DEVELOPING AN INFRASTRUCTURE AND PRODUCT TEST PIPELINE TO DELIVER NOVEL THERAPIES FOR CITRUS GREENING DISEASE

Target Audience
The goal of Objective 1 is: Students increase knowledge of bioinformatics, genome analysis and database construction: New data visualization tools. The target audience continued to include 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 made available publicly. The analysis of pathways will provide a basis for molecular based studies on the ACP. 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 continued 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. Additionally other target audiences are scientists in the field, citrus growers, citrus industries, regulators (FDA/EPA) and citrus/agricultural researchers. In addition, some of our developments are so novel and broad reaching that, if successfully demonstrated it could target growers of many crops. 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 continued to include social scientists, consumers of citrus and citrus products, citrus industry representatives including growers, producers, distributors and retailers.

Changes / Problems
Nothing Reported

Training & Professional Development
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 experienced annotators mentor and train new students. Peers and mentors met once a week and all local annotators at IRSC met with their instructor 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 ACP 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 - Guest scientists joined in that were experts on the students pathways to help provide insights into completing the annotation (bioinformatics) and reporting of results - Mentored 17 students that completed over 400 gene models for genome version 3. Students presented at 2 state conferences (8 posters total) and 1 nation conference (5 posters). Students prepared 7 pathway reports for bioRxiv preprint. - 5 IRSC, 2 KSU undergraduate student annotators and 1 KSU senior annotator attended the 2019 Arthropod Genomics Symposium Pre-Symposium Workshop: CURATION CLINIC: Gene Annotation using Apollo held in Manhattan, Kansas on June 12, 2019 - 5 students prepared a poster to present the results of their genome annotation and to showcase the skills they learned in bioinformatics at the 2019 Arthropod Genomics Symposium - 4 students presented posters and attended scientific talks at the 2019 Florida Academy of Sciences meeting and the 2019 Florida Phytopathological Society meeting - Max Reynolds, IRSC undergraduate intern, won 1st place for his Outstanding Undergraduate Student Poster Presentation at the 2019 FAS meeting in Melbourne, FL - Crissy Massimino, IRSC program coordinator, won 1st place for her Honorable Mention Undergraduate Student Poster Presentation at the 2019 FAS meeting in Melbourne, FL - University of Florida graduate student, Julie Boswell, received 1st place graduate presentation for Agricultural and Natural Resources at the 83rd Annual Meeting Florida Academy of Sciences held in Melbourne, FL March 8-9, 2019 - University of Florida graduate student, Julie Boswell, was a Symposia Organizer for session AGRI/Natural Resources Symposia at the Florida Academy of Sciences in Melbourne, FL on March 6-8, 2019 - Students have used their knowledge gained through participation in annotation to advance to careers and graduate school. Several students have changed their career path because of the interest and knowledge gained with interning for this project. - Former IRSC intern Chad Vosburg, started a Masters degree at Penn State University. His experience as an intern helped secure his acceptance to the Department of Plant Pathology & Environmental Microbiology. - Former IRSC intern Yesmarie De la Flor, started a career at the Smithsonian Marine Station as a Research Technician working on coral diseases caused by bacteria. Her bioassay experience as an intern helped solidify her career decision. - Former IRSC interns Kristine Aguirre, Denisse Hasan, Max Reynolds & Margaryta Rzhevska Jernigan, are now Research Technicians at USDA-ARS researching citrus. Their experience as an intern provided