce psyllid RNAi responses that block HLB transmission or kill the psyllid (or both). The proposed delivery system has negligible environmental impact, is economical in comparison to current control strategies and is highly tractable, allowing it to function as a delivery vehicle for different solution strategies. Co-delivery with bactericides (previously shown to be effective against HLB through laborious injection methods) will be evaluated as complementary methods of control. To translate these therapeutic treatments into long-term solutions, transgenic research will be initiated to produce interdiction molecules (peptides and dsRNA) expressed in the phloem. By engaging stakeholders in design and testing stages, and educating the public, we will deliver acceptable solutions that are applicable to citrus greening and extensible to a wide variety of related economically important pathogens of citrus and other specialty crops.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2162499104020%
2163110104070%
2164010104010%
Goals / Objectives
Research:Improved understanding of HLB transmission and interactions in psyllid and plant phloem.Grove deployable solutions and suite of effective management practices identified to keep current citrus production, maintain high fruit quality, and prevent fruit drop.Extension/OutreachIncreased growers/public understanding of biotechnology methods of disease management.Researchers understand consumer attitudes towards biotechnologies.Increased awareness of novel therapeutic application technologies and products by industry segments.Industry understands economic feasibility and practicality of management recommendations.Students increase knowledge of bioinformatics, genome analysis, database construction: New data visualization tools.Understanding of industry/ consumer knowledge about HLB and its impact on U.S. citrus production.
Project Methods
Objective 1: Data integration / AnalysisCombine -omics data into single platform.Manually curate target gene sets in psyllid.Model biochemical pathways.Create visual digital library to access organ or organ system data.Objective 2:HLB Science to SolutionPerform PPI and proteomic studies on gut proteins.Mine interactome and screen dsRNA, RNA aptamer and nontoxic small molecule libraries for blocker molecules.Test candidate molecules in soil application, RNAi and transgenic plant.Test grove-deployable strategies.Conduct metabolomics analysis of treatment efficacy.Evaluate delivery strategies in greenhouse experiments.Objective 3 Engagement, Extension, EducationUtilize industry-relevant Extension and Engagement Methods to introduce emerging agricultural strategies to the citrus industryInvestigate consumer attitudes toward and acceptance of HLB reduction and prevention technologies to inform consumer engagement and outreach

Progress 03/01/16 to 02/28/17

Outputs
Target Audience:Our diverse target audience includes scientists working on plant, insect and bacterial genomics, proteomics and metabolomics. Our integrated analysis platform, designed to facilitate studies of interactions between the plant host, bacterial pathogen and insect vector is of general interest to basic scientists and bioinformaticians. Interested students include undergraduates who are being trained in gene annotation, web portal design, graphics and video production, as well as graduate students and post-doctoral trainees involved in basic and applied research. In addition, outcomes of this project that effect citrus production are of interest to stakeholders in the citrus industry including citrus growers, citrus packers, citrus processors (processing fresh fruit and juices) and consumers of citrus products. Changes/Problems: At the 2nd annual project meeting, new approaches relevant to one of the research goals: Improve understanding of HLB transmission and interactions in plant phloem, were discussed. These experiments will provide detailed visualization of CLas in the psyllid and will require continuing the MicroCT scanning beyond the first two years. As suggested by reviewers of the original project proposal, we are using our pipeline to test the evaluate a protein-based nanoparticle for delivery of dsRNA and RNA aptamers to psyllids and citrus. We have already shown that this new biodelivery system is effective in delivering dsRNA to pyllids in their diet and fluorescent markers to virtually all cell of a citrus leaf. We have added Dr. John Tomich, Director of the Biotechnology center at KSU, who created this biodelivery system, as key personnel to collaborate on experiments in years 3, 4, 5. An additional graduate student at the PIE Center at University of Florida has been added to increase outreach/engagement with industry representatives. What opportunities for training and professional development has the project provided? Danielle Dixon was a summer 2016 intern at Cornell University/Boyce Thompson Institute Three undergraduate students at Indian River State College presented posters for the 2016 Florida Academy of Sciences Dr. Tom D'Elia incorporated molecular analysis of ACP into BSC3465L Introduction to Biotechnology II Lab course at Indian River State College for 23 undergraduate students Bi-weekly video conference annotation lab meetings provided mentoring for 18 undergraduate students by 6 annotation experts, 2 Apollo experts and 3 guest annotators Students involved in research include: graduates (1 at University of California-Davis, 1 at Kansas State University, 1 at Cornell University/Boyce Thompson Institute, 2 at University of Florida-PIE Center); undergraduates (3 at Kansas State University, 9 at Indian River State College, 6 at University of California-Davis, 2 at Cornell University/Boyce Thompson Institute); high school students (2 at ARS USDA US Horticultural Research Lab) Undergraduate students at IRSC worked with a media consultant to produce educational videos explaining citrus greening disease Graphic design student at IRSC created the logo and layout for the project's web portal Computer science student at IRSC implemented the layout to create the web portal Student researchers and project members received relevant feedback at the 2nd Annual Citrus Greening Solutions Meeting from participating members of the Scientific and Stakeholder Advisory Council. 2nd Annual Citrus Greening Solutions Meeting was held at Indian River State College in Ft Pierce, FL. 6 undergraduate students, 3 graduate students, 8 key personnel, 7 co-PIs, 11 project members and 7 Scientific and Stakeholder Advisory Council members attended. Dr. Carolyn Slupsky received the Kinsella Endowed Chair in Food, Nutrition, and Health. Graduate student, Laurynne Chetelat, attended two RNAseq workshops. Several undergraduate, graduate and post-doctoral trainee project participants had the opportunity to present their work at conferences and symposia. These are listed below: *Indicates Undergraduate, Graduate or Post-Doctoral Trainee Project Members in bold font Cilia, M. and *Kruse, A. Citrus Greening Solutions: Cilia Lab Update for Objective Two. 2nd Annual Citrus Greening Solutions Meeting. Ft Pierce, FL *D'Angelo, J., Rumble, J., Lamm, A., Taylor, M., and Ellis, J. A comparative study of GM science diffusion among agricultural undergraduate students. Association for Communication Excellence in Agriculture, Natural Resources, and Life and Human Sciences Conference. Memphis, TN D'Elia, T., *Cordola, C., *Bell, T., *Villalobos-Ayala, K., Hunter, W., and Wiersma-Koch, H. Analysis and characterization of the cathepsin gene family in the ACP, Diaphorina citri (Hemiptera: Liviidae). Florida Academy of Sciences Conference. St. Petersburg, FL. D'Elia, T., *Cordola, C., *Bell, T., *Villalobos-Ayala, K., Hunter, W., and Wiersma-Koch, H. Identification and analyses of Rab genes in the genome of ACP (Hemiptera: Liviidae). Florida Academy of Sciences Conference. St. Petersburg, FL *Dixon, D., *Kruse, A., *Ramsey, J., *Hosmani, P., Saha, S., Ammar, E., Hall, D., Cilia, M., and Mueller, L. Elucidating genetic variation in Candidatus Liberibacter asiaticus transmission between ACP isofemale lines. 