Progress 09/01/12 to 08/31/17

Outputs
Target Audience:Target audiences include the primary benefactors of the research, the U.S. citrus growers. In addition, the target is the scientific community who is engaged in developmental research that has allowed this research project to be envisioned, and on whose progress we will continue to move forward. The general public is a target of our outreach. As consumers, they are interested and concerned about how research solutions are implemented to solve practical problems, and have shown interest in the foundations of this research project. Finally, policy-makers who often are involved in funding research for Florida citrus, need to be appraised of the project, its goals, and expectations that come from progress. The Outreach Team has determined that 1) because the effector and driver systems are all progressing equally it will not be possible to eliminate one or more from the outreach efforts and 2) an educational program should focus on the context of genetic technologies in general so that the nuPsyllid option for disease management is contrasted for example with a genetically modified citrus host and other technology options. Together with molecular biologist and extension expert Peggy Lemaux, Powerpoint presentation materials based on 3 grower interview events in California have been drafted, adapted as handouts and incorporated into the website "Science for Citrus Health." Changes/Problems:Our team recognized from the outset that creating and releasing a psyllid population that was refractory to disease spread would be a high-risk but potentially a high-payoff intervention that was complimentary to other research. To mitigate technical risk, we implemented a strategy that launched parallel research strategies both with respect to manipulating the genotype of the population (Driver Systems) and the phenotype of blocking disease spread (Effector Mechanism). To anticipate downstream regulatory requirements for phased testing, we refined methods of rearing, releasing and monitoring nuPsyllid populations. Central to the phased testing approach to biological release is the need to build, validate and refine models to predict effects in space and time across real landscapes. The fundamental difference in this type of program is the notion of a Driver System, a biotechnological tool to spread a trait in a population. The total genetic structure of a population includes, nuclear-encoded chromosomal genes but also endosymbiont and pathogenic bacteria which in the case of the genus Wolbachia in insects, are inherited almost like the DNA of cytoplasmic organelles. Likewise, any biological population hosts a plurality of viral genomes, including many that induce little or no symptoms. These viral genomes are naturally-occurring sequences that can be manipulated as part of a system of pest management. The strategy with this concurrent development plan was to be able to adapt our budgets and focus if there was a clear intervention opportunity that became available with any of the three Driver Systems. It was the consensus of the Team Leaders, Advisors and Stakeholders in several interim meetings that each of these approaches had good merit and progress. We enhanced the effort on achieving psyllid transgenesis, obtaining useful insect/psyllid cell lines and on modeling effects of RNAi on a landscape scale that are directly related to the goals of this project. We updated project objectives across all sub-award budgets to optimize the remaining use of funds with the need to provide continuity to other funded programs and to generalize the biotechnology tools, models and outreach goals. At the end of this research project a robust infrastructure was established for communicating innovations to growers and the general public and in modeling the spread and adoption of nuPsyllid and related technologies. There were no major difficulties encountered with the outreach and socio-economic modeling portions of the project. Likewise, the efforts to rear, release and monitor psyllids in the population developed in this and related work and likely sufficient for the nuPsyllid application. Future improvements in this technical area are likely to reduce costs of an intervention. The foundational knowledge obtained by both Effector-Mechanism teams has yielded many candidate genes that can be further evaluated in Driver Systems as they become available. There is a comprehensive database of high quality annotated sequences (protein, RNA, DNA) and a detailed molecular description of the complete life-cycle of CLas moving through a psyllid from one citrus site of infection to another. Both of these teams have generated new actives (RNAi and peptides) and small molecule targets for intervention. These are important leads for product development but it is unclear whether there are practical delivery methods except delivery in transgenic trees. This will require further innovation and especially cost reduction before they are available for growers. A major bottleneck in this program, was to develop a high throughput method to rank Effector candidates. The work with RNAi was adequate for the purpose but it would be better to express sequences directly in the Driver system of choice. Therefore, the major emphasis of the project remained on the challenges of finding Drivers that could create the desired nuPsyllid phenotype, refractoriness to spread of HLB. We set out to survey Wolbachia bacteria within psyllids to look for and/or create combinations that might result in material interference with acquisition or transmission of CLas. However, these bacteria are part of the Florida psyllid population structure and any adequate description of the behavior of the population must include these endosymbionts as part of the system. This description is now completed and has led to new concepts of manipulating endosymbiont interactions to potentially create the refractory phenotype. This line of research is well supported in ongoing funded programs. The ability to transform Wolbachia would be an important advance. Likewise, the survey of psyllid viruses from global collections has uncovered a rich diversity of viral