valuable experience before accepting jobs with USDA-ARS. - Former IRSC intern Jordan Norus, is now a Research Technician at the University of Florida Medical Entomology Lab. His experience as an intern annotating the ACP helped secure this job. - Angela Kruse, graduate student at Cornell/BTI, completed her Ph.D. under the direction of Dr. Michelle Heck - Marina Mann, graduate student at Cornell/BTI, is being advised by Dr. Michelle Heck. Marina has been working on completing the transcriptomics analysis for the ACP expression network. -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 *Boswell J, Hunter WB, & Lopez S. (March 2019). Passive phloem release of Candidatus Liberibacter asiaticus from sweet orange [AGR-01]. 83rd Annual Meeting Florida Academy of Science. Melbourne, FL *Coates L, Mahoney J, Ramsey J, Warwick E, Johnson R, MacCoss M, Krasnoff S, Howe K, Moulton K, Saha S, Mueller L, Hall D, Shatters R, Heck M, Slupsky C. (March 2019). Sex-dependent effects of CLas exposure on Diaphorina citri. IRCHLB. Riverside, CA *Coates L, Padhi E, Leveau J, Heck M, & Slupsky C. (January 2020). The use of multi-omics approaches to understand the effects of CLas on citrus and its insect vector Diaphorina citri. CEND. Berkeley, CA D'Elia T & *Vosburg C. (June 2019). Genomic structure and characterization of Wnt signaling genes in Diaphorina citri. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Kercher K, & *Tamayo B. (June 2019). A metabolic insight into the Asian citrus psyllid: Annotation of glycolysis and gluconeogenesis pathways. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Kercher K, & *Tamayo B. (March 2019). Glycolysis and Gluconeogenesis Pathway Annotation in Diaphorina citri. Florida Academy of Sciences. Melbourne, FL D'Elia T, Wiersma-Koch H, & *Rahmoune A. (March 2019). Identification and Annotation of Chromatin Remodeling Complexes in Diaphorina citri. Florida Academy of Sciences. Melbourne, FL D'Elia T, Wiersma-Koch H, *Vosburg C, & *DeLaFlor Y. (June 2019). Annotation of genes from the melanin biosynthesis pathway in the citrus greening vector, Diaphorina citri. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Vosburg C, *DeLaFlor Y, & *Massimino E. (March 2019). A Pathway to Recovery: Evolutionary analysis of the yellow gene family in the citrus greening vector, Diaphorina citri. Florida Academy of Sciences. Melbourne, FL D'Elia T, Wiersma-Koch H, *Vosburg C, *DeLaFlor Y, & *Massimino E. (March 2019). Melanization pathway annotation in the Asian citrus psyllid. Florida Academy of Sciences. Melbourne, FL D'Elia T, Wiersma-Koch H, *Vosburg C, & *Massimino E. (June 2019). Molecular evolution of yellow genes in Diaphorina citri reveals duplications and losses. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Vosburg C, & *Reynolds M. (June 2019). Shedding Light on Cryptochrome and the Circadian Rhythm Pathway in Diaphorina citri. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Vosburg C, & *Reynolds M. (March 2019). Annotation of the Circadian Rhythm Pathway in Diaphorina citri. Florida Academy of Sciences. Melbourne, FL Heck M & *Hosseinzaedh S. (December 2019). Small RNA signaling in CLas-ACP interactions. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Heck M & *Kruse A. (December 2019). Proteomic and morphological studies guide understanding and interdiction of the Asian citrus psyllid stylet sheath. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Heck M & *Mann M. (December 2019). Elucidating tissue specific CLas-ACP interactions using transcriptomics. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Saha S, Mueller L, Hosmani P, Flores M, & *Mann M. (December 2019). New resources at the Citrusgreening.org portal and future work. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Slupsky C & *Coates L. (December 2019). Optimizing FANA ASO for Gene Silencing in Diaphorina citri. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Slupsky C & *McNeil C. (December 2019). Comparing the metabolic response of Citrus sinensis to diverse pathogens to identify unique responses to CLas. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL

Dissemination Streams
In addition to scientific publications listed in the Product Section, dissemination of our results has been described below: - 5th Annual Citrus Greening Solutions (CGS) Meeting. IRSC, Ft. Pierce, FL. December 10-12, 2019. Attended: 7 graduate students, 19 undergraduate students, 5 key personnel, 5 co-PIs, 19 project members, 5 SSAC members, 2 coordinators - 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 182 times by 503 authors in the world across 28 different countries, 206 of those authors being within the U.S. in 26 different states spanning across the disciplines of Biology, Agriculture, Engineering, Chemistry, Medical Sciences, and Social Science. -Project publications have been featured in 51 news stories, tweeted approximately 165 times with an audience reach of over 382,000 followers, and have over 700 Mendeley readers. Objective 1 activities include: - Meeting were held with researchers in the potato psyllid and pear psylla community from Texas and Washington in order to establish collaborations. The potato psyllid genome and transcriptome will be an important resource for comparative genomics of the ACP. - Researchers from the Mueller lab presented 1 talk and 2 posters about the web portal at the IRCHLB meeting in Riverside, CA (http://irchlb.org/hlb.aspx) Objective 2 activities include: - Dr. Carolyn Slupsky and lab members prepared and released an informational video on HLB. It was shared with media outlets and UC Davis media: https://youtu.be/dzKIigG24uE - Dr. Carolyn Slupsky was interviewed for the informational video called "Can Science Save California Citrus From Greening Disease?" https://www.youtube.com/watch?v=_KkHBFnmSW4 - Dr. Carolyn Slupsky talked with various news agencies about HLB. - Dr. Robert Shatters gave presentations at the annual Citrus Show in Fort Pierce, FL in January 2020 to describe the discovery and delivery of biologically based molecules. - Dr. 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. (December 2019). Micro-CT achievements, new discoveries, and presentation of the anatomical micro-CT atlas of the Asian Citrus Psyllid. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Brown S, Saha S, Hunter W, Mueller L, Alba-Tercedor J, Cano L, & Cicero J. (March 2019). Interactive digital video animation promotes accessibility of complex insect anatomy for cytological and molecular audiences in pursuit of solving the citrus greening problem in Florida. ToScA North American Symposium Gainesville, FL D'Elia T. (December 2019). Updates from manual curation and student engagement through genome annotation. 5th Annual CGS Meeting. Fort Pierce, FL Ellis J. (December 2019). Outcomes of multi-modal consumer research. 5th Annual CGS Meeting. Fort Pierce, FL Gupta G. (December 2019). HLB therapy: citrus derived antibacterial peptides and proteins. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Gupta G. (March 2019). HLB treatment by citrus-derived helix-turn-helix peptides. 6th IRCHLB. Riverside, CA Hunter W. (December 2019). Moving Biotech to Fields Trials - Citrus. 5th Annual CGS Meeting. Fort Pierce, FL Hunter W. (December 2019). Design and Efficacy Targeting CLas and Psyllid. 5th Annual CGS Meeting. Fort Pierce, FL Hunter W, Aishwarya V, Pelz-Stelinski K, Paris T, Miles G, McCollum G, Boyle M, Altman S, Holland C, & Sandoval-Mojica A. (March 2019). Advances in treatments to manage Huanglongbing: Targeting Liberibacter bacteria and psyllid endosymbionts. 257th American Chemical Society National Meeting & Expo. Orlando, FL Hunter W & Manthey F. (March 2019). The Effect of Iron on Prodigiosin Production in S. odorifera. 83rd Annual Meeting Florida Academy of Science. Melbourne, FL Hunter W & Tomich J. (January 2020). Adult Insect Gene Editing: BAPC-assisted-CRISPR-Cas9 Delivery into Adults for Heritable Gene Editing (Hemiptera). XXVIII International Plant & Animal Genome Conference. San Diego, CA Hunter W, Pelz-Stelinski K, & Sandoval-Mojica A. (March 2019). Antisense Oligonucleotides Targeting HLB in its Vector and Host. Joint 21st Conference of the IOCV Virologist and the 6th IRCHLB. Riverside, CA Hunter W, Tomich J, & Braswell E. (March 2019). BAPC-assisted-CRISPR-Cas9 Delivery into Nymphs and Adults for Heritable Gene Editing (Hemiptera). Joint 21st Conference of the IOCV and the 6th IRCHLB. Riverside, CA. Voted biggest advancement in psyllid pest management. Hunter W, Tomich J, & Gonzalez M. (April 2019). BAPC-assisted-CRISPR-Cas9 Delivery into Nymphs and Adults for Heritable Gene Editing (Hemiptera). American Society of Biochemistry & Microbiology Annual Meeting. Orlando, FL Hunter W, Tomich J, & Gonzalez M. (March 2019). BAPC-assisted CRISPR/Cas9 System: Targeted Delivery into Psyllid Adult Ovaries for Heritable Germline Gene Editing (Arthropoda: Hemiptera). 83rd Annual Meeting FAS. Melbourne, FL Hunter W, Tomich J, Gonzalez M, & Braswell E. (March 2019). BAPC-assisted CRISPR/Cas9 System: Targeted Delivery into Psyllid Adult Ovaries for Heritable Germline Gene Editing (Arthropoda: Hemiptera). 83rd Annual Meeting FAS. Melbourne, FL Saha S, Hosmani P, & Flores M. (December 2019). ACP version 3 genome, official gene set version 3 and Isoseq transcriptome. 5th Annual CGS Meeting. Fort Pierce, FL Shatters R. (January 2020). Screening and Discovery of Bactericidal Molecules Active Against Clas, Whole Tree Application Studies. Florida Citrus Show. Fort Pierce, FL Shatters R. (December 2019). Topical and Cell Transplant Technologies for Molecule Discovery. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Shatters R. (December 2019). Use of Bioassays for Molecule Discovery (lab bench to greenhouse). 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Shatters R, Stover E, & Gupta G. (December 2019). USDA progress toward developing HLB-tolerant/resistant Citrus scions. Florida Citrus Research Foundation. Leesburg, FL Shatters R, Stover E, & Gupta G. (November 2019). USDA progress toward developing HLB-tolerant/resistant Citrus scions. Citrus Research and Education Center, University of Florida seminar. Lake Alfred, FL Shatters R, Stover E, & Gupta G. (September 2019). USDA progress toward developing HLB-tolerant/resistant Citrus scions. Citrus Research and Development Foundation. Fort Pierce, FL Shatters R, Stover E, & Gupta G. (March 2019). Identifying and Developing Resistance and Tolerance to HLB. International Research Conference on Huanglongbing. Riverside, CA Stover E. (December 2019). Transgenic citrus targeting ACP (or ACP & CLas): tools for development, and delivery of identified therapeutic molecules. 5th Annual CGS Meeting. Fort Pierce, FL

