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
EXTENDED
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 29, 2020
Grant Year
2017
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
0%
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 03/01/18 to 02/28/19

Outputs
Target Audience:The goals of Objective 1 are: Students increase knowledge of bioinformatics and genome analysis. Database construction: New data visualization tools. The target audience includes undergraduate and post-baccalaureate students. The reports generated from this project target the broader citrus greening research community. The data generated will be used to update the Asian citrus psyllid genome and be made available publicly. The analysis of pathways will provide a basis for molecular based studies on the Asian citrus psyllid. The goals of Objective 2 are: 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 quality, and prevent fruit drop. Increased awareness of novel therapeutic application technologies and products by industry segments. Industry understands economic feasibility and practicality of management recommendations. The target audience continues to include members of the project team, other citrus greening and plant pathology researchers. This work is also of interest to citrus growers groups including citrus grove managers and corporate representatives, industry professionals including agricultural chemical companies who might advance these peptides into commercial production for use in citrus production and other industry representatives. Our findings on shared and sex-specific responses in adult ACP in response to CLas will benefit scientists studying CLas transmission, as well as scientists investigating sex differences in other insect vectors of disease. The target audience is scientists in the field. Citrus growers, Citrus industries, regulators (FDA/EPA), citrus/agricultural researchers. The goals of Objective 3 are: 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. The target audience includes social scientists, consumers of citrus and citrus products, citrus industry representatives including growers, producers, distributors and retailers. Changes/Problems: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 quality, and prevent fruit drop. Researchers understand consumer attitudes towards biotechnologies. Understanding of industry/consumer knowledge about HLB and its impact on U.S. citrus production. 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 and genome analysis. Database construction: New data visualization tools. - We have completed many activities and will continue with no changes to meet the above goals. Increased growers/public understanding of biotechnology methods of disease management. Problems encountered in trying to meet the above goal. - Planned conference in Florida delayed and finally cancelled due to lack of citrus industry support. Venue changed to California. What opportunities for training and professional development has the project provided?In addition to scientific publications listed in the Product Section, the project has provided the training and professional development opportunities below: - Successful annotation was completed by utilizing a peer mentoring system that worked by having 3 experienced annotators mentor and train 7 new students. Peers and mentors met once a week and all local annotators at IRSC met with their instructor (D'Elia) once a week to review progress and complete a gene model as a group. - Bi-weekly zoom video conferences to train students and coordinate activities for the psyllid genome annotation using the WebApollo platform. - Weekly and bi monthly virtual meetings allowed students to communicate their progress to the larger group of annotators and receive help from experts. - Two students presented over annotation and bioassay results at a state conference, the Annual Florida Academy of Sciences. https://fas.fit.edu/media/site-specific/fasfitedu/Outstanding-Student-Awards-FAS-Annual-Meeting-2018FinalVersion20180330.pdf - Chad Vosburg, IRSC undergraduate intern, won 1st place for his Outstanding Poster Presentation in Agriculture Division at the 2018 FAS meeting in Miami Shores, FL. - Rebecca Grace, IRSC undergraduate intern, won 1st place for her Outstanding Oral Presentation in Agriculture Division at the 2018 FAS meeting in Miami Shores, FL. - Students have used their knowledge gained through participation in annotation to advance to careers and graduate school. This includes graduates that have begun PhD graduate studies at Florida State University and Cornell University over the past year. Other recent graduates are now working for the USDA, the American Bee project and teaching middle school science. - Chad Vosburg, Program Coordinator at IRSC, mentored and trained new students on annotation. -Stephanie Hoyt, an undergraduate student at Cornell/BTI, has generated the first complete genome assemblies for the Carsonella and Proftella endosymbiont genomes from a Florida Asian Citrus Psyllid for the first time. She also generated near complete assemblies for two strains of Wolbachia in the ACP. - Former IRSC intern, Tracey Bell, started a PhD degree in biomedical sciences at Florida State University. Her experience as an intern helped secure her acceptance to the program. - Former IRSC intern, Dan DeAvila, started a career as a Science Teacher. His bioassay experience as an intern helped solidify his career decision. - Former IRSC intern, Rebecca Grace, started a PhD degree in biomedical sciences at Cornell University. Her experience as an intern helped secure her acceptance to the program. - Former IRSC intern, Rita Noel, is now a Research Technician at USDA-ARS. Her experience as an intern provided valuable experience before accepting a job with USDA-ARS. - Dr. Javier Alba Tercedor received the award to the best presentation during the conference Bruker Micro-CT Users Meeting 2018 celebrated in Ghent from April 16-19th 2018. The presentation demonstrated how Micro-CT can be used to observe the life of the Coffee Borer Beetle inside the coffee berries, and how micro-CT helped to the entertainment industry to the visual effect that appeared in the movie "Blade Runner 2019". This movie received the Oscar award for the best visual effects in 2018. -Undergraduate and graduate student project participants had the opportunity to present their work at conferences and symposia. These are listed below: *Indicates Undergraduate or Graduate Student Project Members in bold font *Grace R, Wiersma-Koch H, & D'Elia T. (March 2018). Genomic identification, annotation, and comparative analysis of Vacuolar-type ATP synthase subunits in Diaphorina citri. Florida Academy of Sciences. Miami Shores, FL. *Hoyt S, Hosmani P, Flores M, Hunter W, Brown S, Mueller L, & Saha S. (November 2018). Characterization of bacterial endosymbionts and systems biology resources for Diaphorina citri, insect vector for the citrus greening disease. Biological Data Science 2018. Cold Spring Harbor, NY. Rumble J, Lamm A, *Beattie P, & *Ruth T. (April 2018). Attitudes and transparency: A case for communication. Association for International Agricultural and Extension Education. Merida, Yucatan, MEXICO. Saha S, Hosmani P, Flores M, Mann M, *Hoyt S, *Kruse A, Ramsey J, Hunter W, Brown S, Heck M, & Mueller L. (January 2019). Visualization of Insect Vector-Plant Pathogen Interactions in the Citrus Greening Pathosystem using Genomics, Transcriptomics and Proteomics. Plant, Animal & Microbe Genomes XXVII. San Diego, CA. *Vosburg C. & D'Elia T. (March 2018). Annotation of Segment Polarity Genes in Diaphorina citri. Florida Academy of Sciences. Miami Shores, FL. How have the results been disseminated to communities of interest?In addition to scientific publications listed in the Product Section, dissemination of our results has been described below: - 4th Annual Citrus Greening Solutions Meeting. Indian River State College, Ft. Pierce, FL. December 2018. Attended: 4 graduate students, 8 undergraduate students, 6 key personnel, 7 co-PIs, 11 project members, 6 SSAC members, 1 project coordinator, & 2 evaluators - Project members have been published in a number of major journals in their fields, as well as receiving attention on various online platforms. - Project publications have been cited a total of 76 times by 125 authors in the world & 118 within the United States. Additionally, these citations span across the disciplines of Biology, Agriculture, Engineering, and Chemistry. - Project publications have been featured 16 times on 13 different online news outlets, have been tweeted approximately 73 times & are being read by over 300 Mendeley readers. Objective 1 activities include: - Javier Alba-Tercedor collaborated (by scanning & reconstructing images of different beetles species by micro-CT), with the Canadian company BUFF who produced the visual effects for the movie "Blade Runner 2049". The movie obtained an Oscar award for the best visual effects; different media interviewed him to broadcast the news. - https://www.eurekalert.org/pub_releases/2018-02/uog-usd022718.php - https://elpais.com/elpais/2018/02/02/ciencia/1517577303_353028.html - https://www.ideal.es/culturas/escarabajos-oscar-20180306004703-ntvo.html - https://techxplore.com/news/2018-02-scientist-d-scans-beetles-blade.html - https://www.20minutos.es/noticia/3251277/0/imagenes-escarabajos-3d-profesor-ugr-base-efectos-especiales-blade-runner-2049/ - https://www.3ders.org/articles/20180301-3d-scanning-used-for-visual-effects-in-blade-runner-2049.html - https://www.ideal.es/miugr/escarabajo-universidad-granada-20180203003214-nt.html - Meetings were held with researchers in the potato psyllid community from Texas & Washington in order to establish collaborations. - Connected with researchers in Oklahoma State University, University of Idaho & Florida. - Project members attended the Citrus Grand Challenge Meeting to discuss the resources at citrusgreening.org for the research community & explained how published or unpublished data can be included. - An animation crafted to allow for broad scientific audiences to understand basic anatomy & functionality of the ACP. https://citrusgreening.org/microtomography/cicero_alimentarycanal_2018 - An animation crafted to allow others to identify anatomical features of the ACP oral region in their TEM cross-sections. https://citrusgreening.org/microtomography/cicero_stylet_2018 Objective 2 activities include: - Carolyn Slupsky presented "New Strategies to Save California Citrus" to stakeholders & faculty at Seed Central at UC Davis. - Carolyn Slupsky presented "Invasive Species - Asian Citrus Psyllid" to stakeholders invited by the Dean of the College of Agriculture & Environmental Sciences. - Carolyn Slupsky presented "Metabolomics as an EDT for HLB" to stakeholders at the California Citrus Research Board in Visalia. - Robert Shatters has presented results to multiple grower meetings in Florida. Objective 3 activities include: - Multiple conference papers and posters - Guest lectures to students and industry organizations including feedback and discussion - Outcomes of this project have also been disseminated to the greater scientific community through local, national and international conference and symposium presentations listed below: Project Members in bold font Alba-Tercedor J, Alba-Alejandre I, & Hunter W. (December 2018). Advances in the micro-CT procedures to study the Asian Citrus Psyllid. 4th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Alba-Tercedor J, Alba-Alejandre I, & Vega F. (April 2018). Micro-CT unveils the secret life of the coffee berry borer (Hypothenemus hampei; Coleoptera, Curculionidae: Scolytinae) inside coffee berries. Bruker Micro-CT Users Meeting 2018. Ghent, BELGIUM Alba-Tercedor J, Buffin P, Wise C, & Lepine P. (April 2018). Micro-CT as a valuable base tool for the industry of visual effects: the case of the animated beetles appearing in the film "Blade Runner 2049". Bruker Micro-CT Users Meeting 2018. Ghent, BELGIUM Flores M, Fernandez-Pozo N, Mann M, Hosmani P, Brown S, Heck M, Mueller L, & Saha S. (January 2019). Diaphorina citri resources for exploring the citrus greening disease complex. Plant, Animal & Microbe Genomes XXVII. San Diego, CA Flores M, Fernandez-Pozo N, Mann M, Hosmani P, Brown S, Heck M, Mueller L, & Saha S. (November 2018). A tissue and host-specific expression atlas for Citrus sinensis and Diaphorina citri for exploring the citrus greening disease complex. Biological Data Science 2018. Cold Spring Harbor, NY Flores M, Hosmani P, Fernandez-Pozo N, Mann M, Brown S, Heck M, Mueller L, & Saha S. (January 2019). A tissue and host-specific expression atlas for Citrus sinensis and Diaphorina citri for exploring the citrus greening disease complex. Plant, Animal & Microbe Genomes XXVII. San Diego, CA Hunter W. (November 2018). Antisense oligos (ASO): Advanced RNA silencing technology to reduce pathogens and ectoparasites in livestock. ESA, ESC, and ESBC Joint Annual Meeting. Vancouver, CANADA Hunter W. (November 2018). Reducing psyllid vectors and bacterial pathogens: RNA suppression Technologies. ESA, ESC, and ESBC Joint Annual Meeting. Vancouver, CANADA Hunter W. (August 2018). Biotechnology: RNAi, Antisense Oligonucleotides and CRISPR Strategies to Reduce Psyllids and Bacterial Pathogens in Citrus Trees. ACS National Meeting & Expo. Boston, MA Hunter W. (July 2018). BAPC-CRISPR-Cas9 Gene-Knock OUT: Asian Citrus Psyllid. The 101st Annual Meeting International Florida Entomology Society. St. Augustine, FL Hunter W. (June 2018). RNA suppressing technologies which improve activity and persistence in plants and animals: dsRNA, Antisense oligonucleotides, and CRISPR-Cas9. National AGR Teleconference. Ft. Pierce, FL Hunter W. (June 2018). RNA Suppressing Technologies: Improve Activity and Persistence in Plants and Animals: RNAi, Antisense Oligonucleotides, BAPC-CRISPR-Cas9. Innovative Research. Ft. Pierce, FL Hunter W. (March 2018). Biotechnology: RNAi, Antisense Oligonucleotides and CRISPR Strategies to Reduce Psyllids and Bacterial Pathogens in Citrus Trees. Annual Conference Entomological Society of America. Orlando, FL Saha S. (December 2018). AgriVectors: a portal for plant diseases transmitted by insect vectors. International Phytobiomes Conference 2018. Montpelier, FRANCE Saha S. (July 2018). Developing scalable and accessible databases for agricultural disease systems and crops. Driving Innovation through Data in Agriculture. Beltsville, MD Saha S. (June 2018). Insights into genome organization and non-coding genes of Diaphorina citri, the vector of citrus greening disease. 11th Annual Arthropod Genomics Symposium. Urbana, IL Saha S, Chin E, Ramsey J, Flores M, Mishchuk D, Chavez J, Howe K, Zhong X, Humann J, Foster E, Polek ML, Godfrey K, Main D, Mueller L, Bruce J, Heck M, & Slupsky C. (January 2019). Novel Bioinformatic Resources and Multi-Omics Approaches enable the Characterization of Early Response to Ca. Liberibacter asiaticus Infection in Citrus. Plant, Animal & Microbe Genomes XXVII. San Diego, CA Saha S, Hunter W, Hosmani P, Flores M, & Mueller L. (January 2019). AgriVectors: A Data and Systems Resource for Arthropod Vectors of Plant Diseases. Plant, Animal & Microbe Genomes XXVII. San Diego, CA Stover E. (December 2018). Transgenic Citrus to Disrupt Clas/ACP. 4th Annual Citrus Greening Solutions Meeting. Fort 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 quality, and prevent fruit drop. Objective 2 activities planned to meet the above goals. - We are planning on submitting the multi-omics, systems-level findings on the sex-specific and non-specific responses of adult ACP to CLas by March. - We will present our findings at the UC Davis microbiology graduate group research symposium, and have submitted an abstract for consideration for presentation at the 2019 International Research Conference on HLB. - We are working to complete experiments directly interrogating defense responses in ACP to CLas to determine those that are critical to limiting CLas propagation. - As part of our outreach, we are planning a music video on HLB and prevention to raise consumer awareness of the disease with the target audience being the general public. - We will further refine the effectiveness of the HTH peptides in HLB treatment. - We will demonstrate that they also augment citrus innate immune defense. - We will collect field and greenhouse efficacy data. - We will replicate plants and test effects of transgenics vs controls on ACP colonization and development of CLas. 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. - We are abandoning the idea for Florida, looking into viability of organizing a similar conference in California. - We will analyze focus group data and present, publish with our focus at national research meetings and scholarly journals. - We will publish research findings at conferences,and/or in journals. 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. -Industry is conducting production cost evaluations and developing effective dose studies to support the initiation of regulatory approval phase. -Industry has hired Engineering firm to transition from prototyping delivery devices to commercial design and manufacturing. Students increase knowledge of bioinformatics and genome analysis. Database construction: New data visualization tools. Objective 1 activities planned to meet the above goals. - Students are completing reports on pathways to be used in the second peer reviewed publication on the Asian citrus psyllid genome. Students will continue annotation of genes. - Current students will train new students in genome analysis over the course of the next year to meet objectives. They will utilize a peer training system that has been developed over the course of the grant and has efficiently trained new students to quickly learn the required skills for genome analysis. - As new students join the annotation team, additional training will be provided to help them learn the standard operating procedures. - The endosymbiont genomes will be released with the new v3 genome assembly. We will validate some of the regions using genomics PCR. - All the genes annotated on the v2 genome will be incorporated in the Official gene set v3.0 that will be created for the new genome v3.0. -We will continue working with the scientist performing experiments to leverage their knowledge and expertise to target genes of functional importance for manual curation. - We will explore the contigs that were not assembled into the reference assembly due to structural variation and duplication.