genomes. These also are part of the endogenous psyllid population dynamics in Florida and elsewhere. Full-length clones have been obtained and several of these viruses are likely to prove useful as tools to introduce genes in to psyllids and understand their function in the search for a refractory phenotype and may well prove useful as novel biocontrol agents for population suppression. This line of research is also well supported in ongoing funded programs but there was not a paratransgenesis system developed in the timeframe of the research funded. The Driver System based on manipulation of chromosomal DNA is likely to be the most robust and effectively deployed. All the necessary components have been assembled and shown to validate multiple theoretical predictions in model systems. There are versatile variations that provide options for confinement and reversal that may be required for initial use in phased testing. The only barrier to progress here is the lack of transgenesis in the psyllid. This the single most important technical challenge. There is a great need for robust and useful insect and psyllid cell lines for use in all the Driver System research. There should be a major effort to bring a variety of new technologies to focus on insect transformation in general, including psyllids. A breakthrough tool or process here would likely make testing the nuPsyllid concept feasible with various chromosomal driver concepts and existing candidate effectors. The transgenesis system needs to perform with reasonably high frequency to be of practical use. Early ACP embryos can be micro-injected with foreign DNA with a reasonable level of survival that resulted in nymphal hatching. However, while injected and control non-injected nymphs were able to hatch on leaves, only a few have been able to continue nymphal development, and those surviving to adulthood have accounted for less than 1% of hatched nymphs. Thus, the current state of the ACP transformation project at the termination of the grant funding period is that a foundation of knowledge and methodology has been created that should allow continued efforts in evaluating potential transformants. The major roadblock at this time is the inability of newly laid eggs to survive after transfer from flush for injections, and therefore efforts are necessary to improve viability after removal from flush, potentially using artificial media. The ability to transform insect pests of agriculture such as psyllids, will enable gene drive approaches to population eradication, replacement and suppression. It will also facilitate the emerging commercial applications of sterile insect technologies based on genetics. The production and release of sterile males by biotechnology is less costly and more effective than a population approach based on irradiation. There is a high cost of production because of the expense of the radiation source and centralized production and the insects released will be much less fit than those created by biotechnology methods. A more efficient, decentralized, low-capital, biotechnology approach should make this population suppression approach applicable to additional insect pests. What opportunities for training and professional development has the project provided?Sub-project elements of this project continue in University and USDA laboratories in a number of states, providing considerable professional training to undergraduate and graduate students through direct involvement in the nuPsyllid project. The project employed a number of Post-Doctoral trainees in the labs, whose contributions to the research objectives provided them additional professional training. All involved in this project were exposed to the approaches and mechanics of team research on a large scale, with team meetings that outlined how the component research objectives fit into the larger picture. The project put the Quantitative Biology & Epidemiology (QBE) lab at UC Davis in the center of some of the most difficult issues facing US citrus industries in the struggle against ACP/HLB. QBE's role in the project raised our profile as an interdisciplinary research team resulting in an almost continuous series of invitations to perform similar roles for other teams making applications to CDRE and to other NIFA programs. Considering just CDRE, as an example we are involved in two proposals for the current round of funding, where our role will (if the projects are supported) be essentially to coordinate interaction between technologists and social scientists and to perform prospective analysis of the probable uptake of technology and its impact if deployed. QBE's capacity to act as an effective coordinating hub for projects involving technology and social science has led to receiving close to $2M in MAC funding to coordinate research on Early Detection Technologies and on ACP sampling and population dynamics in CA, and to playing a coordinating role in industry-funded research and outreach in California; this is all built on the ground-work done in the nupsyllid project. In addition to the direct research funded by the nuPsyllid grant, D. citri viruses identified under the nuPsyllid project allowed for additional and expanded research opportunities towards HLB and D. citri. Dr. Bryce Falk (UC Davis) and William Dawson (Univ. of Florida, CREC), PIs, and eight Co-PIs were awarded a 3-year $4.7 million grant from USDA NIFA SCRI. This grant entitled "Non-transgenic, near term RNA interference-based application strategies for managing Diaphorina citri and citrus greening/Huanglongbing (HLB)", and has the option for 2 additional years of funding at $2.7 million. This grant was only possible because of our research successes demonstrated from the nuPsyllid grant and CRDF funded research by Dawson. This project will build on successes and expand efforts for using D. citri and citrus viruses to induce desirable RNAi effects in D. citri, focusing the effort to use other D. citri viruses discovered in the original virus discovery part of the nuPsyllid grant. At the Univ. of Arizona Dr. Brown's lab's involvement in Nu-Psyllid led to cooperation between the Brown lab and the Arizona Department of Agriculture