Next Reporting Steps
Nothing Reported

Target Audience
The goal of Objective 1 is: Students increase knowledge of bioinformatics, genome analysis and database construction: New data visualization tools. The target audience continued to include 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 made available publicly. The analysis of pathways will provide a basis for molecular based studies on the ACP. 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 continued 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. Additionally other target audiences are scientists in the field, citrus growers, citrus industries, regulators (FDA/EPA) and citrus/agricultural researchers. In addition, some of our developments are so novel and broad reaching that, if successfully demonstrated it could target growers of many crops. 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 continued to include social scientists, consumers of citrus and citrus products, citrus industry representatives including growers, producers, distributors and retailers.

Changes / Problems
Nothing Reported

Training & Professional Development
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 experienced annotators mentor and train new students. Peers and mentors met once a week and all local annotators at IRSC met with their instructor 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 ACP 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 - Guest scientists joined in that were experts on the students pathways to help provide insights into completing the annotation (bioinformatics) and reporting of results - Mentored 17 students that completed over 400 gene models for genome version 3. Students presented at 2 state conferences (8 posters total) and 1 nation conference (5 posters). Students prepared 7 pathway reports for bioRxiv preprint. - 5 IRSC, 2 KSU undergraduate student annotators and 1 KSU senior annotator attended the 2019 Arthropod Genomics Symposium Pre-Symposium Workshop: CURATION CLINIC: Gene Annotation using Apollo held in Manhattan, Kansas on June 12, 2019 - 5 students prepared a poster to present the results of their genome annotation and to showcase the skills they learned in bioinformatics at the 2019 Arthropod Genomics Symposium - 4 students presented posters and attended scientific talks at the 2019 Florida Academy of Sciences meeting and the 2019 Florida Phytopathological Society meeting - Max Reynolds, IRSC undergraduate intern, won 1st place for his Outstanding Undergraduate Student Poster Presentation at the 2019 FAS meeting in Melbourne, FL - Crissy Massimino, IRSC program coordinator, won 1st place for her Honorable Mention Undergraduate Student Poster Presentation at the 2019 FAS meeting in Melbourne, FL - University of Florida graduate student, Julie Boswell, received 1st place graduate presentation for Agricultural and Natural Resources at the 83rd Annual Meeting Florida Academy of Sciences held in Melbourne, FL March 8-9, 2019 - University of Florida graduate student, Julie Boswell, was a Symposia Organizer for session AGRI/Natural Resources Symposia at the Florida Academy of Sciences in Melbourne, FL on March 6-8, 2019 - Students have used their knowledge gained through participation in annotation to advance to careers and graduate school. Several students have changed their career path because of the interest and knowledge gained with interning for this project. - Former IRSC intern Chad Vosburg, started a Masters degree at Penn State University. His experience as an intern helped secure his acceptance to the Department of Plant Pathology & Environmental Microbiology. - Former IRSC intern Yesmarie De la Flor, started a career at the Smithsonian Marine Station as a Research Technician working on coral diseases caused by bacteria. Her bioassay experience as an intern helped solidify her career decision. - Former IRSC interns Kristine Aguirre, Denisse Hasan, Max Reynolds & Margaryta Rzhevska Jernigan, are now Research Technicians at USDA-ARS researching citrus. Their experience as an intern provided valuable experience before accepting jobs with