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. - We have integrated our findings from the microbiota, transcriptomics, proteomics and metabolomics responses in male and female adult ACP to CLas. - We have begun writing a manuscript on the sex-specific responses of ACP to CLas, and have completed all figures for the manuscript. - Based on our detailed understanding of the similarities and differences between males and females in response to CLas, we have planned and begun preparation for experiments to investigate critical defense mechanisms in ACP against CLas which we hope will be completed in the coming year. 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. - Two biological peptides have been identified that kill CLas within citrus leaves (i) proven in excised leaf assays (ii) One has demonstrated in plant activity by expressing using a Citrus Tristeza Virus (CTV) delivery method. (iii) Invention disclosures are being submitted to protect IP (iv) commercial interests have developed CRADA with USDA to advance to commercial product (v) transgenic plants expressing these peptides have been developed and one induces significant psyllid mortality when the insects feed on these plants. - We designed the citrus-derived HTH peptides that are: (i) highly effective in rapidly clearing CLas (ii) produced in large-scale at a low cost (iii) not susceptible to bacterial resistance, which often defeats antibiotics (iv) amenable to topical delivery for the treatment of already infected citrus - Numerous transgenics targeting ACP have been created. - In early trials with detached leaves, some have displayed remarkable ACP mortality. - More targets have been identified by other project members and transgenics are being created. 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. - Investigated potential to initiate a social science research conference for Florida citrus industry to share findings from consumer research with citrus industry stakeholders. - We completed focus group research with consumers in four cities, two focus groups per city. - After analysis we presented and/or published research results from national and state-specific survey project data collected in years 2 & 3. - We completed focus groups with consumers in four cities, two focus groups per city. Increased awareness of novel therapeutic application technologies and products by industry segment. Industry understands economic feasibility and practicality of management recommendations. Objective 2 activities highlighted below pertain to the research goals listed above. -As a result of our interactions with the Ag Chemical community, one company has entered into a CRADA to develop 1) novel delivery system we have demonstrated and 2) new bactericides based on antimicrobial peptides we have discovered with activity against CLas. -We are currently engaged with an engineering firm that is developing our novel delivery devices into a commercializable product (i.e. optimizing design to meet performance, manufacturing and cost requirements). Students increase knowledge of bioinformatics and genome analysis. Objective 1 activities highlighted below pertain to the research goal listed above. - In this reporting year, more than 684 genes from 17 pathways in the psyllid genome have been manually curated using Apollo annotation tool by 19 student annotators at IRSC, Cornell/BTI and KSU. - The students have analyzed their data and prepared pathway reports to be used for the second publication of the Asian citrus psyllid genome. Students utilized a wide range of bioinformatic tools to perform both comparative and phylogenetic analysis. - During the annotation process, students have learned and mastered the use of genome browsers (WebApollo), accessing molecular data (Citrusgreening.org, NCBI, Flybase, etc.), performing pairwise and multiple sequence analysis (NCBI, BLAST, MEGA, EBI) and construction of phylogenetic trees (MEGA). - Manual annotations from the second round of curation were based on Official Gene Set v2.0. - Manual curation focused on pathways of experimental interest to foster interactions between the scientists working in the laboratory and the undergraduate annotators. The open forum of Basecamp and video conferences allowed for constant feedback. - General guidelines were established to support student driven genome annotation, along with measurable objectives, which were published in a preprint article and have been accepted for peer review publication. -New assemblies and annotations of the Florida strains of the ACP bacterial endosymbionts, Wolbachia, Profftella, and Carsonellawere also characterized from the genome sequencing data. Database construction: New data visualization tools. Objective 1 activities highlighted below pertain to the research goal listed above. - ACP genome, annotation and endosymbionts: Advances in psyllid genome sequencing were utilized to improve the ACP genome assembly, including using Pacbio, Dovetail Chicago and long-range Hi-C scaffolding. This resulted in the identification of 13 psyllid chromosomes in the new version of the reference genome (v3). This is the first description of chromosome number for this economically important hemipteran insect vector. - Together with Pacbio IsoSeq technology to sequence psyllid transcripts from different life stages and those reared on CLas infected and uninfected trees, approximately 20,000 putative full-length protein coding psyllid genes were identified. This high quality automated annotation for the ACP v2 reference genome assembly was designated Official Gene Set v2.0. - Isoseq transcriptome: We combined Illumina RNA sequencing data from ACP gut tissue, male and female whole body, salivary glands, antennae and terminal abdomen along with Pacbio Iso seq transcript data from adult and nymph psyllids to create the new comprehensive Isoseq transcriptome. This contains 22,357 genes, 166,705 transcripts with an average length of 1762.4 bp. This transcriptome database will replace the MCOT transcriptome generated previously. - Psyllid Expression Network (PEN): New data sets were added to PEN. We created a expression atlas for the new Official Gene Set v2.0 in the Psyllid Expression Network (PEN). This is a user-friendly web-based tool for mining gene and protein expression patterns. - Citrus Expression Network: CEN contains public expression data from NCBI Sequence Read Archive for C. sinensis and C. clementina. PEN and CEN facilitate effective data analysis by enabling simultaneous visualization of correlated genes to develop novel hypothesis in addition to candidate gene identification. These tools are a useful resource not only for citrus greening research but also other tritrophic disease systems. - Sample preparation research: We have also developed library preparation methods for ACP and microbial transcriptome sequencing where we can get both the poly-A enriched fraction from the psyllid and the poly-A depleted (flow through) fraction representing the microbai transcripts. - A metabolic pathway database using BioCyc Pathway Tools software for the C. sinensis v2 reference genome was developed to visualize and analyze large-scale genomics, transcriptomics, proteomics and metabolomics data.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hunter W, Pelz-Stelinski K, Sandoval-Mojica A, Paris T, Miles G, Metz J, & Aishwarya V. (March 2018). Biotechnology: RNAi, Antisense Oligonucleotides and CRISPR Strategies to Reduce Psyllids and Bacterial Pathogens in Citrus Trees. Southeastern Branch Annual Conference Entomological Society of America. Orlando, FL.
  • Type: Book Chapters Status: Published Year Published: 2019 Citation: Saha S. (2019). Long Range Sequencing Improves Insect Genome Assemblies, Insect Genomics (pp.33). Humana Press, 1858, New York. 978-1-4939-8774-0
  • Type: Book Chapters Status: Published Year Published: 2018 Citation: Cicero J, Alba-Tercedor J, Hunter W, Cano L, Saha S, Mueller L, & Brown S. (2018). Asian citrus psyllid stylet morphology and applicability to the model for inter-instar stylet replacement in the potato psyllid, Arthropod Structure & Development (pp.542-551). Elsevier Ltd, 47. doi:10.1016/j.asd.2018.06.007
  • Type: Book Chapters Status: Published Year Published: 2018 Citation: Sinisterra-Hunter X. & Hunter W. (2018). Towards a holistic integrated pest management: Lessons Learned from Plant-Insect Mechanisms in the Field. The Biology of Plant-Insect Interactions: A Compendium for the Plant Biotechnologist (pp.204-226). CRC Press, USA. 9781498709736-CAT#K25008
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Rumble J, Wu Y, Tully K, Ruth T, Ellis J, & Lamm A. (February 2019). A Mixed-Methods Comparison of Self-Reported and Conversational Trust in Science. National Agricultural Communications Symposium. Birmingham, AL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Hunter W, Sandoval-Mojica A, Metz J, Holland C, Godfrey M, Paris T, McCollum G, Boyle M, Cano L, Altman S, & Pelz-Stelinski K. (January 2019). Advances in RNA suppression of the Asian citrus psyllid vector and bacteria (Huanglongbing Pathosystem). Florida Citrus Show. Ft. Pierce, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hunter W, Sandoval-Mojica A, Paris T, Miles G, Metz J, McCollum G, Boyle M, Altman S, Aishwarya V, & Qureshi J. (March 2018). Antisense Oligonucleotides, F-ASO, and PPMO, new tools to reduce pests and pathogens in citrus and other agricultural crops. Southeastern Branch Entomological Society of America Annual Conference. Orlando, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Coates B, Fabrik J, Garczynski S, Geib S, Gundersen-Rindal D, Hunter WB, Perera OP, Sim S, Oppert B, James R, & Hackett KJ (September 2018). Applications of CRISPR/gene editing technology within the ARS Arthropod Genomics Research working group. USDA, Office of the Chief Scientist (OCS), Symposium on CRISPR and Gene Editing Technologies in Agriculture. Washington DC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Clarke S, Paris T, Hunter W, Brown S, & Qureshi J. (March 2018). Assessment of Psyllid-dsRNA Off Target Effects on a Ladybird Beetle Predator. 82nd Annual Meeting Florida Academy of Sciences. Miami Shores, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hunter W, Gonzalez M, & Tomich J. (July 2018). BAPC-CRISPR/Cas9 System for Heritable Embryonic Gene Editing: Gene Knock-Out, Asian Citrus Psyllid. The 101st Annual Meeting of the International Florida Entomological Society. St. Augustine, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hunter W, Paris T, Sandoval-Mojica A, Garzyncki S, Qureshi J, & Pelz-Stelinski K. (March 2018). Gene Editing, CRISPR knockouts in Asian Citrus Psyllid, Diaphorina citri, Kuwayama (Hemiptera: Liviidae). Southeastern Branch Annual Conference Entomological Society of America. Orlando, FL.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Ruth T, Rumble J, Lamm A, & Ellis J. (February 2019). How consumers contrast and assimilate information about agricultural biotechnology. National Agricultural Communications Symposium. Birmingham, AL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Beattie P, Lamm A, Rumble J, & Ellis J. (March 2018). Identifying generational differences to target extension programming when discussing genetic modification. American Association for Agricultural Education Southern Region Conference. Jacksonville, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Alba-Tercedor J, Buffin P, Wise C, & Lepine P. (April 2018). Micro-CT as a valuable base tool for the industry of visual effects: the case of the animated beetles appearing in the film ⿿Blade Runner 2049⿝. Bruker Micro-CT Users Meeting 2018. Ghent, Belgium