to establish HLB qPCR diagnostics and assist with detection of the bacterium in citrus leaf and psyllid samples collected in the Arizona survey/quarantine effort. Subsequently, overflow psyllid samples are now being processed and analyzed for CLas presence by qPCR. The lab is certified annually by APHIS-PPQ. As a result of Dr. Shatters' USDA lab finding that learned interdiction peptides identified to kill Liberibacter in citrus would not work in the direct application of creating a 'nuPsyllid', other strategies for use of these peptides in controlling HLB are being evaluated as part of two subsequent NIFA SCRI grants: a. As part of the UC-Davis NIFA SCRI grant (Non-transgenic, near-term RNA interference-based application strategies for managing Diaphorina citri and citrus greening/Huanglongbing (HLB) the use of these peptides in conjunction with RNAi inducing dsRNA molecules that target essential psyllid genes. The idea here is that by combining control strategies wit very different modes of action we will develop robust psyllid/HLB controls method. Delivery via nuclear transformation of citrus and via expression in the Citrus tristeza virus vector are being evaluated b. Evaluation of direct delivery of these peptides to the citrus using injection and novel delivery strategies as part of the KSU NIFA SCRI grant (Developing an infrastructure and product test pipeline to deliver novel therapies for citrus greening). Additional benefits from the project have enhanced other collaborative funding prospects and involvement with HLB community: (1) New collaborative basis with certain team members (Kirsten, Nabil). (2) Psyllid Omics data will be made available to the ACP omics-databases (after publishing key papers); ACP transcript mapping to ACP genome. (3) Genome sequence drafts for endosymbionts/dissected ovaries (ACP). (4) Expansion beyond nuPsyllid: (a.) Arizona Dept of Agriculture and California Dept. of Food and Agriculture: qPCR monitoring of psyllids for CLas; for AZ-Dept. Agric. qPCR monitoring of citrus trees; certified testers 4 years+. (b.) Consortium participant: HLB Data Analytic Tactical Operations Cell (DATOC). Survey data, risk models, early detection Technology (EDT) and other data sources are available to inform decision makers on management and mitigation of the spread of HLB. It is imperative to establish an analytic group to process incoming information and intelligence, promote cross-analysis and comparison of diverse data sources and utilize our diverse expertise as the epidemic intensifies. This group will seek to use sound science and other data-based information streams to inform planning as well as operational strategies and logistics in a cross disciplinary environment. The group will meet virtually or in person to exchange analytic approaches and datasets, when feasible, to collaborate in making tactical recommendations to regulatory, operational and organization decision makers on tactics to mitigate HLB. The focus will be citrus in California, but also potentially other locations. The California Citrus Research Board is funding this project to provide dedicated staff, IT infrastructure and regular meetings (travel to in-person or virtual meetings). The objective of these meetings is to provide a forum where experts can combine their aggregated expertise to address complex questions from regulators, industry and other citrus stakeholders in mitigating the threat of Huanglongbing. The project established important pieces of modeling infrastructure that are continuing to be used in on-going research and in providing tactical support to disease management programs in Florida and California. The agent-based model of ACP dynamics, developed in the collaboration between UC Davis and USDA-ARS is being used to find optimal disease management approaches, while the ACP phenology model developed by OSU Corvallis in collaboration with UC Davis is helping the CPDPC in California evaluate the effectiveness of its ACP suppression program by providing a simulated baseline of development rates against which field observations in areas with active ACP control can be compared. The ability to transform insect pests of agriculture such as psyllids, will enable gene drive approaches to population eradication, replacement and suppression. It will also facilitate the emerging commercial applications of sterile insect technologies based on genetics. The production and release of sterile males by biotechnology is less costly and more effective than a population approach based on irradiation. There is a high cost of production because of the expense of the radiation source and centralized production and the insects released will be much less fit than those created by biotechnology methods. This method has proved effective in some cases such as the medfly and screwworm. A more efficient, decentralized, low-capital, biotechnology approach should make this population suppression approach applicable to additional insect pests. How have the results been disseminated to communities of interest?Annual Team meetings of stakeholders, administrative management and advisors as well as numerous meetings of citrus growers in California, Texas and Florida at which presentations on project goals & objectives were presented. Psyllid bioinformatics database - major accomplishment from Dr. Brown's (UAZ) objective - six annotated transcriptome datasets that can be mined to identify candidate effectors, design primers for validation and qPCR quantification (of gene expression), and select dsRNA targets for RNA interference knockdown experiments. Publicly available (www.sohomoptera.org/ACPPoP ; Vyas et al. 2014, Fisher et al. 2015); additional databases to be released upon publication of other submitted manuscripts. Important pieces of modeling infrastructure continue to be used in on-going research and provide tactical support to disease management programs in FL and CA using the agent-based model of ACP dynamics, developed in collaboration between UC Davis & USDA-ARS to find optimal disease management approaches, while the ACP phenology model developed by OSU Corvallis in collaboration with UC Davis is helping the CPDPC in CA evaluate