16th Annual PGRP Symposium. Ithaca, NY. (Received special recognition) Ellis, J., Rumble, J., *D'Angelo, J., and *Beattie, P. Objective 3: Education and Outreach - Consumer & Industry. 2nd Annual Citrus Greening Solutions Meeting. Ft Pierce, FL *Flores, M., Saha, S., *Hosmani, P., Brown, S., and Mueller, L. Systems biology resources for the Citrusgreening disease complex. International Plant & Animal XXV Conference. San Diego, CA Humann, J., Piaskowski, J., Jung, S., Cheng, C., Lee, T., Frank, M., Scott, K., *Flores, M., Saha, S., Mueller, L., Gmitter, F., Abott, A., and Main, D. Citrus Genome Database (CGD): a resource for genomics, genetics and breeding research. International Plant & Animal Genome XXV Conference. San Diego, CA *Kruse, A., Saha, S., Johnson, R., Fattaha-hosseini, S., Warwick, E., Sturgeon, K., MacCoss, M., Shatters, R., and Cilia, M. Comparative proteomics to identify critical proteins for transmission of Candidatus Liberibacter asiacticus by the ACP. 76th Annual Meeting of the Northeastern Division of The American Phytopathological Society. Ithaca, NY Rumble, J., Ruth, T., Lamm, A., and *Beattie, P. Citrus greening solutions: Extension's role in Florida, California and Texas. American Association for Agricultural Education Southern Region Conference. Mobile, AL Rumble, J., Lamm, A., *Beattie, P., and Bird, B. Communicating about genetic modification: Desired information and trusted sources. Southern Association of Agricultural Scientists Conference, Agricultural Communication Section. Mobile, AL Saha, S., *Flores, M., and Mueller, L. Citruscyc: Metabolic Pathway Databases for the C. Clementina and C. sinensis Genomes. International Plant & Animal Genome XXV Conference. San Diego, CA Saha, S., *Flores, M., *Hosmani, P., and Muller, L. Citrusgreening.org - A systems biology resource for vector biologists. 76th Annual Meeting of the Northeastern Division of The American Phytopathological Society. Ithaca, NY Saha, S., *Ramsey, J., *Kruse, A., and Cilia, M. Exploring the Dynamic Interplay between ACP and CLas using -Omics Technologies. California ACP and Huanglongbing Research and Extension Summit. Riverside, CA Slupsky, C. and *Chetelat, L. Objective 2: HLB Science to Solution - Interactome Studies. 2nd Annual Citrus Greening Solutions Meeting. Ft Pierce, FL *Villalobos-Ayala, K., *Cordola, C., *Bell, T., Wiersma-Koch, H., Hunter, W., and D'Elia, T. Genomic analysis of Diaphorina citri, ACP, reveals numerous classes of heat shock proteins. Florida Academy of Sciences Conference. St. Petersburg, FL How have the results been disseminated to communities of interest? Danielle Dixon's work at BTI was reported in the local newspaper, which helped inform the local readers about the impact of citrusgreening disease and the importance of training undergraduates. http://www.ithacajournal.com/story/news/local/2016/08/10/ithaca-students-learn-advanced-biology-research/88512814/ Webinar series presentation titled Student-focused Collaborative Research was presented by Tom D'Elia to the organization Life Science South Florida Three undergraduate students at IRSC presented posters at the 2016 Florida Academy of Sciences based on their annotation progress and analysis of the psyllid genome. Slupsky, C. was interviewed by "Jim Offner" of Farm Journal Media regarding citrus greening. Slupsky, C. was an invited seminar speaker at Cornell University Slupsky, C. spoke at the California Citrus Board External Review Meeting D'Elia, T. gave an overview of research progress to the board of trustees at IRSC Third year PhD student Annie Kruse submitted a first author paper and contributed to another The outcomes of this project have been disseminated to the greater scientific community through local, national and international conference and symposium presentations listed below: *Indicates Undergraduate, Graduate or Post-Doctoral Trainee Project Members in bold font Alba-Tercedor, J. Micro-CT of the ACP Diaphorina citri Kuwayama, 1908. CGS Project Update Meeting. Webinar Alba-Tercedor, J., and Hunter, W. Micro-CT of the ACP-II. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL Ammar, E., Shugart, H., Rogers, M., Hall, D., and Shatters, R. Methods of correlating electropenetrography waveform data to hemipteran probing behavior and pathogen transmission. International Conference of Entomology. Orlando, FL Borovsky, Dov., Powell, C., Shatters, R., Zaritsky, A., and Dov, E. Bioengineering of TMOF to control mosquitoes and other insects. International Conference of Entomology. Orlando, FL *Chetelat, L. and Slupsky, C. ACP metabolize alters in presence of CLas and depends sex and experimental design. CGS Project Update Meeting. Webinar Cilia, M. Citrus greening: From Science to Solutions. Boyce Thompson Institute Symposium. Ithaca, NY Cilia, M. Molecular and genetic interactions between Diaphorina citri and Candidatus Liberibacter asiaticus. IS-MPMI. Portland, OR Cilia, M. Using 'Omics to dissect the molecular interplay between the ACP and CLas to identify targets for interdiction. CGS Project Update Meeting. Webinar D'Elia, T. Objective 3: Education and Outreach - Student Involvement. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL D'Elia, T. Student-focused Collaborative Research. Life Science South Florida. Webinar Ellis, J., Lamm, A., Rumble, J., *D'Angelo, and Taylor, M. A comparative study of GM science diffusion among agricultural undergraduate students. Association for Communication Excellence in Agriculture, Natural Resources, and Life and Human Sciences. Memphis, TN Gupta, G. HLB Pathogenesis: Disease Management Options. CGS Project Update Meeting. Webinar. Gupta, G. Objective 2: HLB Science to Solution - Blocker System, Disease Management Options. 2nd Annual Citrus Greening Solution Meeting. Ft. Pierce, FL Hunter, W. Advances in RNAi Technology to Manage Pests, Pathogens, Plants. Biological Controls Conference and Expo. Monterrey, CA Hunter, W. Food Security by 2050: Insect Vectors of plant pathogens. 71st Annual North Central Branch, Entomological Society of America Conference. Cleveland, OH Hunter, W. IPM-Biotechnology: Biotechnology Meets Biopesticides in Management of Pests and Pathogens. 71st Annual meeting of the North Central Branch of the ESA. Cleveland, OH Hunter, W. Objective 2: HLB Science to Solution - Blocker System, RNAi. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL Hunter, W. Reducing Hemipteran Vectors and Pathogen Spread, Topically Applied RNAi. American Phytopathology Society. Tampa, FL Hunter, W. RNAi to manage pests and pathogens. 2016 Animal Biotech Summit. Washington, DC Saha, S., and *Flores, M. Objective 1: Data Integration & Analysis: Database. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL Saha, S., and *Hosmani, P. Objective 1: Data Integration & Analysis: Genome annotation. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL Saha, S. Objective 1 Update. CGS Project Update Meeting. Webinar Saha, S. Objective 3: Education & Outreach - Student Involvement. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL Saha, S., Brown, S., and Muller, L. Using Long Reads, Optical Maps and Long-Range Scaffolding to Improve the Diaphorina citri Genome. International Plant & Animal Genome XXV Conference. San Diego, CA Shatters, R., Stover, E. and Morgan, K. Commercial Grove Studies On the Effectiveness Of FireLine™ And FireWall™ On HLB Symptoms In Florida Citrus. Florida Citrus Show. Avon Park, FL Shatters, R. Dealing with Disaster - Stories from the Frontline of the War on Citrus Greening Disease. 