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Paris T, George J, Miles G, Clarke S, Vosburg C, Kercher K, Reynolds M, DeAvila D, Cordola C, Grace R, Norus J, Tamayo B, DElia T, Qureshi J, Lapointe S, Hunter W, & Pelz-Stelinski K. (November 2018). Crossing Borders: Entomology in a Changing World. ESA, ESC, and ESBC Joint Annual Meeting. Vancouver, BC, CANADA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Clarke S, Paris T, Hunter W, & Brown S. (November 2018). dsRNA Delivery to Citrus Plants and the Citrus Psyllid. ESA, ESC, and ESBC Joint Annual Meeting. Vancouver, BC, CANADA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Clarke S, Paris T, Hunter W, Brown S, & Quereshi J. (June 2018). Factors affecting citrus tree absorption of double-stranded ribonucleic acid, and RNAi delivery to psyllids. 131st Annual Florida State Horticultural Society Conference. Ft. Lauderdale, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Alba-Alejandre I, Hunter W, Cicero J, Alba-Tercedor J, & Cano L. (July 2018). Micro-CT study of male genitalia and reproductive system of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). 101st Annual Meeting of the Florida Entomological Society. St. Augustine, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Alba-Tercedor J, Alba-Alejandre I, & Vega F. (April 2018). Micro-CT unveils the secret life of the coffee berry borer (Hypothenemus hampei; Coleoptera, Curculionidae: Scolytinae) inside coffee berries. Bruker Micro-CT Users Meeting 2018. Ghent, Belgium.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Clarke S, Paris T, Hunter W, Brown S, & Qureshi J. (March 2018). Parameters on Plant Absorption and Delivery of dsRNA. 82nd Annual Meeting Florida Academy of Sciences. Miami Shores, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Clarke S, Paris T, Hunter W, Brown S, & Qureshi J. (July 2018). Parameters on plant absorption of double-stranded Ribonucleic acid, dsRNA. The 101st Annual Meeting of the International Florida Entomological Society. St. Augustine, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hunter W, Metz J, Sandoval-Mojica A, Altman S, Boyle M, McCollum G, Aishwarya V, & Pelz-Stelinski K. (June 2018). RNA suppressing technologies which improve activity and persistence in plants and animals: dsRNA, Antisense oligonucleotides, and CRISPR-Cas9. National AGR Teleconference. Ft. Pierce, FL.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Ruth T, Rumble J, Lamm A, & Ellis J. A model for understanding decision-making related to agriculture and natural resource science and technology. Journal of Agricultural Education, 59(4), 224-237. https://doi.org/10.5032/jae.2018.04224
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Hosmani P, Shippy T, Miller S, Benoit J, Munoz-Torrez M, Flores M, Mueller L, Wiersma-Koch H, DElia T, Brown S, & Saha S. A quick guide for student-driven community genome annotation. PLOS Computational Biology.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ruth T, Rumble J, Lamm A, Irani T, & Ellis J. Are Americans attitudes toward GM science really negative? An academic examination of willingness to expose attitudes (pp.113). Science Communication, 41(1), Oregon, US. https://doi.org/10.1177/1075547018819935
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Hunter W, & Sinisterra-Hunter X. Emerging RNA Suppression Technologies to Protect Citrus Trees from Citrus Greening Disease Bacteria (pp.163-199). Advances in Insect Physiology, 55, USA. https://doi.org/10.1016/bs.aiip.2018.08.001
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Cornara D, Garzo E, Morente M, Alba-Tercedor J, Moreno A, & Fereres A. EPG combined with micro-CT and video recording reveals new insights on the feeding behavior of Philaenus spumarius (e0199154:). PLos ONE, 13(7), USA. https://doi.org/10.1371/journal.pone.0199154
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Cicero J, Alba-Tercedor J, Hunter W, Cano L, Saha S, Mueller L, & Brown S. Asian citrus psyllid stylet morphology and applicability to the model for inter-instar stylet replacement in the potato psyllid (pp.542-551). Arthropod Structure & Development, 47(5), USA. https://doi.org/10.1016/j.asd.2018.06.007
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Hunter W, Gonzalez M, & Tomich J. BAPC-assisted CRISPR/Cas9 System: Targeted Delivery into Adult Ovaries for Heritable Germline Gene Editing (Arthropoda: Hemiptera). bioRxiv. https://doi.org/10.1101/478743
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Avila L, Brown S, Reeck G, Park Y, & Tomich J. Delivery of Lethal dsRNAs in Insect Diets by Branched Amphiphilic Peptide Capsules. (pp.139-146). Journal of Controlled Release, 273, USA. https://doi.org/10.1016/j.jconrel.2018.01.010
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Mann M, Fattaha-Hosseini S, Ammar E, Strange R, Warwick E, Sturgeon K, Shatters R, & Heck M. Diaphorina citri nymphs are resistant to morphological changes induced by ⿿Candidatus Liberibacter asiaticus⿝ in midgut epithelial cells (e00889-17). Infection and Immunity, 86(4). doi:10.1128/IAI.00889-17
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ghosh S, Gundersen-Rindal D, Park A, & Hunter W. Double-stranded RNA oral Delivery methods to induce RNA Interference in phloem and plant-sap-feeding hemipteran insects (e57390). JoVE Visualized Experiments, 135, USA. doi:10.3791/57390
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Macias-Velasco J, Brunson R, Hunter W, & Bextine B. Evaluation of North American Asian citrus psyllid (Hemiptera: Liviidae) population genetics using cytochrome P450 melt curve analysis. bioRxiv. http://dx.doi.org/10.1101/445734
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Beattie P, Lamm A, Rumble J, & Ellis J. Identifying generational differences to target extension programming when discussing genetic modification. (pp.154). Journal of Agricultural Education, 59(3), West Virginia, US. https://doi.org/10.5032/jae.2018.03154
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: DAngelo J, Ellis J, Burke K, & Ruth T. (March 2018). Media portrayal of GM science and citrus greening in state and national newspapers (pp.5). Journal of Applied Communications, 102(1), USA. https://doi.org/10.4148/1051-0834.1361
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Alba-Alejandre I, Hunter W, & Alba-Tercedor J. Micro-CT study of male genitalia and reproductive system of the Asian citrus psyllid, Diaphorina citri Kuwayama, 1908 (Insecta: Hemiptera, Liviidae) (e0202234). PLoS ONE, 13(8), USA. https://doi.org/10.1371/journal.pone.0202234
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Alba-Alejandre I, Alba-Tercedor J, & Vega F. Micro-CT to Document the Coffee Bean Weevil, Araecerus fasciculatus (Coleoptera: Anthribidae), Inside Field-Collected Coffee Berries (Coffea canephora) (pp.1-9). Insects, 9, Switzerland. http://dx.doi.org/10.3390/insects9030100
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Clarke S, Paris T, Hunter W, Brown S, & Quereshi J. Parameters on plant absorption of double-stranded Ribonucleic acid, dsRNA. Florida State Horticulture Society
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Hunter W, McKenzie C, & Mitchell B. Rapid, Affordable, Collection and Analysis of bioaerosol viral pathogen (Begomoviruses and Whitefly Vectors). bioRxiv. doi:10.1101/451559
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Natarajan P, Sukthankar P, Changstrom J, Holland C, Barry S, Hunter W, Sorensen C, & Tomich J. Synthesis and Characterization of Multifunctional Branched Amphiphilic Peptide Bilayer Conjugated Gold Nanoparticles (pp.11071-1108). ACS Omega, 3(9), USA. doi:10.1021/acsomega.8b01633
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Lamm A, Taylor M, Rumble J, & Ellis J. Targeting extension programs to opinion leaders guiding genetic modification discussions. Journal of Human Sciences and Extension
  • Type: Websites Status: Published Year Published: 2018 Citation: Cicero J, Hunter W, Cano L, Saha S, Mueller L, & Brown S. Asian citrus psyllid stylet biogenesis. https://citrusgreening.org/microtomography/cicero_stylet_2018


Progress 03/01/17 to 02/28/18

Outputs
Target Audience:The goals of Objective 1 are: Students increase knowledge of bioinformatics and genome analysis. Database construction: New data visualization tools. The target audience includes undergraduate and post-baccalaureate students. The databases were constructed for the scientists involved in this project and are publicly available to the scientific community. The goals of Objective 2 are: 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 quality, and prevent fruit drop. Increased awareness of novel therapeutic application technologies and products by industry segments. Industry understands economic feasibility and practicality of management recommendations. The target audience includes members of the project team, other citrus greening and plant pathology researchers. This work is also of interest to citrus growers groups including citrus grove managers and corporate representatives, industry professionals including agricultural chemical companies who might advance these peptides into commercial production for use in citrus production and other industry representatives The goals of Objective 3 are: 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. The target audience includes consumers of citrus and citrus products, citrus industry representatives including growers, producers, distributors and retailers. Changes/Problems: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 quality, and prevent fruit drop. Changes to meet the above goals. The original plan called for RNA aptamer selection against whole tissues, including guts and stylet sheaths. Efforts to produce tissue-specific RNA aptamers were unsuccessful. Thus, the focus was changed to producing RNA aptamers against whole protein targets from the analysis of the gut and salivary sheath proteome analysis. Because the gut-binding peptides only reduced the successful acquisition of CLas by the psyllids by approximately 50%, we included a screening for antimicrobial peptides that target CLas. Discovery of a highly active antimicrobial peptide active against CLas within citrus, we decided to evaluate the combined use of gut-binding and antimicrobial peptides. 