effectiveness of its ACP suppression program by providing a simulated baseline of development rates against which field observations in areas with active ACP control can be compared. Collaboration between UC Davis & UW Madison has yielded an economic surplus model for evaluating the likely financial outcome of deployment of gene drive technology, revealing that low deployment & low benefit equilibria may exist for such technologies, which are generically useful methodological developments for the analysis of technology development and adoption in agriculture. Peggy LeMaux, molecular biology specialist at UC Berkeley, developed a grower seminar "Food fights in the marketplace: is there a way to use genetics to address HLB disease in citrus?" The seminar was presented in several locations in CA as part of the Citrus Research Board Grower Seminar series, and covered explanations of how plants and insects are genetically modified, consumer attitudes towards GM, and potential uses of GM for the citrus industry. A survey was conducted at the Florida Citrus Expo in August of 2016 with the results indicating that although 93% of the respondents practiced what they considered to be "conventional" grove management practices, 46% would definitely accept GMO alternative technologies to solve HLB; another 44% stated they would likely be in favor of accepting such solutions if they were available. Eighty per cent felt the greatest hurdle to overcome in adoption of GMO crops was public perception; 60% favored a technology that would target trees (i.e. HLB-resistant varieties by genetic modification). The web site, "Science for Citrus Health" developed by Peggy Lemaux (UCR) and collaborator Lukasz Stelinski (UF) went live in May 2015, http://ucanr.edu/sites/scienceforcitrushealth/ and has had over 2000 visits to date. The web site describes the ACP/HLB situation and provides resources for growers to better understand the techniques that are being developed to battle the disease. A poster presentation was made for the International Research Conference on HLB in Orlando in 2017, highlighting resources available on the Science for Citrus Health Web pages. Funding has been obtained from other sources to continue the web site after the nuPsyllid project funding ends. The web site has a section called research snapshots that describe approaches currently being studied nationwide to manage HLB (both engineered and non-engineered approaches) in lay terms, and contains downloadable fact sheets for distributing at extension events. These Research Snapshots are divided into four categories: Early Detection Techniques, Established Orchard solutions, Replant solutions and nuPsyllid. An extensive collection of PowerPoint slides covering a diverse array of topics, is posted on the web site and updated as new information comes out. To date there have been nearly 100 downloads from the site. Topics included in the customizable slide set include descriptions of genetic engineering of plants and insects and how these might be used to address HLB, nonengineering approaches, regulatory issues, consumer attitudes, labeling considerations and trade issues. The powerpoint is used by extension and research personnel to educate growers about the approaches researchers are using to combat HLB. The Public Outreach effort has become a hub of communication about citrus technologies for both grower and the general public, critical to building trust that technologies, especially those supported with public funding, are not perceived to have been developed in secret, an important principle in establishing and maintaining trust between scientists and the general public that technology solutions under investigation and development are communicated as they are developed. The outreach team compiled a fact sheet titled 'What makes lemons, oranges and limes look and taste different?', defining crop and insect genetic engineering in the context of citrus and includes the nuPsyllid project as a portion of the examples; it is available on the web site and is used as a handout at grower meetings. The Science for Citrus Health blog alerts the citrus industry and the media of updates to the web site, also utilized by media to showcase research activities and helps communicate the progress being made in the fight against Huanglongbing to growers and the general public. The site is developing twitter followers as well. The following references are IN ADDITION to Presentations listed under the Publications made by J Brown, F Dong, BW Falk, PD Mitchell, S. Nouri in Brazil, France, Hawaii, Mexico, Spain and the United States: Nouri, S. Diverse array of new viral sequences identified in worldwide populations of the Asian citrus psyllid (Diaphorina citri) using viral metagenomics. Society for Invertebrate Pathology, July 27, 2016, Tours, France. Green blog - http://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=24671&sharing=yes Tim Hearnden Capital Press. New UC website explains scientific advances on citrus psyllid, HLB http://www.capitalpress.com/Research/20170726/new-uc-website-explains-scientific-advances-on-citrus-psyllid-hlb California Citrus Mutual https://www.cacitrusmutual.com/just-facts-maam-uc-launches-readers-digest-website-share-important-research-updates-citrus-growers/ AgAlert, California Agricultural Newspaper http://agalert.com/story/?id=11043 FreshPlaza http://www.freshplaza.com/article/179124/New-website-simplifies-latest-citrus-greening-research-for-farmers ANR Report blog - http://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=24865 Pests in the Urban Landscape blog - http://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=24818 Retail and Garden Center IPM news http://ipm.ucanr.edu/PDF/PUBS/Summer_2017_Retail_Newsletter.pdf http://campaign.r20.constantcontact.com/render?m=1102549393270&ca=35747d32-97cc-416e-bf20-5b722072ef32 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our first objective was to develop modified ACP colonies (nuPsyllid) that could be reared and released to replace the wild populations and that would be unable to move CLas in the environment. It is not possible to rely on selection alone to spread this trait into a