2016 48th Annual APRES Meeting. Clearwater Beach, FL Shatters, R., Borovsky, D. and Ammar, E. Objective 2: HLB Science to Solution - Blocker System, Peptide Blockers. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL Shatters, R. Research Advances in Interdiction Molecule Discovery and Delivery for Therapeutic Treatment of Citrus Greening Disease. CGS Project Update Meeting. Webinar Shatters, R., Stover, E. and Morgan, K. USDA/Agricultural Research Service Bactericide Field Trial Results. 2016 Florida Citrus Expo. Ft. Myers, FL Slupsky, C. Using metabolomics to dissect the pathogenicity of C. Liberibacter asiaticus in citrus. The 3rd Annual SECIM Metabolomics Symposium. Gainesville, FL Stover, E. Advances in Identifying & Developing Resistance and Tolerance to Huanglongbing in Citrus. International Citrus Congress. Brazil Stover, E., and Shatters, B. Objective 2: HLB Science to Solution - Delivery Approach. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL Stover, E. Rapid Assay of Huanglongbing Resistance With Conetainer Seedlings Exposed To No-Choice Asian Citrus Psyllid Inoculation. American Society for Horticultural Sciences Annual Meeting. Atlanta, GA Stover, E. Resistance and Tolerance to Citrus Greening Disease AKA Huanglongbing or HLB. Arcadia Citrus School. Arcadia, FL Stover, E. Resistance and Tolerance to Citrus Greening Disease AKA Huanglongbing or HLB. California Citrus Show. Visalia, CA Stover, E. Resistance and Tolerance to Citrus Greening Disease AKA Huanglongbing or HLB. Florida Citrus Growers' Institute. Avon Park, FL Stover, E. Scion Resistance and Tolerance to Citrus Greening Disease AKA Huanglongbing or HLB. Citrus Growers. Immokalee, FL Villalobos-Ayala, K. Objective 1: Data Integration & Analysis - Web Portal. 2nd Annual Citrus Greening Solutions Meeting. Ft. Pierce, FL What do you plan to do during the next reporting period to accomplish the goals?Improve understanding of HLB transmission and interactions in psyllid and plant phloem. Grove deployable solutions and suite of effective management practices identified to keep current citrus production, maintain high fruit quality, and prevent fruit drop. Objective 2 activities planned to meet the above goals. Use proteomic tools developed in the first phase of this grant to identify the gut receptors to which the three gut binding peptides bind and evaluate these as potential bacterial interacting proteins that facilitate bacterial systemic movement in the psyllid Continue research objectives of graduate student Annie Kruse, including: optimization of microscopy methods, RNA aptamer technology development, and understanding the differences in bacterial localization in nymph versus adult insects Collection and analysis of samples for the Asian citrus psyllid expression atlas Complete systems biology characterization of the effect of CLas on male and female ACP and publish the results Contribute proteomics data to systems biology database and information integration platform Study the effect of interdiction molecules on ACP metabolism to understand how they may stop transmission / acquisition of CLas Evaluate CTV paratransgenic as a strategy to deliver peptides that block CLas acquisition and induce psyllid mortality (constructs are currently being developed) Evaluate transgenic citrus plants with phloem specific expression of peptides that block CLas acquisition and induce psyllid mortality (transformant shoots are already produced) Conduct topical application experiments (greenhouse pilot studies and research field applications) to determine if direct peptide delivery to citrus is a viable delivery strategy. Work with industry to evaluate delivery strategies Further improve activity of discovered antimicrobial peptide and small molecule in ex planta testing Evaluate In planta efficacy of nano-capsule delivery systems Evaluate combination therapy using (i) membrane-targeting antimicrobials and conventional antibiotics and (ii) efflux pump inhibitor small molecules and conventional antibiotics. Evaluate efficacy of combination therapies in green house trials in cooperation with a commercial partner, with expectation that the CLas pathogen will be reduced or eliminated from infected citrus trees. Create transgenic citrus expressing therapeutic molecules to provide HLB resistance Initiate new cooperative projects that emerged from the annual project meeting in December 2016. 1. Examine labeling and visualization of CLas in the psyllid using microCT scanning. 2. Feeding dense materials to differentiate alimentary tract. Increased growers/public understanding of biotechnology methods of disease management. Researchers understand consumer attitudes towards biotechnologies. Understanding of industry/consumer knowledge about HLB and its impact on U.S. citrus production. Objective 3 activities planned to meet the above goals. Develop an engagement strategy to keep the scientific and stakeholder advisory board current on grant research progress. Collaborate with citrus extension specialists in Florida to further disseminate progress of this project. Use survey data to develop potential key messages about GM sciences and other Citrus Greening solutions. These messages will be tested with consumers using focus groups in both citrus- and non-citrus-producing states. With the new improved genome and transcriptome (Official gene set), new gene-based targets will be designed and evaluated. New technologies to increase delivery of dsRNA as topical spray and soil treatments, already developed, will be optimized for citrus trees. Evaluate several biotechnologies that permit targeting of microbes to determine if CLas can be reduced in citrus trees or psyllids to disrupt acquisition and transmission. Increased awareness of novel therapeutic application technologies and products by industry segments. Industry understands economic feasibility and practicality of management recommendations. Objective 2 activities planned to meet the above goals. Continue to speak at citrus industry conventions during 2017. Updates on new molecules and delivery strategies and transgenic work will be provided. Field delivery systems, currently being established and evaluated at the USHRL research farm, if successful, will be used in field day demonstrations to growers. Continue stakeholder education through talks and articles Publish a citrograph article describing results of graduate student Annie Kruse To meet goals specific to years 3,4 and 5 we will hire a postdoctoral research associate with expertise in field management to test application strategies. Collaborate in field testing of CTV paratransgenic delivery of dsRNA molecules that could control the ACP. This is being done with the company that owns the commercialization rights this CTV system. Similar field performance will be done with CTV expression of active peptides discovered in this research. Students increase knowledge of bioinformatics and genome analysis. Database construction: New data visualization tools. Objective 1 activities planned to meet the above goals. Continue genome annotation and comparative genomics projects with students. The genes in the Official Gene Set based on the current genome will be ported over to the new genome. Experimental data published by partner labs will be leveraged to target genes of functional importance for manual curation. As new students join the annotation team, they will be trained in standard operating procedures. Student annotators will submit a manuscript to BioRxiv that focuses on the mortality assays. Present work on combined annotation and RNAi lab experience in six student posters at the Florida Academy of Sciences meeting in 2017. Expand undergraduate outreach for additional summer scholars Integrate MCOT database with the latest genome annotations into proteomics workflows Generate haplotype phasing for the ACP genome using the 10X instrument. The new Pacbio genome will be loaded into the web portal database and made available to the community. Generate RNAseq data from CLas-exposed/healthy psyllids from multiple samples for differential expression experiments. Use differential expression data in PEN database to develop atlas of all interactions between genes and metabolites in ACP. Increased breadth of RNAseq data is essential for identifying co-expression networks. Establish meta-DiaphorinaCyc database highlighting co-expression pathways generated from ACP and its endosymbionts.