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. Changes to meet the above goals. Data collection states for focus groups changed. Originally planned locations were in Florida, Texas, and California. Data will be collected in Florida, Illinois, New Jersey and California to provide representation of major media markets in both citrus and non-citrus producing states. 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 and genome analysis. Database construction: New data visualization tools. No changes to meet the above goals. What opportunities for training and professional development has the project provided?In addition to scientific publications listed in the Product Section, the project has provided the training and professional development opportunities below: 6 posters over annotation and bioassay results were presented by IRSC students at the FAS in March 2017 Chris Cordola, IRSC undergraduate intern, won 1st place for his poster presentation at the 2017 FAS meeting in the undergraduate agriculture category Former IRSC intern, Krystal Villalobos-Ayala, started a master's degree in biomedical sciences at the University of South Florida. Her experience as an intern provided valuable experience and helped secure her acceptance to the program. Former IRSC intern, Gabe DeAvila started a career at the Moffitt Cancer Institute in Tampa, Florida. His analytical experience on the citrus project helped him secure his new position. Two teams of two IRSC citrus greening undergraduate students participated in a National Science Foundation funded STEM Entrepreneurial competition and won first and third place. The teams developed business plans and performed market research. One team, Gabe and Daniel DeAvila, designed actual lab supplies and make them on a 3-D printer. This team formed an LLC and sold their lab supplies to USDA scientists. The other team, Chris Cordola and Hannah Mann, created a business plan and market research related to development of RNAi therapeutics for use in citrus industry. Anna Yaschenko, summer 2017 intern at Cornell University/Boyce Thompson Institute from University of Maryland, Baltimore County, presented her work on characterizing long non-coding RNA in the ACP through genome annotation and molecular biology at the PGRP symposium for all summer interns at BTI. She validated the presence of specific long non-coding RNA in nymphs and discovered that host plant affects long non-coding RNA expression. She was awarded the best poster presentation prize for this work. She also presented this poster at local meetings in Maryland. Annie Kruse, graduate student at BTI, was Anna's mentor. Stephanie Hoyt, summer 2017 intern at Cornell University/Boyce Thompson Institute, analyzed whole-body ACP DNA sequences for differences in taxonomy and abundance of endosymbionts. She presented her work on taxonomic analysis of the endosymbionts in the ACP using metagenomics classification methods at the PGRP symposium. Annie Kruse, graduate student at Cornell University/Boyce Thompson Institute, attended an intensive electron microscopy training course Annie Kruse taught courses on introductory plant biology to 5th graders in the Ithaca, NY area Tracey Bell, Program Coordinator at IRSC, mentored and trained new students on annotation Dr. Joseph Cicero took up training in computed tomography using the General Electric nanoCT V unit and its accompanying reconstruction software at the Nanoscale Research Facility, University of Florida. Determined its limitations and expectations for use in cutting-edge elucidations of ACP anatomy. Determined time and cost effectiveness of this approach, including specialized specimen processing techniques, advantages and disadvantages over alternative imaging appliances such as electron microscopy and confocal microscopy. Dr. Javier Alba-Tercedor received an award for the best micro-CT picture during the contest celebrated at the International Bruker-microCT users meeting held in Brussel in June 2017. 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 *Beattie, P., Lamm, A., & Rumble, J. Generational perceptions of the relative advantages of genetic modification. American Association for Agricultural Education National Conference. San Luis Obisbo, CA *Bell, T., *Cordola, C., *DeAvila, G., *DeAvila, D., *Mann, H., Hunter, W., Wiersma-Koch, H., D'Elia, T., & *Villalobos-Ayala, K. RNAi Treatments Targeting Heat Shock Proteins in Diaphorina citri (Hemiptera: Liviidae). Florida Academy of Sciences. Lakeland, FL *Coates, L., Mahoney, J., Warwick, E., MacCoss, M., Johnson, R., *Ramsey, J., Krasnoff, S., Howe, K., Moulton, K., Shatters, R., Hall, D., Cilia, M., & Slupsky, C. Employing multi 'omics to assess effects of sex and citrus variety on D. citri - CLas interactions. Microbiology Graduate Group Research Symposium, University of California. Davis, CA *Coates, L., Mahoney, J., Warwick, E., Johnson, R., MacCoss, M., *Ramsey, J., Krasnoff, S., Howe, K., Moulton, K., Shatters, R., Hall., Cilia, M., & Slupsky, C. Employing Multi 'omics to Assess Effects of Sex and Citrus Variety on D. citri - CLas Interactions. 3rd Hemipteran-Plant Interactions Symposium. Madrid, Spain *Cordola, C., *Bell, T., *Villalobos-Ayala, K., *Mann, H., *DeAvila, G., DeAvila, D., Wiersma-Koch, H., D'Elia, T., & Hunter, W. Annotation and phylogenetic analysis of three immunity-related gene groups in D. citri (Hemiptera: Liviidae). Florida Academy of Science. Lakeland, FL D'Elia, T., Wiersma-Koch, H., Hunter, W., *Villalobos-Ayala, K., *Cordola, C., & *Bell, T. Analysis and characterization of FREP and cathepsin gene families in the ACP (Hemiptera: Liviidae). Florida Academy of Sciences. Lakeland, FL *DeAvila, D., *Cordola, C., *Villalobos-Ayala, K., *Bell, T., *Mann, H., *DeAvila, G., Hunter, W., Wiersma-Koch, H., & D'Elia, T. Genomic analysis of CLIP-domain serine protease subfamilies in D. citri (Hemiptera: Liviidae). Florida Academy of Sciences. Lakeland, FL *DeAvila, D., *DeAvila, G., *Bell, T., *Villalobos-Ayala, K., *Cordola, C., *Mann, H., Wiersma-Koch, H., Hunter, W., & D'Elia, T. Rab11A RNA interference in D. citri (Hemiptera: Liviidae): Analysis of mortality. Florida Academy of Sciences. Lakeland, FL *DeAvila, G., *DeAvila, D., *Bell, T., *Villalobos-Ayala, K., *Cordola, C., *Mann, H., Hunter, W., D'Elia, T., & Wiersma-Koch, H. Core RNAi Pathway Genes and Auxiliary Factors in D. citri (Hemiptera: Liviidae). Florida Academy of Sciences. Lakeland, FL *Hoyt, S., *Ramsey, J., Mueller, L., Cilia, M., & Saha, S. Taxonomic Analysis of the Endosymbionts in the ACP through Metagenomics. 16th Annual PGRP Symposium. Ithaca, NY *Kruse, A., Shatters, R., Cilia, M., MacCoss, M., & Saha, S. Comparative proteomics and microscopy provide insights into transmission of CLas by the ACP. 5th IRCHLB. Orlando, FL *Yashenko, A., *Kruse, A., Hosmani, P., Mueller, L., Cilia, M., & Saha, S. Characterizing long non-coding RNA in the ACP through genome annotation and molecular biology. 16th Annual PGRP Symposium. Ithaca, NY How have the results been disseminated to communities of interest?In addition to scientific publications listed in the Product Section, dissemination of results are described below: 3rd Annual Citrus Greening Solutions Meeting. UC Davis. December 2017. Attended: 4 graduate students, 7 key personnel, 6 co-PIs, 14 project members, & 9 SSAC members Objective 1 activities include: 12 videos on youtube related to MicroCT of the ACP. Alba-Tercedor, J https://www.youtube.com/user/albatercedor Discussions with psyllid research community in Texas & Washington. Saha, S D. citri genome assembly DIACI 1.1. Ag Data Commons. http://dx.doi.org/10.15482/USDA.ADC/1342728 D. citri genome assembly DIACI 1.9. Ag Data Commons. http://dx.doi.org/10.15482/USDA.ADC/1342727 Objective 2 activities include: Understanding Biology Through Structure. Symposium. Gupta Lab Synthetic Immunity. Symposium. Gupta Lab The Battle Within & the Battle Without! When Insect, Bacterium and Plant Interactions Attack an Industry: Citrus Greening Disease. Community Science Festival. Shatters Lab Citrus Greening. Interview. College of Agriculture & Environmental Sciences. Slupsky, C 3rd Annual CGS Meeting. Interview. CBS 13 News. Slupsky, C Citrus Greening. Interview. Nature. Slupsky, C Citrus Greening. Interview. California Aggie. Slupsky, C Systems Biology of CLas Interaction with Citrus & ACP, and Metabolomics as an EDT. Seminar. Citrus Research & Development Foundation. Slupsky, C Insect transmission of plant pathogens: lessons from systems biology. Seminar. Entomology Society of America. Cilia, M Insect transmission of plant pathogens: lessons from proteomics. Seminar. Pacific Northwest Mass Spectrometry Group Meeting. Cilia, M Citrus greening web portal. Seminar. 5th IRCHLB meeting. Mueller lab Current Situation & Ongoing Research on HLB: a Devastating Disease of Citrus. Seminar. New Mexico Consortium. Stover, E HLB & the US citrus industry: Status & ongoing research. Seminar. University of Maryland. Stover, E Objective 3 activities include: Multiple conference papers & posters Focus groups held in CA, FL, IL & NJ Guest lectures to students & industry organizations including feedback and discussion Outcomes of this project have also been disseminated to the greater scientific community through local, national & international conference & symposium presentations listed below: Project Members in bold font Alba-Tercedor, J, Hunter, W, Cicero, J, & Brown, S. Micro-CT scanning of ACP, D. citri, anatomy & feeding. 5th IRCHLB. Orlando, FL Flores, M, Hosmani, P, Brown, S, Fernandez-Pozo, N, & Mueller, L. Community portal for the Citrus Greening disease resources. 10th AGS. Notre Dame, IN Flores, M, Hosmani, P, Brown, S, Fernandez-Pozo, N, Mueller, L, & Saha, S. Systems biology resources for D. citri, a vector for the Citrus Greening disease. 10th AGS. Notre Dame, IN Flores, M, Saha, S, Hosmani, P, Brown, S, & Mueller, L. Systems biology resources for the citrus greening disease complex. 5th IRCHLB. Orlando, FL Gupta, G. Understanding of plant innate immune response and engineering of novel innate immunity for pathogen clearance & disease protection. GPMB. Valencia, Spain Hosmani, P, Flores, M, D'Elia, T, Hunter, W, Brown, S, Mueller, L, & Saha, S. Biocuration: Deciphering the draft genome of ACP one gene at a time. 5th IRCHLB. Orlando, FL Hosmani, P, Flores, M, Fernandez-Pozo, N, Brown, S, Mueller, L, & Saha, S. Utilizing system biology resources to decipher a tritrophic disease complex. ESA 2017. Denver, CO Hosmani, P, Flores, M, Hunter, W, Brown, S, Mueller, L, & Saha, S. Why we need to improve the D. citri genome. 5th IRCHLB. Orlando, FL Hosmani, P, Flores, M, Hunter, W, Brown, S, Mueller, L, & Saha, S. Using long reads, optical maps & long-range scaffolding to improve the D. citri genome. 