wild population of psyllids because the inability to spread disease would itself offer no survival advantage. Success with this approach requires a non-Mendelian method to drive the genes into the population, a Driver System. Six psyllid viruses and one psyllid bacteria were described in this work that could be adapted for this purpose. Ideally, the inability to spread disease would be a trait encoded in the chromosomal DNA of the insect. We demonstrated two generally applicable methods to drive a trait into an insect population from components assembled into the genome. In the first case, population replacement is achieved only if the population is released above a dominant numerical threshold. For example, if the 60% or greater of the released population is the desired nuPsyllid type (incapable of spreading disease) in 10 generations it would locally replace essential all of the wild psyllids. Whereas, if 40% or less of the released population was the nuPsyllid type, then the wild type would predominate and the nuPsyllid population would disappear. This is a very attractive feature of gene drive because it is essentially reversible. In the second case, population replacement is achieved with release of a nuPsyllid population that continues to spread in the population. This is desirable for citrus disease management because of the large number of trees that occupy both the urban and commercial lands. Initial assessments have not identified the required variation in CLas transmission to occur naturally in ACP populations. The effector is the content of the phenotypic change we aim to introduce, an Effector Mechanism. Candidate effectors have been identified through multiple parallel methods of investigation including bioinformatics, proteomics, yeast two-hybrid (Y2H), peptide-ligand and antibody fragment scFV-ligand libraries. Experimental results and theory based on models of CLas movement within ACP have guided selection of priority effectors for future studies. This work also provides a detailed molecular description of how the CLas bacterium becomes established in and moves from the gut of the ACP, into the body of the insect and then the salivary gland. Several genes were identified that block the ability of the insect to spread disease when they are disrupted. Data is made available publically through at the website (www.sohomoptera.org/ACPPoP). Our second objective was to provide optimized management strategies for integration of the proposed population displacement technique into current management practices. Detailed models of the conditions for dissemination of psyllids and the adoption of this technology by society were established. Our team improved the ability to rear, release and monitor psyllids in detailed studies on the composition artificial media, shipping conditions, attractants and improved traps designs. Analytical infrastructure was developed to coordinate interaction between technologists and social scientists and to perform prospective analysis of the probable uptake of technology and its impact if deployed. The agent-based model of ACP dynamics is being used to find optimal disease management approaches, while the ACP phenology model is being used to evaluate the effectiveness of its ACP suppression program by providing a simulated baseline of development rates against which field observations in areas with active ACP control can be compared. A new economic surplus model developed for the work evaluates the likely financial outcome of deployment of gene drive technology and reveals that low deployment, low benefit equilibria may exist for such technologies under plausible assumptions about knowledge spillover effects among technology developers. We embedded the somewhat technical elements of the model formulation in a more accessible systems analysis approach that also yielded useful qualitative insights into factors that are likely to determine the sustainable development of this, or similar, technologies. These are generically useful methodological developments for the analysis of technology development and adoption in agriculture. For the third objective, the project's outreach plan was developed to help the public understand the release of a modified psyllid. Because a modified psyllid was not developed during the project period, our outreach emphasis shifted to developing information that would help growers and the general public better understand the techniques that are being used by scientists to attempt to develop a nuPsyllid population, the importance of genetic engineering, and the other tactics that scientists are using to solve the Huanglongbing problem. It is an important principle in establishing and maintaining trust between scientists and the general public that technology solutions under investigation and development are communicated as they are developed. The web site, "Science for Citrus Health" was developed as a UC ANR web site and it went live in May 2015, http://ucanr.edu/sites/scienceforcitrushealth/. The web site describes the ACP/HLB situation and provides resources for growers to better understand the techniques that are being developed to battle the disease. Postcards announcing the web site have been distributed to citrus growers at meetings in California and Florida. A poster presentation was made for the International Research Conference on HLB in Orlando in 2017, highlighting resources available on the Science for Citrus Health Web pages. The web site has a section called research snapshots that describe approaches that are currently being pursued nationwide to manage HLB (both engineered and non-engineered approaches). The team worked with researchers to write up descriptions of their research strategies and accomplishments and we then translate that material into language that can be readily understood by general readers. These Research Snapshots are divided into four categories, Early Detection Techniques (3 entries), Established Orchard solutions (5 entries), Replant solutions (2 entries) and nuPsyllid (3 entries). Each of the snapshots is written in a form that can be used as a fact sheet to be passed out at extension events including using insect viruses