Impacts
What was accomplished under these goals? Improve understanding of HLB transmission and interactions in psyllid and plant phloem Objective 2 activities highlighted below pertain to the research goal listed above. Completed and published study on the influence of acquisition period on acquisition, replication and inoculation of CLas Analyzed ACP gut transcriptome and proteome using Illumina RNAseq and high resolution mass spectrometry Identified ACP gut molecular targets for conventional RNA aptamer development Enriched for RNA aptamers with affinity to ACP stylets Developed electron and confocal microscopy methods to visualize and distinguish among different bacteria in ACP Identified 12 protein interactions between ACP and CLas as potential interdiction targets Performed proteomics and titer analysis of psyllid samples, CLas+ and CLas-, males and females Developed method for sequential isolation of metabolites, RNA, DNA and protein from the same unique ACP sample Performed metabolomic and transcriptomic anlaysis on adult female and male ACP reared on CLas+ or CLas- citrus. Identified changes in the metabolome and transcriptome in response to CLas infection (diaphorin - an important toxic chemical produced by an ACP endosymbiont - is upregulated in response to infection) Grove deployable solutions and suite of effective management practices identified to keep current citrus production, maintain high fruit quality, and prevent fruit drop. Objective 2 activities highlighted below pertain to the research goal listed above. Identified peptide combinations that when delivered to psyllids in planta, induced 95% mortality and 100% inhibition of pathogen acquisition by surviving psyllids Demonstrated effectiveness Citrus tristeza virus as a paratransgenic delivery system for dsRNA that induces mortality in developing psyllid nymphs Aphis permit awarded for field testing CTV-dsRNA in citrus and experimental setup underway Cellular uptake of interdiction peptides demonstrated in seedling-based assay developed as platform for root delivery Identified small molecules and peptides with acceptable MICs against surrogate bacteria Provided identified 2 peptides that disrupt alpha-proteobacteria membranes for CLas analysis in planta Gene-based targeting products, microsugars, and phytochemicals evaluated in field trees for decreasing fruit drop, and/or decreasing CLas titers in infected citrus trees. Projects underway will run another 5-12 months Excellent phloem-specific expression demonstrated in citrus when a target gene is expressed using a SCAmpP promoter and associated sequences Promoter studies identified regions in conserved intron and 5' region necessary for high levels of phloem specific expression Plasmid constructs comparing SCAmpP and ubiquitin promoters expressing short peptides that induce nymphal psyllid mortality and kill CLas were constructed and over 1000 putative transformed shoots have been generated Increased growers/public understanding of biotechnology methods of disease management. Researchers understand consumer attitudes towards biotechnologies. Understanding of industry/consumer knowledge about HLB and its impact on U.S. citrus production. Objective 3 activities highlighted below pertain to the research goals listed above. Scientific and Stakeholder Advisory Board Survey conducted to understand how they're communicating about HLB; what they see as challenges to implementing solutions in citrus industry Quantifying various strategies grant collaborators are using to communicate about their research, disseminate research outcomes to build strategy for keeping stakeholders informed of progress Completed citrus-state and national surveys to understand consumer acceptance of GM sciences as a technology Several news-type internet reports have emerged this year which may help increase understanding of the specificity of gene-based targeting technologies Increased awareness of novel therapeutic application technologies and products by industry segments. Industry understands economic feasibility and practicality of management recommendations. Objective 2 activities highlighted below pertain to the research goals listed above. Therapeutic delivery strategies were presented to growers at 4 industry directed meetings/workshops, 2 scientific research meetings, and at the Fort Pierce Indian River Lagoon Science Festival where general public attendance was estimated at over 8,000 HLB resistant transgenic citrus were described at 2 national/international scientific meetings and 5 grower meetings/workshops in FL and CA Under confidentiality agreement, delivery strategies were demonstrated to citrus growers who provided feedback optimize system for grower acceptance CRADA agreement developed with agricultural company to evaluate commercialization of these techniques. Regulatory hurdles and required supporting data were identified Students increase knowledge of bioinformatics and genome analysis. Objective 1 activities highlighted below pertain to the research goal listed above. 18 undergraduate students, 6 annotation experts, 2 Apollo experts and 3 guest annotators used Basecamp to manage documents and met biweekly via videoconferencing, which provided timely feedback to student curators Using WebApollo, 322 genes from 21 families were manually curated by student annotators at IRSC, Cornell/BTI, UC and KSU as well as community experts, to develop the Official Gene Set 7 students at IRSC developed RNA interference mortality assays in the lab. Students analyzed gene models, to design PCR primers matching to their target sequence. Students improved bioinformatics skills and connected these skills to lab-based applications Database construction: New data visualization tools. Objective 1 activities highlighted below pertain to the research goal listed above. An MCOT gene set was generated that encodes 30,562 functional annotated proteins. MCOT gene models improve on those misassembled or missing in the genome A new genome assembly based on PacBio, 10x and Dovetail scaffolding, and BNG optical maps is in progress RNA from CLas-exposed/healthy adults and nymphs was sequenced A visualization platform for modeling transcriptomics, proteomics and metabolomics data has been designed to discover novel associations CitrusgreeningCyc which includes pathway databases for D citri, CLas psy62, CLas gxpsy and others was created and has been updated in collaboration with the Gmitter and Ware labs MicroCT scans of ACP, linked with genomic and other data, will improve understanding of ACP and CLas acquisition, movement, and transmission into and from citrus trees

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Arp A, Hunter W, Pelz-Stelinski K, Annotation of the Asian citrus psyllid genome reveals a reduced innate immune system, Frontiers in Physiology, 7:1-18, 2016.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Ammar E, Ramos J, Hall D, Dawson W, Shatters R, Acquisition, replication and inoculation of Candidatus Liberibacter asiaticus following various acquisition periods on huanglongbing-infected citrus by nymphs and adults of the Asian citrus psyllid, PloS one 11(7):e0159594, 2016.