USDA-ARS. - Former IRSC intern Jordan Norus, is now a Research Technician at the University of Florida Medical Entomology Lab. His experience as an intern annotating the ACP helped secure this job. - Angela Kruse, graduate student at Cornell/BTI, completed her Ph.D. under the direction of Dr. Michelle Heck - Marina Mann, graduate student at Cornell/BTI, is being advised by Dr. Michelle Heck. Marina has been working on completing the transcriptomics analysis for the ACP expression network. -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 *Boswell J, Hunter WB, & Lopez S. (March 2019). Passive phloem release of Candidatus Liberibacter asiaticus from sweet orange [AGR-01]. 83rd Annual Meeting Florida Academy of Science. Melbourne, FL *Coates L, Mahoney J, Ramsey J, Warwick E, Johnson R, MacCoss M, Krasnoff S, Howe K, Moulton K, Saha S, Mueller L, Hall D, Shatters R, Heck M, Slupsky C. (March 2019). Sex-dependent effects of CLas exposure on Diaphorina citri. IRCHLB. Riverside, CA *Coates L, Padhi E, Leveau J, Heck M, & Slupsky C. (January 2020). The use of multi-omics approaches to understand the effects of CLas on citrus and its insect vector Diaphorina citri. CEND. Berkeley, CA D'Elia T & *Vosburg C. (June 2019). Genomic structure and characterization of Wnt signaling genes in Diaphorina citri. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Kercher K, & *Tamayo B. (June 2019). A metabolic insight into the Asian citrus psyllid: Annotation of glycolysis and gluconeogenesis pathways. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Kercher K, & *Tamayo B. (March 2019). Glycolysis and Gluconeogenesis Pathway Annotation in Diaphorina citri. Florida Academy of Sciences. Melbourne, FL D'Elia T, Wiersma-Koch H, & *Rahmoune A. (March 2019). Identification and Annotation of Chromatin Remodeling Complexes in Diaphorina citri. Florida Academy of Sciences. Melbourne, FL D'Elia T, Wiersma-Koch H, *Vosburg C, & *DeLaFlor Y. (June 2019). Annotation of genes from the melanin biosynthesis pathway in the citrus greening vector, Diaphorina citri. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Vosburg C, *DeLaFlor Y, & *Massimino E. (March 2019). A Pathway to Recovery: Evolutionary analysis of the yellow gene family in the citrus greening vector, Diaphorina citri. Florida Academy of Sciences. Melbourne, FL D'Elia T, Wiersma-Koch H, *Vosburg C, *DeLaFlor Y, & *Massimino E. (March 2019). Melanization pathway annotation in the Asian citrus psyllid. Florida Academy of Sciences. Melbourne, FL D'Elia T, Wiersma-Koch H, *Vosburg C, & *Massimino E. (June 2019). Molecular evolution of yellow genes in Diaphorina citri reveals duplications and losses. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Vosburg C, & *Reynolds M. (June 2019). Shedding Light on Cryptochrome and the Circadian Rhythm Pathway in Diaphorina citri. Arthropod Genomics Symposium. Manhattan, KS D'Elia T, Wiersma-Koch H, *Vosburg C, & *Reynolds M. (March 2019). Annotation of the Circadian Rhythm Pathway in Diaphorina citri. Florida Academy of Sciences. Melbourne, FL Heck M & *Hosseinzaedh S. (December 2019). Small RNA signaling in CLas-ACP interactions. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Heck M & *Kruse A. (December 2019). Proteomic and morphological studies guide understanding and interdiction of the Asian citrus psyllid stylet sheath. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Heck M & *Mann M. (December 2019). Elucidating tissue specific CLas-ACP interactions using transcriptomics. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Saha S, Mueller L, Hosmani P, Flores M, & *Mann M. (December 2019). New resources at the Citrusgreening.org portal and future work. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Slupsky C & *Coates L. (December 2019). Optimizing FANA ASO for Gene Silencing in Diaphorina citri. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Slupsky C & *McNeil C. (December 2019). Comparing the metabolic response of Citrus sinensis to diverse pathogens to identify unique responses to CLas. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL

Dissemination Streams
In addition to scientific publications listed in the Product Section, dissemination of our results has been described below: - 5th Annual Citrus Greening Solutions (CGS) Meeting. IRSC, Ft. Pierce, FL. December 10-12, 2019. Attended: 7 graduate students, 19 undergraduate students, 5 key personnel, 5 co-PIs, 19 project members, 5 SSAC members, 2 coordinators - 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 182 times by 503 authors in the world across 28 different countries, 206 of those authors being within the U.S. in 26 different states spanning across the disciplines of Biology, Agriculture, Engineering, Chemistry, Medical Sciences, and Social Science. -Project publications have been featured in 51 news stories, tweeted approximately 165 times with an audience reach of over 382,000 followers, and have over 700 Mendeley readers. Objective 1 activities include: - Meeting were held with researchers in the potato psyllid and pear psylla community from Texas and Washington in order to establish collaborations. The potato psyllid genome and transcriptome will be an important resource for comparative genomics of the ACP. - Researchers from the Mueller lab presented 1 talk and 2 posters about the web portal at the IRCHLB meeting in Riverside, CA (http://irchlb.org/hlb.aspx) Objective 2 activities include: - Dr. Carolyn Slupsky and lab members prepared and released an informational video on HLB. It was shared with media outlets and UC Davis media: https://youtu.be/dzKIigG24uE - Dr. Carolyn Slupsky was interviewed for the informational video called "Can Science Save California Citrus From Greening Disease?" https://www.youtube.com/watch?v=_KkHBFnmSW4 - Dr. Carolyn Slupsky talked with various news agencies about HLB. - Dr. Robert Shatters gave presentations at the annual Citrus Show in Fort Pierce, FL in January 2020 to describe the discovery and delivery of biologically based molecules. - Dr. 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. (December 2019). Micro-CT achievements, new discoveries, and presentation of the anatomical micro-CT atlas of the Asian Citrus Psyllid. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Brown S, Saha S, Hunter W, Mueller L, Alba-Tercedor J, Cano L, & Cicero J. (March 2019). Interactive digital video animation promotes accessibility of complex insect anatomy for cytological and molecular audiences in pursuit of solving the citrus greening problem in Florida. ToScA North American Symposium Gainesville, FL D'Elia T. (December 2019). Updates from manual curation and student engagement through genome annotation. 5th Annual CGS Meeting. Fort Pierce, FL Ellis J. (December 2019). Outcomes of multi-modal consumer research. 5th Annual CGS Meeting. Fort Pierce, FL Gupta G. (December 2019). HLB therapy: citrus derived antibacterial peptides and proteins. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Gupta G. (March 2019). HLB treatment by citrus-derived helix-turn-helix peptides. 6th IRCHLB. Riverside, CA Hunter W. (December 2019). Moving Biotech to Fields Trials - Citrus. 5th Annual CGS Meeting. Fort Pierce, FL Hunter W. (December 2019). Design and Efficacy Targeting CLas and Psyllid. 5th Annual CGS Meeting. Fort Pierce, FL Hunter W, Aishwarya V, Pelz-Stelinski K, Paris T, Miles G, McCollum G, Boyle M, Altman S, Holland C, & Sandoval-Mojica A. (March 2019). Advances in treatments to manage Huanglongbing: Targeting Liberibacter bacteria and psyllid endosymbionts. 