to combat the psyllid and stopping spread of CLas using beneficial bacteria. The Science for Citrus Health blog alerts the citrus industry and the media to updates of the web site. The web site is utilized by media to showcase research activities and helps communicate the progress being made in the fight against Huanglongbing to growers and the general public. The site is developing twitter followers as well. The outreach team completed the fact sheet titled 'What makes lemons, oranges and limes look and taste different?' The fact sheet defines crop and insect genetic engineering in the context of citrus and includes the nuPsyllid project as a portion of the examples. The fact sheet is available on the web site and is used as a handout at grower meetings. An extensive collection of PowerPoint slides, covering a diverse array of topics, is posted on the web site and updated as new information comes out. The PowerPoint is used by extension and research personnel to educate growers about the approaches researchers are using to combat HLB. A grower seminar "Food fights in the marketplace: is there a way to use genetics to address HLB disease in citrus?" was developed and presented in several locations. The seminar covers explanations of how plants and insects are genetically modified, consumer attitudes towards GM, and potential uses of GM for the citrus industry. Grower preferences and opinions were surveyed in polls at industry conferences.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yuan, Qing, Jordan, Ramon, Brlansky, Ronald H., Minenkova, Olga, Hartung, John. 2015. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display. J. Microbiological Methods 117:148-154. http://dx.doi.org/10.1016/j.mimet.2015.07.020.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Fang Ding, YongPing Duan, Cristina Paul, Ronald H. Brlansky and John S. Hartung 2015. Localization and distribution of Candidatus Liberibacter asiaticus in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody. PLoS One: 10(5):e0123939.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yuan, Qing, Jordan, Ramon, Brlansky, Ronald H., Minenkova, Olga, Hartung, John. 2016. Development of single chain variable fragment (scFv) antibodies against surface proteins of Ca. Liberibacter asiaticus. J. Microbiological Methods. 122:1-7. http://dx.doi.org/10.1016/j.mimet.2015.12.015.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Fang Ding, Yongping Duan, Qing Yuan, Jonathan Shao and John S. Hartung. 2016. Serological detection of Candidatus Liberibacter asiaticus in citrus and the identification of a promising secreted chaperone protein responding to cellular pathogens. Scientific Reports 6:29272. DOI: 10.1038/srep29272.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Huawei Liu, Sagheer Atta, and John S. Hartung. 2017. Characterization and purification of proteins of used for the production of antibodies against Ca. Liberibacter asiaticus. Protein Expression and Purification 139: 36-42
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fang Ding, Cristina Paul, Ronald H. Brlansky and John S. Hartung 2017. Anti-Omp A polyclonal Antibody-based in Situ Immuno Tissue Print and Immune Capture-PCR, Diagnosis and Detection of Candidatus Liberibacter asiaticus. Scientific Reports 7:46467. DOI:10.1038/srep46467
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Stover, E., Hall, D.G., Shatters, R.G. and Moore, G.A., 2016. Influence of citrus source and test genotypes on inoculations with Candidatus Liberibacter asiaticus. HortScience, 51(7), pp.805-809.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Soderlund, C.A., Nelson W.M., and Goff, S.A. 2014. Allele Workbench: Transcriptome pipeline and interactive graphics for allele-specific expression. PLoS ONE 9(12): e115740. doi:10.
• Type: Journal Articles Status: Other Year Published: 2017 Citation: Brown, J.K., Saberi, E., Brown, C.C., and Rast, TJ. 201x. Gene expression AAP time course.
• Type: Journal Articles Status: Other Year Published: 2017 Citation: Fisher, T.W., Rast, T.J., Soderlund, C., He, R, Gang, D. R. and Brown, J.K. 201x. Interactions between Ca. Liberibacter effector-psyllid proteins, and a model for systemic invasion of the psyllid host and vector. Parasite and Vector (in prep).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Cicero, J.C., and Brown, J.K. 2013. SEM- and TEM-Informed Anatomical Observations of Ca. Liberibacter parasite localization in its psyllid host. Third International Research Conference on Huanglongbing-IRCHLB III. Orlando, Florida, February 4-7, 2013 (poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fisher, T., Cicero, J.M., and Brown, J.K. 2013. Connecting anatomical and molecular data of Candidatus Liberibacter asiaticus and solanacearum during vector-mediated transmission. Third International Research Conference on Huanglongbing-IRCHLB III. Orlando, Florida, February 4-7, 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fisher, T., He, R., Soderlund, C., Smith, W., Vyas, M., Gang, D., and Brown, J.K. 2013. A comparative transcriptomics approach to elucidate psyllid-Ca. Liberibacter interactions. Third International Research Conference on Huanglongbing-IRCHLB III. Orlando, FL, February 4-7, 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fisher, T., Nelson, W., Vyas, M., He, R., Willer, M., Gang, D.R., Soderlund, C., and Brown, J.K. 2013. Publicly available website for the identification of psyllid-Ca. Liberibacter interactors using comparative transcriptome analysis. Phytopathol. 103(6S): 44. APS-MSA meeting, August 8-14, 2013. Austin, TX
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Brown, J.K. 2015. Assessment of detection methods and the threat of Candidatus Liberibacter in seed and other propagative plant materials. APS Symposium presentation, Pasadena, CA Aug 1-5, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Brown, J.K., Rast, T.J., and Fisher, T.W. 2015. Transcriptomics, proteomics, and yeast-2-hybrid analyses reveal genes pathways important for Candidatus Liberibacter sp. circulative, propagative transmission. International Research Conference on HLB, Caribe Royale, Orlando, FL February 9-13, 2015 (oral presentation).