5th IRCHLB. Orlando, FL Hosmani, P, Flores, M, Hunter, W, D'Elia, T, Brown, S, Mueller, L, & Saha, S. Biocuration & improvement of the D. citri draft genome assembly with long reads, optical maps & long-range scaffolding. 10th AGS. Notre Dame, IN Hosmani, P, Flores, M, Miller, S, Shippy, T, Hunter, W, D'Elia, T, Cilia, M, Brown, S, Mueller, L, & Saha, S. Deciphering the genome of the insect vector D. citri to develop solutions for the citrus greening disease. PAG XXVI. San Diego, CA Hosseinzadeh, S, Ramsey, J, Hunter, W, Mahoney, J, Hall, D, & Cilia, M. Expression of D. citri hemocyanin protein correlates with color morphology & exposure to the HLB pathogen CLas. 5th IRCHLB. Orlando, FL Hunter, W, Metz, J, Lopez, S, Altman, S, Aishwarya, V, & Pelz-Stelinski, K. Method for Detecting Binding Efficiencies of synthetic Oligonucleotides: Targeting Bacteria & Insects. 10th AGS. Notre Dame, IN Hunter, W, Metz, J, Mojica, A, Altman, S, Boyle, M, Aishwarya, V, McCollum, G, & Pelz-Stelinski. K. FANA & Morpholino Treatments for Targeting Pathogens in Citrus Trees & ACP. 5th IRCHLB. Orlando, FL Mahoney, J, Warwick, E, Johnson, R, MacCoss, M, Ramsey, J, Howe, K, Hall, D, Shatters, R, Cilia, M, & Slupsky, C. Multi'omics reveal adult female & male D. citri respond similarly to CLas exposure. HPIS 2017. Madrid, Spain Mahoney, J, Warwick, E, MacCoss, M, Johnson, R, Ramsey, J, Krasnoff, S, Howe, K, Moulton, K, Shatters, R, Hall, D, Cilia, M, & Slupsky, C. Employing multi'omics to assess effects of sex & citrus variety on D. citri - CLas interactions. Microbiology Graduate Research Symposium. Davis, CA Metz, J, Hunter, W, Boyle, M, Mojica, A, McCollum, G, Pelz-Stelinski, K, & Aishwarya, V. Antisense Oligonucleotides, FANA_ASO Reduces Pathogens & Pest of Fruit Crops: Citrus & Grapevine. PAG XXVI. San Diego, CA Metz, J, Hunter, W, Mojica, A, Altman, S, Boyle, M, McCollum, G, Aishwarya, V, & Pelz-Stelinski, K. Delivery of Gene BioTechnologies to Plants: Pathogen & Pest Control. 10th AGS. Notre Dame, IN Saha, S. Community resources for all y'all Omics. 5th IRCHLB. Orlando, FL Saha, S. Sequencing an arthropod genome using single molecule reads & long-range scaffolding technologies. ESA 2017. Denver, CO Shatters, R. Citrus Greening Disease: Using Studies on Molecular Interactions to Develop Novel Interdiction Strategies in the Tritrophic Relationship between Citrus, the ACP & the CLas Bacterium. 34th MAPMBS. Laurel, MD Shatters, R, Borovsky, D, Ammar, E, Hall, D, Sturgeon, K, Warwick, E, Giulianotti, M, Santos, R, & Pinilla, C. Identification of gut epithelium binding peptides that reduce systemic movement of CLas within the ACP vector. 5th IRCHLB. Orlando, FL Shatters, R, Borovsky, D, Sturgeon, K, Warwick, E, & Powell, C. Development of a screening bioassay for in planta evaluation of bactericidal compounds & it's use in discovery of new bactericidal compounds active against CLas within citrus. 5th IRCHLB. Orlando, FL Shi, Q, Andrade, E, Zhang, S, Hunter, W, Shatters, R, & Stover, E. Regulation of gene expression through root application of double-stranded RNA in citrus. 5th IRCHLB. Orlando, FL Slupsky, C. HLB detection using plant metabolites. Citrus Greening Research Review: HLB Diagnostics & Detection. Webinar Stover, E. Advances in Identifying & Developing Resistance & Tolerance to HLB in Citrus. Florida Grower Meetings & University of California Citrus Extension Seminars. FL & CA. Stover, E. Advances in Identifying & Developing Resistance & Tolerance to HLB in Citrus. 5th IRCHLB. Orlando, FL Stover, E. Update on USDA Citrus Scion Breeding Focusing on "Sweet Orange". Florida Processing Orange Board. Lake Wales, FL Stover, E. Breeding Citrus Scion Cultivars: New Opportunities & Overcoming Obstacles. American Society for Horticultural Sciences. Wiakoloa, HI Stover, E. HLB & the US Citrus Industry. Hawaii Tropical Fruit Growers Annual Meetings. HI 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 quality, and prevent fruit drop. Objective 2 activities planned to meet the above goals. ACP exhibits three different color morph variants, blue, gray and yellow. Blue morphs have a greater capacity for long-distance flight as compared to non-blue morphs, but little else is known about how color morphology influences vector characteristics. We show that the color morphology of the insect is derived from pigmented cells of the fat body. Blue morphs acquire a lower level of CLas in their bodies from infected trees as compared to their grey and yellow conspecifics. Accordingly, CLas titer in citrus leaves inoculated by yellow or grey insects was 6-fold higher than in leaves inoculated by blue insects. A greater proportion of blue insects can be observed on CLas infected plants as compared to healthy plants on two citrus host varieties, suggesting some degree of plasticity in the color morph phenotype dependent on the presence of CLas infection in the plant. Blue color morphs also harbored lower titers of Wolbachia and 'Ca. Profftella armatura,' two of the D. citri bacterial symbionts. Expression of hemocyanin, a copper-binding oxygen transport protein responsible for the blue coloration of hemolymph of other arthropods and mollusks, was previously correlated with blue color morphology and is highly up-regulated in insects continuously reared on 'Ca. L. asiaticus' infected citrus trees. Our future plans include an 'omics analysis comparing different color morphs of the ACP, sex, and the influence of the host plant. These data will be invaluable for the Psyllid Expression Network (part of Objective 1) and for our understanding of the influence of color morphology on CLas acquisition and transmission. Gut-binding peptides provide only an approximately 50% reduction in successful acquisition of CLas by the ACP. We therefore have to plan for using this information in development of new solutions to citrus greening. Use them as tags to identify the gut molecules they bind to and use this information to identify more potent inhibitors. Combine these with other peptides we have identified (antimicrobial peptides active against CLas) to provide a solution that results in both reduce CLas titer in citrus and in psyllids and to greatly reduce the ability of the psyllids to acquire CLas so that transmission to other citrus trees in significantly reduced. We have synthesized large batches of these peptides and are initiating whole tree trials of topically applied peptides. We will use the results of our study as the foundation on which to build an understanding of the effects of the interdiction molecules on ACP health as well as citrus health being developed by the Shatters, Gupta, Hunter, and Stover labs. Specifically, our results have led to several hypotheses that we will be able to test with interdiction molecules for direct involvement in CLas transmission and interrogate when comparing good vs poor CLas transmitters. This may be important for development of novel molecules or ecological strategies to reduce transmission of CLas in both nymphs vs adults. Our finding that peptides can be taken up in an effective manner by citrus trees has created a strategy to evaluate the use of topically applied peptides with biological activity targeting both the psyllids and the CLas bacterium. We will conduct application experiment to evaluate the effect of antimicrobial and gut-binding peptides on CLas and ACP when applied directly to intact citrus trees. Test the safety and efficacy of various interdiction/antimicrobial molecules in reducing and preventing CLas transmission, including its effect on the insect vector and plant. Continue creating constructs and transgenics Testing of therapies delivered exogenously and through transgenics Larger trial being repeated, to determine if treatments can be improved. If these can be improved researchers will move towards discussions with the citrus industry representatives, and initiate review on how to register products through EPA for use in citrus. 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 industry outreach plan based on the outcomes of the focus groups Outline efforts to increase industry awareness of progress from the grant and the potential of forthcoming solutions being investigated in the other objectives Reach out to industry effectively and efficiently Identify target messages for the industry audiences Finish focus groups research using key messages from survey data. Focus group results will be published via scientific journals and conferences. Recommendations for communications to consumers by the industry will be developed and disseminated as part of the first objective's plan. Research will include aspects of both attitudes toward biotechnology and knowledge of HLB and its impact on the U.S. citrus industry. 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. Establishing more field trials to develop delivery systems for optimal plant uptake. This work is being done in conjunction with industry that will support delivery of the system to growers. Initiate field trials to evaluate delivery methods. A 3D printer was purchased to synthesize prototype delivery devices that will be tested in greenhouse and field experiments. Students increase knowledge of bioinformatics and genome analysis. Database construction: New data visualization tools. Objective 1 activities planned to meet the above goals. Recruit a new cohort of student annotators to join the annotation team More experienced peers will train new student annotators on the standard operating processes through the spring and summer 2018 Undergraduate students will generate reports to contribute to the publication of the Official Gene Set v2.0 based on the updated genome. Continue working with the scientists performing experiments to leverage their knowledge and expertise to target genes found to be of functional importance for manual curation The new genome and annotation (v2.0) will be loaded into the Citrus Greening web portal database to create pages for all genes connected to relevant databases to enable researchers to analyze each gene in detail Jbrowse genome browser and all blast databases will be updated with the new datasets Generate new version of the DiaphorinaCyc pathway database based on the new gene set New experiments will be added to the Psyllid Expression Network for expression data generated for proteomics and transcriptomics experiments when mapped to the new annotation Generate full-length cDNA transcripts from diseased and healthy ACP tissue from multiple life stages with the Pacbio IsoSeq technology. This will be combined with Illumina data generated from RNA sequencing for Psyllid Expression Network to create a updated and comprehensive transcriptome for the ACP. Public webinar will be held in March 2018 to unveil the new ACP v2.0 genome and annotation for the citrus greening community. Tools like Psyllid Expression Network and DiaphorinaCyc will be demonstrated for new users to encourage more researchers to utilize the data generated by this NIFA project and also submit their own data to the portal.