257th American Chemical Society National Meeting & Expo. Orlando, FL Hunter W & Manthey F. (March 2019). The Effect of Iron on Prodigiosin Production in S. odorifera. 83rd Annual Meeting Florida Academy of Science. Melbourne, FL Hunter W & Tomich J. (January 2020). Adult Insect Gene Editing: BAPC-assisted-CRISPR-Cas9 Delivery into Adults for Heritable Gene Editing (Hemiptera). XXVIII International Plant & Animal Genome Conference. San Diego, CA Hunter W, Pelz-Stelinski K, & Sandoval-Mojica A. (March 2019). Antisense Oligonucleotides Targeting HLB in its Vector and Host. Joint 21st Conference of the IOCV Virologist and the 6th IRCHLB. Riverside, CA Hunter W, Tomich J, & Braswell E. (March 2019). BAPC-assisted-CRISPR-Cas9 Delivery into Nymphs and Adults for Heritable Gene Editing (Hemiptera). Joint 21st Conference of the IOCV and the 6th IRCHLB. Riverside, CA. Voted biggest advancement in psyllid pest management. Hunter W, Tomich J, & Gonzalez M. (April 2019). BAPC-assisted-CRISPR-Cas9 Delivery into Nymphs and Adults for Heritable Gene Editing (Hemiptera). American Society of Biochemistry & Microbiology Annual Meeting. Orlando, FL Hunter W, Tomich J, & Gonzalez M. (March 2019). BAPC-assisted CRISPR/Cas9 System: Targeted Delivery into Psyllid Adult Ovaries for Heritable Germline Gene Editing (Arthropoda: Hemiptera). 83rd Annual Meeting FAS. Melbourne, FL Hunter W, Tomich J, Gonzalez M, & Braswell E. (March 2019). BAPC-assisted CRISPR/Cas9 System: Targeted Delivery into Psyllid Adult Ovaries for Heritable Germline Gene Editing (Arthropoda: Hemiptera). 83rd Annual Meeting FAS. Melbourne, FL Saha S, Hosmani P, & Flores M. (December 2019). ACP version 3 genome, official gene set version 3 and Isoseq transcriptome. 5th Annual CGS Meeting. Fort Pierce, FL Shatters R. (January 2020). Screening and Discovery of Bactericidal Molecules Active Against Clas, Whole Tree Application Studies. Florida Citrus Show. Fort Pierce, FL Shatters R. (December 2019). Topical and Cell Transplant Technologies for Molecule Discovery. 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Shatters R. (December 2019). Use of Bioassays for Molecule Discovery (lab bench to greenhouse). 5th Annual Citrus Greening Solutions Meeting. Fort Pierce, FL Shatters R, Stover E, & Gupta G. (December 2019). USDA progress toward developing HLB-tolerant/resistant Citrus scions. Florida Citrus Research Foundation. Leesburg, FL Shatters R, Stover E, & Gupta G. (November 2019). USDA progress toward developing HLB-tolerant/resistant Citrus scions. Citrus Research and Education Center, University of Florida seminar. Lake Alfred, FL Shatters R, Stover E, & Gupta G. (September 2019). USDA progress toward developing HLB-tolerant/resistant Citrus scions. Citrus Research and Development Foundation. Fort Pierce, FL Shatters R, Stover E, & Gupta G. (March 2019). Identifying and Developing Resistance and Tolerance to HLB. International Research Conference on Huanglongbing. Riverside, CA Stover E. (December 2019). Transgenic citrus targeting ACP (or ACP & CLas): tools for development, and delivery of identified therapeutic molecules. 5th Annual CGS Meeting. Fort Pierce, FL

Next Reporting Steps
Nothing Reported <br><br>

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.

Training & Professional Development
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.

Dissemination Streams
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

Next Reporting Steps
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. <br><br>