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Fisher, T.W., and Brown, J.K. 2015. Optimization of dsRNA knockdown assays to evaluate RNAi efficacy for gene silencing in the potato psyllid. International Research Conference on HLB, Caribe Royale, Orlando, FL February 9-13, 2015 (oral presentation).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Brown, J.K., Rast, T.J., Cicero, J.E., and Fisher, T.W. 2016. Proposed model for Candidatus Liberibacter asiaticus and solanacearum systemic invasion and multiplication in the psyllid host and vector. American Phytopathological Society, Tampa FL, July 28-Aug. 4, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Brown, J.K., Rast, T.J., and Fisher, T.W. 2016. Psyllid vector-Liberibacter interactions at cellular and molecular interfaces. Symposium: Biology, Ecology and Management of the Asian Citrus Psyllid Diaphorina Citri, Vector of Huanglongbing International Congress of Entomology (ICE). Orlando, FL. September 26, 2016.
• Type: Book Chapters Status: Published Year Published: 2016 Citation: Brown, J.K., Cicero, J.M., and Fisher, T.J. 2016. Psyllid-transmitted Candidatus Liberibacter species infecting citrus and solanaceous hosts. Pages 399-422 in: Vector-Mediated Transmission of Plant Pathogens, (ed.) Brown, J.K. American Phytopathological Society Press, St. Paul, MN. 496 pp.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Cicero, J.M., Stansly, P.A., and Brown, J.K. 2015. Functional anatomy of the oral region of the potato psyllid (Hemiptera: Triozidae) Ann. Entomol. Soc. Am. 108: 743-761.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: He, R., Willis, M., Fisher, T.W., Soderlund, C., Pelz-Stelinski, K., Brown, J.K., and Gang, D.R. 2017. Impact of Candidatus Liberibacter asiaticus infection on Asian citrus psyllid transcriptome. Fifth International Research Conference on Huanglongbing-IRCHLB III. Orlando, FL, March 13-17, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Brown, J.K., Biofilms and yellow dragons. College of Agriculture, Spring Awards Luncheon. Featured Speaker, University of Arizona, Tucson, AZ, March 22, 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Brown, J.K., Psyllid-Liberibacter complexes: Emerging vector-pathogens. National Plant Disease Recovery System, Arlington, VA, April 15, 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Brown, J.K., Invited presentation: Emergent and Invasive Psyllid-Liberibacter complexes. Western Plant Board 95th Annual Conference. Tucson, Arizona. March 24-27, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Brown, J.K., Symposium: Ca. Liberibacter solanacearum: a psyllid-transmitted, endemic and exotic, emergent fastidious prokaryote. Plant Virus-Vector Complexes in the Western US, APS-Pacific Division Meeting. Vector Symposium, Bozeman, MT July 8-11, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wang, X., R. He, J. Wang, A. Berim, J.-J. Park, T.W. Fisher, J.K. Brown, D. R. Gang. 2017. Combined UPLC-MS/MS and MALDI-MSI analyses identify metabolic differences between CLso-infected and uninfected psyllids. Fifth Internl Res. Conf. on HLB. March 14-17, 2017. Orlando, FL (oral).
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Brown, J.K., Invited seminar: Psyllid transcripts with potential involvement in Ca. Liberibacter invasion and propagative transmission: Toward RNAi mediated abatement of citrus greening and zebra chip diseases. Vet-Science Microbiology Seminar, University of Arizona Nov 10, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Brown, J.K., Invited Workshop: Citrus greening: biofilms and yellow dragons. USDA-APHIS Workshop, Phoenix AZ. Jan 29, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Brown, J.K., Invited Seminar: Application of RNAi to interfere with insect transmission of citrus greening disease. UF Whitney Marine Biosciences Laboratory, Augustine, FL Feb 13, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Brown, J.K., Maricopa County Master Gardeners (K. Young, host). Insect vectors of plant and fastidious bacterial pathogens in Arizona. Maricopa Co. Extension Center, Phoenix, AZ. April 20, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Brown, J.K., Invited speaker: Pacific Branch-ESA symposium Animal and Plant Vector Biology: Addressing Old Questions with New Technologies. A cumulative model for Liberibacter invasion and circulation based on electron microscopy, functional genomics, proteomics, and yeast-2-hybrid analyses. April 6, 2016. Honolulu, HI. April 2-7, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Brown, J.K. 2017. Ca. Liberibacter tactics enabling invasion and circulative-propagative transmission by the psyllid host. US Arid-Land Agricultural Research Center, Maricopa, AZ March 27, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Brown, J.K. 2017. Psyllid vector biology, Liberibacter-psyllid interactions. Psyllid-Liberibacter Workshop, Guatemala May 16-18, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Brown, J.K., Rast, T.J., Cicero, J.E., and Fisher, T.W. 2017. Psyllid vector-Ca. Liberibacter interactions at cellular and molecular interfaces. Third Hemiptera-Plant Interactions Symp., Madrid, Spain, June 4-8, 2017.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kandul, Nikolay, M. Guo, and B. Hay. July 2017. A positive readout for site-specific MRNA cleavage. PeerJ, DOI 10.7717/peerj.3602.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Falk, B. W. 2016. RNAi-based strategies against insect vectors of plant pathogens. Plenary lecture. Sept 20, 2016, International Citrus Congress. Foz do Iguacu, Brazil.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Falk, B. W. 2016. RNAi-based strategies against the Asian citrus psyllid, Diaphorina citri. Fundecitrus, Araraquara, Brazil. Dec. 16, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Nouri, S. 2017. Applying viral metagenomics to discover new viruses of insect vectors of plant pathogens: the Asian citrus psyllid. APS Annual Meeting Hot Topic. San Antonio, TX, August, 2017.