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. To investigate the effects of CLas exposure at the gut-pathogen interface, we performed RNAseq and mass spectrometry-based proteomics to analyze the transcriptome and/or proteome of ACP whole body nymph and adults, gut and hemolymph tissue, and insects sorted by color, sex, and plant variety. Mass spectrometry analysis of salivary sheath proteome identified candidate proteins for RNA aptamer selection. Fluorescent in situ hybridization was used to demonstrate that Wolbachia and CLas inhabit the same ACP gut cells, but do not co-localize within those cells. Wolbachia titer is more variable in the guts of CLas exposed insects and was positively correlated with CLas titer in the insects. CLas is detected on the luminal membrane, in puncta within the gut epithelial cell cytoplasm, along actin filaments in the gut visceral muscles, and rarely, in association with gut cell nuclei. Karyorrhexis, (fragmentation of the nucleus), in midgut epithelial cells is increased in severity and frequency in response to CLas. Nymphs exhibit lower levels of early-stage karyorrhexis compared to adults and are refractory to the induction of advanced karyorrhexis by CLas in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experience oxidative stress in response to CLas. Resistance of the nymph midgut to late stage karyorrhexis through as yet unknown molecular mechanisms benefits CLas for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of CLas acquisition by this vector. Identified a primary inhibitor peptide from a cocktail of gut-binding peptides that reduces ACP transmission of CLas. Applied these directly to citrus leaves and demonstrated similar activity in ACP that feed on these leaves. Shown systemic uptake of these peptides after exogenous application directly to citrus trees. These results demonstrate the possibility of direct delivery of peptides to plants to control HLB. An antimicrobial peptide with activity against CLas within citrus leaves was identified and also shown to reduce the acquisition of CLas in ACP fed on leaves treated with this peptide. A topical application method that allows the peptide to move systemically within citrus was identified. Up to 90% reduction in the bacterial population within treated citrus leaves and almost complete inhibition of acquisition by the ACP occured after one week treatment. This peptide has demonstrated toxicity to ACP perhaps due to its effect on the ACP endosymbionts. To understand how CLas affects the ACP vector, we completed analysis of the transcriptome, metabolome, and microbiome of adult male and female ACP +/- CLas. 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. Initial field tests have shown localized topical application to specific vegetative tissues results in effective systemic movement of bactericide molecules throughout the citrus tree canopy. This delivery method was subsequently shown to work for peptide uptake. Our work on citrus and ACP exposed to CLas has revealed specific responses to the bacterium. -To develop transgenic citrus, verified phloem-specific promoters have been used to construct CLas and ACP directed constructs. These potential therapies were tested in detached leaf assays. Created transgenic citrus trees expressing priority transgenes to test in glasshouse and field conditions. Developed antisense oligonucleotides and showed that they reduce microbial loads in ACP, and CLas infected citrus trees. Engineered helix-turn-helix endogenous amphipathic peptide which showed higher anti-Liberibacter activity than streptomycin. This peptide is used in spray treatment of HLB in greenhouse. 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. National and citrus-state survey data were analyzed to increase understanding of consumer attitudes. Survey results were used to develop key messages for testing consumer audiences via targeted survey and focus group research in four key media markets across the country. Conducted focus group with the SSAC to identify their understanding of what the grant efforts are doing to address the disease. The data is being analyzed to better inform the planning of industry communications efforts. Increased awareness of novel therapeutic application technologies and products by industry segment. Industry understands economic feasibility and practicality of management recommendations. Objective 2 activities highlighted below pertain to the research goals listed above. Increased awareness of our novel therapeutic methods in both producer and agricultural chemical company audiences. Field demonstrations were presented to select groups of growers and chemical company representatives under confidentiality agreements to obtain feedback from these audiences about concerns regarding our technology and adapting our methods to meet these concerns. As part of a new Cooperative Research and Development Agreement (CRADA) to support advancing the technology to a commercial product, economic impacts were evaluated, and management recommendations were made through interaction with commercial grove managers. Students increase knowledge of bioinformatics and genome analysis. Database construction: New data visualization tools. Objective 1 activities highlighted below pertain to the research goals listed above. 15 undergraduate students and 5 annotation experts used Basecamp to manage documents and met biweekly via videoconferencing, which provided timely feedback to student curators Focusing on pathways of interest fostered interactions between the lab scientists and undergraduate annotators. More than 180 genes from 11 pathways in the ACP genome have been manually curated using Apollo annotation tool by student annotators at IRSC, Cornell/BTI and KSU. 2 undergraduate student interns started in the summer at BTI. One validated the presence of specific long non-coding RNA in nymphs and discovered that the host plant effects long non-coding RNA expression. The other analyzed whole-body ACP DNA sequences for differences in taxonomy and abundance of endosymbionts. The community-driven genome annotation for Official Gene Set v1.0 identified a number of misassemblies and missing genes in the current v1.1 genome. Therefore, we created a new annotation platform with the a new v1.91 genome and the latest version of Apollo curation tool. The new Pacbio v1.91 genome assembly was polished with Pacbio and Illumina reads. Dovetail Chicago libraries were used to scaffold contigs. We removed redundant scaffolds from the assembly to create a reference and alternate set of contigs. The improved v2.0 reference assembly has 1906 contigs and a contig N50 of 759Kb while the current v1.1 genome has 161,988 contigs with a contig N50 of 34Kb. The Psyllid Expression Network was extended by adding scatter plots for visualizing the correlation of expression values of genes in two samples in the correlation matrix. MicroCT videos are now embedded in the psyllid genome page.