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: International Psyllid Sequencing and Annotation Consortium, Saha S, Hosmani P, Villalobos Ayala K, Miller S, Shippy T, Rosendale A, Flores M, Hunter W, Cilia M, Mueller L, Munoz-Torres M, Benoit J, D'Elia T, Brown S, Biocuration as a student training experience: Deciphering the draft genome of Asian Citrus Psyllid one gene at a time, Database, 2016.
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Kruse A, Fattaha-hosseini S, Saha S, Johnson R, Warwick E, Sturgeon K, Mueller L, MacCoss M, Shatters R, Cilia M, Combining omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut, Molecular and Cellular Proteomics, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Yang C, Powell C, Duan Y, Shatters R, Fang J, Zhang M, Deciphering the Bacterial Microbiome in Huanglongbing-Affected Citrus Treated with Thermotherapy and Sulfonamide Antibiotics, PloS one 11(7):e0155472, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Slupsky C, Grafton-Cardwell B, Irey M, Bartels D, McRoberts N, Immediate action is needed: Summary of the HLB summit morning session, Citrograph 7:24, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Taning C, Andrade E, Hunter W, Christiaens O, Smagghe G, Asian Citrus Psyllid RNAi Pathway - RNAi evidence, Scientific Reports, 6:38082, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Alba-Tercedor J, Microtomographic study on the anatomy of adult male eyes of two mayfly species, Zoosymposia 11:101-120, 2016
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Alba-Tercedor J, Sainz-Bariain M, Poquet J, Rodriguez-Lopez R, Predicting River Macroinvertebrate Communities Distributional Shifts Under Future Global Change Scenarios in the Spanish Mediterranean Area, PloS one, 2016.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Rumble J, Ruth T, Owens C, Lamm A, Taylor M, Ellis J, Saving citrus: Does the next generation see GM science as a solution?, Journal of Agricultural Education, 2016.
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Avila L, Chandrasekar R, Brown S, Tomich J, Reeck G, Delivery of Lethal dsRNAs in Insect Diets by Branched Amphiphilic Peptide Capsules, Nature Methods, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Chetelat L, Chin E, Mishchuk D, Slupsky C, California Citrus Threats, Citrograph, 7(2):34, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ben-Mahmoud S, Ramos J, Shatters R, Hall D, Lapointe S, Niedz R, Rouge P, Cave R, Borovsky D, Expression of Bacillus thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae, Pesticide Biochemistry and Physiology e0155472, 2016.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Alba-Tercedor J, Bartomeu I. 2016. Micro-CT as a tool straddling scientist research, art and education. Study of Osmia sp., a mason bee (Insecta, Hymenoptera: Megachilidae). In proceedings: Bruket Micro-CT Users Meeting 2016. p 74-9. Ed. Bruker MicroCT, Belgium (ISBN: 9789081678100).
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Andrade E, Hunter W. 2016. RNA Interference - Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC). In proceedings: RNA Interference. p 391-409. Ed.Ibrokhim Y. Abdurakhmonov (ISBN 978-953-51-2272-2).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Ruth, T., Lamm, A., and Rumble, J. Extension's Role in Saving Citrus: Entering the Genetic Modification Science Conversation. American Association for Agricultural Education Southern Region Conference. Mobile, AL.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Rumble, J., Ruth, T., and Lamm, A. Identifying Publics in Florida, California, and Texas as they Relate to Citrus Greening. Southern Association of Agricultural Scientists Annual Convention - Agricultural Communications Section. Mobile, AL.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Rumble, J., Ruth, T., and Lamm, A. The Sound of Silence: Exploring why Supporters of Genetic Modification do not Expose their Attitudes. Southern Association of Agricultural Scientists Annual Convention - Agricultural Communications Section. Mobile, AL.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Rumble, J., Lamm, A., Beattie, P., and Bird, B. Communicating about genetic modification: Desired information and trusted sources. Southern Association of Agricultural Scientists Conference, Agricultural Communication Section. Mobile, AL.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Rumble, J., Ruth, T., Lamm, A., and *Beattie, P. Citrus greening solutions: Extensions role in Florida, California and Texas. American Association for Agricultural Education Southern Region Conference. Mobile, AL.
  • Type: Other Status: Published Year Published: 2016 Citation: Alba-Tercedor J, Brown S. Insect scans could help control citrus killing bug. Reuters Tech Videos. 2016
  • Type: Websites Status: Published Year Published: 2016 Citation: Saha S, Flores M, Fernandez-Pozo N, Mueller L. CitrusgreeningCyc. 2016. http://ptools.citrusgreening.org/
  • Type: Websites Status: Published Year Published: 2016 Citation: Saha S, Flores M, Fernandez-Pozo N, Mueller L. DiaphorinaCyc. 2016. http://ptools.citrusgreening.org/


Progress 03/01/15 to 02/29/16

Outputs
Target Audience:The target audience for our project is diverse. Scientists interested in the outcomes of our project include those working on plant, insect and bacterial genomics, proteomics and metabolomics. Interested students include undergraduates who are being trained in gene annotation, web portal design, graphics and video production, as well as graduate students and post-docs involved in basic and applied research. In addition, outcomes of this project that effect citrus production are of interest to stakeholders in the citrus industry including citrus growers, citrus packers , citrus processors (processing fresh fruit and juices) and consumers of citrus products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? What opportunities for training and professional development has the project provided? Undergraduate student Allison Izsak from the Cilia lab was awarded the prestigious American Phytopathological Society (APS) Frank L. Howard Undergraduate Fellowship Award for 2016 for her project "Gene discovery, annotation and orthology in the Asian citrus psyllid genome"under the mentorship of Angela Kruse and Surya Saha. Several project participants have had the opportunity to present their work at conferences and symposia. These are listed below: *Indicates Undergraduate, Graduate or Post-Doctoral Trainee Project Members in bold font *Bell, T., *Cordola, C., *Villalobos-Ayala, K., Wiersma-Koch, H., Hunter, W., and D'Elia, T. Analysis and characterization of the cathepsin gene family in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). Florida Academy of Sciences Conference. St. Petersburg, FL. *Cordola, C., *Villalobos-Ayala, K., *Bell, T., Wiersma-Koch, H., Hunter, W., and D'Elia, T. Identification and analyses of Rab genes in the genome of Asian citrus psyllid (Hemiptera: Liviidae). Florida Academy of Sciences Conference. St. Petersburg, FL. *Izsak, A., Kruse, A., Saha, S., Cilia, M., and Mueller, L. Gene discovery, annotation and orthology in the Asian citrus psyllid genome. Plant Genome Research Program Symposium. Ithaca, NY. *Kruse, A., Ozer, A., Johnson, R., Ghanim, M., Lis, J., Shatters, R., MacCoss, M., and Cilia, M. Development of interdiction molecules that block Asian citrus psyllid transmission of Candidatus Liberibacter asiaticus. American Society for Cell Biology. San Diego, CA. *Kruse, A., Ozer, A., Sturgeon, K., Warwick, E., Lis, J., Shatters, R., and Cilia, M. Development of RNA Aptamers that block CLas transmission by the Asian citrus psyllid. International Arthropod Genomics Symposium. Manhattan, KS. Rumble, J., *Ruth, T., *Owens, C., Lamm, A., Taylor, M., and Ellis, J. Undergraduate Student Thoughts on Using the Science of Genetic Modification as a Solution to Citrus Greening. American Association for Agricultural Education - Southern Region Meeting, San Antonio, TX. *Villalobos-Ayala, K., *Cordola, C., *Bell, T., Wiersma-Koch, H., Hunter, W., and D'Elia, T. Genomic analysis of Diaphorina citri, Asian citrus psyllid, reveals numerous classes of heat shock proteins. Florida Academy of Sciences Conference, St. Petersburg, FL. Project Update Meeting presentations: video conference presentations to all participants of this project. *Chetelat, L., Slupsky, C., Mishchuk, D., Chin, E, and Cilia, M. Metabolomic Analysis of Citrus Greening. *D'Angelo, J., and Ellis, J. Objective 3 - Engagement & Outreach. *Kruse, A., Ozer, A., Sturgeon, K., Warwick, E., Lis, J., Shatters, R., and Cilia, M. Development of RNA Aptamers that block CLas transmission by the Asian citrus psyllid. Students at IRSC listed below, with advise from KSU graduate student Jeremy D'Angelo I the Dept of Agricultural Communication, are developing videos to inform the public about this project. *Villalobos-Ayala, K., *Cordola C, *Bell T, Wiersma-Koch H, * *Castillo, W., *Richardson, J., *Mann, H., and D'Elia, T. Citrus Greening Video: Participant Perspectivies, Ft. Pierce, FL. Students listed below, participated in a Genome Train workshop and a gene annotation café at first annual meeting/International Arthropod Genomics Symposium at Kansas State University in June. IRSC Faculty (Tom D'Elia) and students (Christopher Cordola, Tracey Bell, Krystal Villalobos Ayala) attended the symposium and received annotation training from WebApollo experts Monica Munoz-Torres and Monica Poelchau. Gene annotation video conferences met biweekly over the zoom.us conference system. Undergraduate Students: Tracey Bell Gabriela Chavez Chris Cordola David Hunter Allison Izsak Hannah Mann Taylar Morgan Krystal Villalobos-Ayala Gene Annotation Experts: Sue Brown Surya Saha Prashant Hosmani Web Apollo Experts: Monica Munoz-Torres Monica Poelchau Guest Gene Annotators: Josh Benoit David Nelson How have the results been disseminated to communities of interest? How have the results been disseminated to communities of interest? Data integration at citrusgreening.org: We are collecting data from transcriptomic, genomic, proteomic and metabolomics studies on psyllids in an integration and analysis platform. Having all the data in one place in compatible formats will significantly improve the discovery to application efforts of the entire project team. In addition, we are developing matched transcriptome/proteome datasets from the Asian citrus psyllid digestive tract and comparison of 'Candidatus' Liberibacter asiaticus (CLas) infected psyllids and uninfected psyllids. We have developed matched colonies of the Asian citrus psyllid feeding on citron (Citrus medica) that are either healthy or CLas infected. We chose citron because of its ability to continually flush even when infected with CLas. Once established, the digestive tracts were extracted from 2,000 adults of each colony. One-thousand of each were provided to the laboratory of Dr. Michelle Cilia for proteomic profiling and the other 1,000 will be used in my laboratory for RNAseq analysis. This information will be used to create a matched transcriptome/proteome profile of the adult insect digestive tract and provided to the research community through our web interface. Several gene families (llsted below) in the Asian Citrus Psyllid genome have been annotated and these data included in the Integration and analysis platform: CATs: Catalases CASPAs: Caspase Activators BGBPs: 1,3-beta-D Glucan Binding Proteins TEPs: Thio-Ester Containing Proteins SRRPs: Small Regulatory RNA Pathway Members