Publications

  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. & Alba-Alejandre, I. (2017). Comparing micro-CT results of insects with classical anatomical studies: The European honey bee (Apis mellifera Linnaeus, 1758) as a benchmark (Insecta : Hymenoptera , Apidae), Bruker Micro-CT Users Meeting 2017 (pp.147-167). Bruker-microCT-Skyscan, Kontich, Belgium. 9789081678100
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Alba-Tercedor, J., Hunter, W., Cicero, J., Sainz-Bariain, M., & Brown, S. (2017). Use of micro-CT to elucidate details of the anatomy and feeding of the Asian Citrus Psyllid Diaphorina citri Kuwayama, 1908 (Insecta: Hemiptera, Liviidae), Bruker Micro-CT Users Meeting 2017 (pp.270-285). Bruker-microCT-Skyscan, Kontich, Belgium. 9789081678100
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Alba-Tercedor, J., Hunter, W., Cicero, J., S�inz-Bari�in, M., & Brown, S. (June 2017). Use of micro-CT to elucidate details of the anatomy and feeding of the Asian Citrus Psyllid Diaphorina citri Kuwayama, 1908 (Insecta: Hemiptera, Liviidae). Bruker Micro-CT Users Meeting 2017, http://bruker-microct.com/company/UM2017/AbstractB
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Basu, S., Rabara, R., Huynh, L., Zhang, S., Nguyen, H., Guzman, J., Hao, G., Shi, Q., Stover, E., & Gupta, G. (January 2018). Investigation of Liberibacter-citrus protein-protein interactions. 2018 Plant and Animal Genome XXVI Conference XXVI, San Diego, CA
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Beattie, P., Lamm, A., Rumble, J., & Ellis, J. (February 2018). Identifying generational differences to target extension programming when discussing genetic modification. American Association for Agricultural Education Southern Region Conference, Jacksonville, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Coates, L., Mahoney, J., Warwick, E., Johnson, R., MacCoss, M., Ramsey, J., Howe, K., Hall, D., Shatters, R., Cilia, M., & Slupsky, C. (June 2017). Employing multi'omics to assess effects of sex and citrus variety on Diaphorina citri - CLas interactions. Hemiptera-Plant Interactions Symposium, Madrid, Spain
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Hao, G., Gupta, G., Duan, Y., & Stover, E. (March 2017). Production of transgenic citrus resistant to citrus canker and Huanglongbing diseases. 5th International Research Conference on Huanglongbing, Orlando, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Huynh, L., Rabara, R., Basu, S., Nguyen, H., & Gupta, G. (May 2017). Rational design of new antimicrobial peptides with reduced susceptibility to bacterial resistance. Symposium on Understanding Biology Through Structure, Santa Fe, NM
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Huynh, L., Rabara, R., Vela?squez Guzma?n, J., Basu, S., Nguyen, H., & Gupta, G. (February 2018). Structure-Activity Correlations of Amphipathic Cationic AMPs: MD simulations and antibacterial measurement. The 62nd annual meeting Biophysical meeting, San Francisco, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rabara, R., Huynh, L., Vela?squez Guzma?n, J., Nguyen, H., Basu, S., Zhang, S., & Gupta, G. (January 2018). Understanding the mechanism of E. coli resistance to membrane-targeting antimicrobial through genomic approach. 2018 Plant and Animal Genome Conference XXVI, San Diego, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Ruth, T., Rumble, J., Lamm, A., & Ellis, J. (May 2017). A theoretical model for decision-making related to agriculture and natural resource science and technology. American Association for Agricultural Education National Conference, San Luis Obisbo, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Velasquez Guzman, J., Basu, S., Huynh, L., Nguyen, H., & Gupta, G. (February 2018). Liposome Delivery System of Antimicrobial Peptides against Huanglongbing (HLB) Citrus Disease. The 62nd annual meeting Biophysical meeting, San Francisco, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Velasquez Guzman, J., Basu, S., Huynh, L., Nguyen, H., & Gupta, G. (February 2018). Structure-Activity Correlations of Amphipathic Cationic AMPs: MD simulations and antibacterial measurement. The 62nd annual meeting Biophysical meeting, San Francisco, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Velasquez Guzman, J., Basu, S., Rabara, R., Huynh, L., Nguyen, H., Shaw, J., Zhang, S., Shi, Q., Stover, E., & Gupta, G. (January 2018). Liposome Delivery System of Antimicrobial Peptides against Huanglongbing (HLB) Citrus Disease. 2018 Plant and Animal Genome XXVI Conference XXVI, San Diego, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zhang, S., Hao, G., Shi, Q., Stover, E., Huynh, L., Velasquez Guzman, J., Nguyen, H., Basu, S., & Gupta, G. (November 2017). HLB Protection and Treatment by Engineering of Novel Innate Immunity. 2017 Materials Innovation for Sustainable Agriculture Annual Symposium, Orlando, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zhang, S., Shaw, J., Shi, Q., Stover, E., Huynh, L., Rabara, R., Velasquez Guzman, J., Nguyen, H., Basu, S., & Gupta, G. (November 2017). Design and Testing Membrane - Targeting Antimicrobials for Liberibacter Clearance. 2017 Materials Innovation for Sustainable Agriculture Annual Symposium, Orlando, FL
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ammar, E., Hall, D., & Shatters, R. (March 2017). Ultrastructure of the salivary glands, alimentary canal and bacteria-like organisms in the Asian citrus psyllid, vector of citrus huanglongbing disease bacteria (pp.9-20). Journal of Microscopy and Ultrastructure, 5(1), Amsterdam, The Netherlands. doi:j.jmau.2016.01.005
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Avila, L., Brown, S., Reeck, G., Park, Y., & Tomich, J. Delivery of Lethal dsRNAs in Insect Diets by Branched Amphiphilic Peptide Capsules. Journal of Controlled Release
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Brown, S., Cilia, M. L., Saha, S., Munoz-Torres, M., Cordola, C., Bell, T., Villalobos-Ayala, K., Guzman, V., Hunter, D., Shatters, R., Hunter, W., Mueller, L., Izsak, A., Benoit, J., Hosmani, P., Flores, M., Dunlevy, M., Morgan, T., Mann, H., Wiersma-Koch, H., Shore, K., D'Elia, T., DeAvila, G., DeAvila, D., Dixon, D., Miller, S., Shippy, T., Rosendale, A., Cano, L., Moore, Z., Buller, K., Ciolkevich, K., Nandyal, S., Mahoney, R., Van Voorhis, J., Farrow, D., Cridge, A., Cao, X., Jiang, H., Leng, N., Johnson, S., Cantarel, B., Richards, S., English, A., Childers, C., Chen, M., Nelson, D., & Poelchau, M. (June 2017). Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community (pp.bax032). Database, 2017, Oxford , UK. doi:10.1093/database/bax032
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Cicero, J., Adair, M., Adair, Jr., R., Hunter, W., Avery, P., & Mizell III, R. (April 2017). Predatory behavior of long-legged flies (Diptera: Dolichopodidae) and their potential negative effects on the parasitoid biological control agent of the Asian citrus psyllid (Hemiptera: Liviidae).(pp.485-487). Florida Entomologist, 100 (2), USA. doi:https://doi.org/10.1653/024.100.0243
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: D'Angelo, J., Ellis, J., Burke, K., & Ruth, T. Media portrayal of GM science and citrus greening in state and national newspapers. Journal of Applied Communications
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kruse, A., Saha, S., Fattaha-hosseini, S., Warwick, E., Sturgeon, K., Johnson, R., MacCoss, M., Mueller, L., Shatters, R., & Cilia, M. (June 2017). Combining 'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut. (e0179531). PlosOne, 12(6), Ithaca, NY. 1932-6203
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Lamm, A., Melissa, T., Rumble, J., & Ellis, J. Targeting extension programs to opinion leaders guiding genetic modification discussions. Journal of Human Sciences and Extension
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Mann, M., Fattaha-hosseini, S., Ammar, E., Strange, R., Sturgeon, K., Warwick, E., Shatters, R., & Cilia, M. Diaphorina citri nymphs are resistant to morphological changes induced by Candidatus Liberibacter asiaticus in midgut epithelial cells. Infection and Immunity. doi:10.1128/IAI.00889-17
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ruth, T., Lamm, A., Rumble, J., & Ellis, J. (December 2017). Identifying publics in citrus producing states to address the issue of citrus greening.(pp.1-14). Journal of Applied Communications, 101(3), Manhattan, KS. doi:https://doi.org/10.4148/1051-0834.1847
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Ruth, T., Lamm, A., Rumble, J., & Ellis, J. Conversing about citrus greening: Extensions role in educating about genetic modification science as a solution.(pp.35-50). Journal of Agricultural Education, 58(4). doi:10.5032/jae.2017.04035
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Ruth, T., Rumble, J., Lamm, A., & Ellis, J. A model for understanding decision making related to agriculture and natural resource science and technology. Journal of Agricultural Education
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Digital Video Library of Asian Citrus Psyllid (Diaphorina citri): 1. https://youtu.be/Vu0h6TH9btA
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Digital Video Library of Asian Citrus Psyllid (Diaphorina citri): 3. https://youtu.be/s0epsA2SKPY
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Digital Video Library of Asian Citrus Psyllid (Diaphorina citri): 4. https://youtu.be/GNfu-3Yzd40
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Digital Video Library of Asian Citrus Psyllid (Diaphorina citri): 5 (Abdominal tip and Spermatheca). https://youtu.be/oKLTtonOwV0
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Digital Video Library of Asian Citrus Psyllid (Diaphorina citri): 5b (Abdominal tip and Spermatheca). https://youtu.be/kaoXkWMcwF8
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Digital Video Library of Asian Citrus Psyllid (Diaphorina citri): 6 (Cross sections). https://youtu.be/-vy0HtA8cR0
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Micro CT study of the male adult of the Asian Citrus Psyllid (Diaphorina citri): 1. https://youtu.be/wpUOWibrbOw
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Micro CT study of anatomy of the male of the Asian Citrus Psyliid (Diaphorina citri): 2. https://youtu.be/C_PVrAfQREs
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Micro-CT of the Asian Citrus Psyllid feeding on a leaf of an orange tree. https://youtu.be/qpKUNDKG08M
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Micro-CT study of anatomy of the male of the Asian Citrus Psyliid (Diaphorina citri) I. https://youtu.be/dW3IqO8ELfA
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Micro-CT study of the male adult of the Asian Citrus Psyllid (Diaphorina citri). II. https://youtu.be/BscpgXkyg6s
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Micro-CT study of the Asian Citrus Psyllid (Diaphorina citri): feeding apparatus and stylets. https://youtu.be/baWuwRmO7Lg
  • Type: Other Status: Published Year Published: 2017 Citation: Alba-Tercedor, J. (December 2017). Testing "in vivo" contrast agent for micro-CT to enhance the digestive in Ceratitis capitata. https://youtu.be/Q8TQyCxRPdk


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.