APHAGs: Autophagy Genes PGRPs: Peptidoglycan Recognition Proteins CASPs: Caspases AMP: Anti-microbial peptides IMD Pathway SRPNs: Serine Protease Inhibitors SODs: Superoxide Dismutases LYSs: Lysozymes Toll Pathway and Receptor JAK/STAT pathway: Signal transduction This information will be added to the genome browsers at the citrusgreening.org. These outcomes of this project have been disseminated to the greater scientific community through local, national and international conference and symposium presentations listed below: Alba-Tercedor, J., Sáinz-Bariáin, M., and Zamora-Muñoz, C. Using micro-CT to elucidate the pupal case architecture as a survival strategy of a caddisfly. Bruker Micro-CT Users Meeting. Belgium. Cilia, M. Mechanisms of Microbial Host Cell Manipulation: From Plants to Humans. Germany. Cilia, M. Unraveling interactions among insect vector borne plant pathogens, plants, insect vector and insect vector bacterial symbionts using mass spectrometry. Cornell Chemistry Biology Interface Symposium. Ithaca, NY. Hunter, W., Pearson, B. and Andrade, E. Environmental Fate of dsRNA in Treated Citrus Groves. Florida Academy of Science Annual Meeting. St. Petersburg, FL. Kaur, N. and Hunter, W. RNAi Bioassay Problems and Solutions. International Plant & Animal Genome Conference. San Diego, CA. Pardington, P., Chaudhary, A., Norvell, M., Dandekar, A., Gouran, H., Aguero, C., Uratsu, S., Feldstein, P., Bruening, G., Civerolo, E., Stover, E., Hao, G., Duan, Y.P., and Gupta, G. Pathogen Clearance by Engineering of a Novel Innate Immune Defense. American Association of Immunologists Meeting. New Orleans, LA. Slupsky, C. Early Detection Technologies (EDT) for HLB. HLB Early Detection Technology Summit. Visalia, CA. Slupsky, C. and Davis, C. Metabolomics-based approaches to plant diagnostics. California Asian Citrus Psyllid and HLB Research and Extension Summit. Davis, CA. The outcomes of this project have been disseminated to the greater scientific audience through several journal articles, listed below: Ramsey, J. S., Johnson, R., Hoki, J. S., *Kruse, A., Mahoney, J. M., Hilf, M.E., Hunter, W. B., Hall, D. G., Schroeder, F. C., MacCoss, M.J., and Cilia, M. Metabolic Interplay between the Asian citrus psyllid and its Profftella symbiont: An Achilles' heel of the citrus greening insect vector. PLoS One. 10:e0140826. 2015. Ammar, E.D., Hall, D.G. and Shatters Jr, R.G. Ultrastructure of the salivary glands, alimentary canal and bacteria-like organisms in the Asian citrus psyllid, vector of citrus huanglongbing-disease bacteria. Journal of Microscopy and Ultrastructure. 2016. Ammar, E.D., Hall, D.G. and Shatters Jr, R.G. Ultrastructure and development of the new stylets inside pre-molting first instar nymphs of the Asian citrus psyllid. Florida Entomologist. 98:373. 2015. Van Ekert, E., Shatters, R.G., Rougé, P., Powell, C.A., Smagghe, G., and Borovsky, D. Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama). FEBS open bio. 5:264. 2015 Yang, C., Powell, C.A., Duan, Y., Shatters, R. and Zhang, M. Nanoemulsion Formulation with Improved Penetration of Foliar Spray through Citrus Leaf Cuticles to Control Citrus Huanglongbing. PLoS One. 10:e0133826. 2015. *Chetelat, L., Chin, E., Mishchuk, D., and Slupsky, C. (Accepted) California Citrus Threats. Citrograph. Rumble, J. N., *Ruth, T.K., *Owens, C.T., Lamm, A.J., Taylor, M.R., and Ellis, J.D. Undergraduate student thoughts on using the science of genetic modification as a solution to Citrus Greening. 2016. What do you plan to do during the next reporting period to accomplish the goals? What do you plan to do during the next reporting period to accomplish the goals? As described in the timeline for year 2, we will move forward on several aspects of all three objectives. Some considerations are highlighted below. Understanding the biology of psyllid/CLas interaction: RNA sequencing and analysis of samples from infected and uninfected psyllid gut tissue. Samples were extracted at Shatters lab. The libraries were prepared at BTI and submitted for sequencing at Cornell. We have collected and processed thousands of Asian Citrus Psyllids for which we will determine unique and shared physiological changes, especially in metabolism, among female versus male ACP in response to infection by CLas. ACP pathways differentially regulated in the presence of CLas will be assessed for importance in CLas transmission and use in development of intervention measures We are in the process of building pathway database for each genome in the research database. This will enable biologists to explore the effect of variation in expression of genes involved in different pathways. The research database will be further expanded with a user-friendly tool to view tissue specific gene expression in the psyllid and citrus. The fragmentation and potential mis-assemblies in the current version of the psyllid genome has been an obstacle in the manual curation effort. To improve the psyllid genome assembly for annotation, additional sequencing of the psyllid genome using Pacbio long read technology will be integrated with next generation physical maps.to create a genome assembly with higher contiguity and lower errors. The genome will be assembled on high performance servers at the Mueller lab. Intervention molecule discovery and testing in the pipeline: We will continue our evaluation of four peptides that influence psyllid/CLas interactions and insect viability. To validate previous findings and to identify the gut membrane proteins to which these peptides aptamers bind. dsRNAs shown to increase psyllid mortality when administered orally, will be tested to determine if systemic delivery can induce effects on psyllid biology when they feed on treated citrus. A rapid screening assay for bactericides effective against CLas has been used to identify one GRAS molecule that shows activity against CLas, reducing its titer in infected plant tissues. We have also identified a peptide with similar activity in infected citrus. Further experiments are underway to validate our initial findings and to test on intact citrus plants using various delivery strategies. We will express and functionally test therapeutic proteins that have been transformed into Tobacco BY-2. We will continue designing virus-like nanoparticles for the delivery of the combination therapy (bactericide and efflux pump inhibitor). Extension, training outreach: Wayne Hunter will teach two courses (Health IT and Data management and practice) in Spring and Fall 2016 at USF for both undergraduate and graduate students in Informatics major. Some of the in-class students will be assigned Psyllid genes for annotation practice. Research data management and practice will also be discussed. The conceptual framework developed in year one to inform the consumer surveys will be carried forward to focus groups. IRSC students will interview scientists and growers and produce videos for the web portal.

Impacts
What was accomplished under these goals? Improved understanding of HLB transmission and interactions in psyllid and plant phloem. Project activities in Objective 2 that are highlighted below pertain to the research goals listed above. Understanding psyllid/CLas interactions to discover intervention methods: A comparative gut proteome has been completed to examine impacts of CLas on the insect gut at the protein level. Proteome insights prompted the development of new hypotheses on the interactions between CLas and the insect vector, including the involvement of the insect's microbiota. Protein interactions between CLas and the ACP have been identified. These interacting partners are also serving as RNA aptamer targets. RNA aptamer work is underway. Enriched libraries have been created that bind to psyllid guts and stylet sheath. Library sequencing is ongoing. Constructs have been designed for generating aptamers against specific gut targets and CLas proteins expressed in the gut. Omics Integration for Vector-Pathogen Interactions: We are taking a system-biology approach to understanding ACP-CLas interactions and have adapted a method for sequential isolation of high quality polar and nonpolar metabolites, DNA, RNA, and proteins from the same unique ACP samples. We have collected and processed thousands of ACPs for which we will determine unique and shared physiological changes, especially in metabolism, among female versus male ACP in response to infection by CLas. ACP pathways differentially regulated in the presence of CLas will be assessed for importance in CLas transmission and use in development of intervention measures. A total of 182 genes in the psyllid genome have been manually curated, these genes are potential targets for RNAi approaches. Development and use of bioassays to test potential intervention approaches in the laboratory and greenhouse. Using ACP acquisition/transmission assays, preliminary evidence shows that a combination of three peptides block movement of the CLas bacterium from the gut to the salivary glands. These results are based on three replicated trials, and further evaluation is underway. These combinations of peptide aptamers also induced psyllid mortality. dsRNAs shown to increase psyllid mortality when administered orally, are now being tested to determine if systemic delivery can induce effects on psyllid biology when they feed on treated citrus. A rapid screening assay for bactericides effective against CLas has been used to identify one GRAS molecule that shows activity against CLas, reducing its titer in infected plant tissues. We have also identified a peptide with similar activity in infected citrus. Further experiments are underway to validate our initial findings and to test on intact citrus plants using various delivery strategies. Grove deployable solutions and suite of effective management practices identified to keep current citrus production, maintain high fruit quality, and prevent fruit drop. Project activities in objective 2 that are highlighted below pertain to the research goals listed above. Development of novel delivery strategies and treating HLB in plants: We have developed a novel delivery strategy for delivery of bioactive molecules to citrus in commercial groves. In laboratory and greenhouse studies dsRNAs were successfully introduced in this manner and moved systemically in the plant. We are currently evaluating peptide and other molecule uptake using this method. For HLB protection, we are developing a combination therapy. This strategy involves the use of two different antibacterial agents that act synergically to direct rapid Liberibacter clearance. Initial efforts focus on discovering efflux pump inhibitors (EPI) that will inhibit the extrusion of antibacterial agents from the cell. This will increase their internal concentration leading to increased bacterial clearance. A library of 300 potential EPIs is being screened in vitro using Liberibacter surrogates Agrobacterium tumefaciens and Sinorhizobium meliottii. We have cloned genes encoding therapeutic proteins designed to clear bacteria. Expression studies in E. coli Shuffle B strain, produced low protein yields and displayed low antimicrobial activity. To test these proteins in Tobacco BY-2 cells, these protein chimeras have been cloned in pEAQ-HT vector and transformed into Agrobacterium tumaficiens. Once they are transformed into Tobacco BY-2 we will test protein expression, purify them and perform functional studies. We are also making lipid nano capsules to encapsulate proteins to deliver therapeutic proteins into infected citrus trees. Green fluorescent protein (GFP) is being used as a test protein to characterize the encapsulation rate. Efforts to design liposomes have focused on the inner liposome shell. A phloem-specific CTV coat protein will constitute the outer shell. A combination of drugs will be encapsulated in these nanoparticles for phloem-specific delivery. We have also started our work on the design of virus-like nanoparticles for the delivery of the combination therapy. Extension/Outreach Increased growers/public understanding of biotechnology methods of disease management. Researchers understand consumer attitudes towards biotechnologies. Understanding of industry/ consumer knowledge about HLB and its impact on U.S. citrus production. Project activities in objective 3 that are highlighted below pertain to the research goals listed above. We have developed a conceptual framework grounded in Diffusion of Innovations and Spiral of Silence as well as Elaboration Likelihood Model. This framework informed the development of the first consumer survey that was designed to gain an understanding of their awareness of citrus greening and their acceptance of technologies used to combat citrus greening. The survey was pilot tested with undergraduate students at UF and KSU, before distribution to 1500 people nationally and another 1500 people in FL, CA, and TX. We are conducting a content analysis to understand how the media is presenting GM science to the general public. We are analyzing the top three circulating newspapers in the top three citrus producing states FL, CA, and TX, in addition to the top three national circulating newspapers. We have completed the code book for content analysis related to GM science and Genetic Technologies. Started gathering media artifacts for analysis. Increased awareness of novel therapeutic application technologies and products by industry segments. Industry understands economic feasibility and practicality of management recommendations. On the project timeline, we have dedicated activities in years 3-5 to these research goals. To keep the growers and industry stakeholder aware of our activities leading up to these goals, members of the Scientific and Stakeholder Advisory Council participated in our first annual project meeting at KSU in June. Sue Brown, the PD presented an overview of the project to the Citrus Disease Subcommittee at their annual meeting in February. Students increase knowledge of bioinformatics, genome analysis, database construction: New data visualization tools. Project activities in objectives 1 and 3 that are highlighted below pertain to the research goals listed above. Creation of the citrusgreening.org website (http://citrusgreening.org). The site includes a BLAST server and genome browser (JBrowse) for each type of organism (pathogen, vector, and host). Citrusgreening.org also hosts a Bioinformatics Data Integration and Analysis Platform. Data types being gathered here include genomic, proteomic and metabolic datasets. Using the WebApollo annotation tool, 182 ACP genes were manually curated by student annotators at Cornell, KSU and IRSC trained as a part of this project. IRCS students designed a Web Portal containing information for consumers, growers and scientists. In preparation for its launch, test and production sites for the Joomla-based blog were created.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ammar ED, Hall DG, Shatters Jr RG, Ultrastructure and development of the new stylets inside pre-molting first instar nymphs of the Asian citrus psyllid, Florida Entomologist 98:373, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Van Ekert E, Shatters RG, Roug� P, Powell CA, Smagghe G, Borovsky D, Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama), FEBS open bio 5:264, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Yang, C., Powell, C.A., Duan, Y., Shatters, R. and Zhang, M, Nanoemulsion Formulation with Improved Penetration of Foliar Spray through Citrus Leaf Cuticles to Control Citrus Huanglongbing, PloS one 10:e0133826, 2015.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Chetelat, L., E. Chin, D. Mishchuk, and C. Slupsky, California Citrus Threats, Citrograph, 2016.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ramsey, J. S., Johnson, R., Hoki, J. S., Kruse, A., Mahoney, J. M., Hilf, M.E., Hunter, W. B., Hall, D. G., Schroeder, F. C., M. J. MacCoss, and M. Cilia, Metabolic Interplay between the Asian citrus psyllid and its Profftella symbiont: An Achilles heel of the citrus greening insect vector, PLoS One 10:e0140826, 2015.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ammar, E.D., Hall, D.G. and Shatters Jr, R.G., Ultrastructure of the salivary glands, alimentary canal and bacteria-like organisms in the Asian citrus psyllid, vector of citrus huanglongbing-disease bacteria, Journal of Microscopy and Ultrastructure, 2016.