Progress 09/01/15 to 08/31/16

Outputs
Target Audience:Target audiences include the primary benefactors of the research, the U.S. citrus growers. In addition, the target is the scientific community who is engaged in developmental research that has allowed this research project to be envisioned, and on whose progress we will continue to move forward. The general public is a target of our outreach. As consumers, they are interested and concerned about how research solutions are implemented to solve practical problems, and have shown interest in the foundations of this research project. Finally, policy-makers who often are involved in funding research for Florida citrus, need to be appraised of the project, its goals, and expectations that come from progress. The Outreach Team has determined that 1) because the effector and driver systems are all progressing equally it will not be possible to eliminate one or more from the outreach efforts and 2) an educational program should focus on the context of genetic technologies in general so that the nuPsyllid option for disease management is contrasted for example with a genetically modified citrus host and other technology options. Together with molecular biologist and extension expert Peggy Lemaux, Powerpoint presentation materials based on 3 grower interview events in California have been drafted, adapted as handouts and incorporated into the website "Science for Citrus Health." Changes/Problems:Despite the complexity of communication and reporting between the 15 institutions, the project is on target with its timeline of objectives. There are no major changes to the project in terms of approach and none are anticipated. However, in this continuation year 4 planning we have spent significant administrative effort in cash flow management to direct funds to the most important current objectives within the original proposed scope of work. CRDF, as the primary on this project, requires quarterly written progress reports on its funded projects, and we have included this term in all nuPsyllid project subcontracts. Consequently, we are receiving and posting these brief progress reports that are generated by each participant and coordinated through the team leaders up to a collective quarterly progress report submitted by the Project Director. We feel this keeps the team members focused on the goals and allows us to communicate regularly on progress. The gene drive system could likely be used to deliver a effector mechanism and create the desired nuPsyllid population if ACP could be transformed and therefore this is one current priority area of emphasis. What opportunities for training and professional development has the project provided?Elements of this project are being conducted in University and USDA laboratories in a number of states. These sub-projects are providing considerable professional training to undergraduate and graduate students through direct involvement in the nuPsyllid project. In addition, the project employs a number of Post-Doctoral trainees in the labs, whose contributions to the research objectives serve also to provide them additional professional training. All involved in this project are being exposed to the approaches and mechanics of team research on a large scale, and team meetings involve shared experience on how component research objectives fit into the larger picture. B Falk Team: Emilyn Matsumura was a visiting graduate student from Brazil. She worked on this project for 1 year from July 1, 2015 to June 30, 2016. Luca Nerva is a visiting graduate student from Italy. He is currently working on this project, he arrived in February, 2016. B. Hay Team: Omar Akbari, a former postdoctoral fellow funded by this work is now an Assistant Professor in the department of Entomology, UC Riverside. Tobin Ivy, a graduate student funded by this work, has advanced to candidacy (achieved the equivalent of a Masters degree), and is in progress on his PhD. N. McRoberts Team: Brianna McGuire (Junior Specialist) and Carla Thomas (Senior Analyst) have been added to the project team at UC Davis. Brianna is helping with statistical analysis of psyllid catch data and is learning relevant programing skills for integrating R and GIS as part of this work. Carla will work on the cognitive map modeling of the social risks for diffusion. She has been learning cognitive mapping theory to prepare for this and more recently has started to familiarize herself with the software we currently use for capturing and representing cognitive maps. How have the results been disseminated to communities of interest?The annual Team meeting was held on May 9th, 2016 at the Citrus Research and Education Center in Lake Alfred, FL including stakeholders, administrative management and advisors. This was a great opportunity for the Driver and Effector teams, who are primarily involved in attempting to isolate or build a nuPsyllid colony to present results to those responsible for the ultimate downstream release and adoption of this technology. Presentations on the project goals and objectives, as well as progress to date have occurred at meetings of the citrus growers in California, Texas and Florida numerous times during the year. There is a substantial effort to rear and release any type of nuPsyllid under development: Florida, Texas, and California will each develop and maintain its own colony to provide nuPsyllids, if available, for initial greenhouse studies and pilot field releases within its borders. The decision as to where to house nuPsyllid colonies within each state will be likely have to be made at several administrative levels. Regulatory agencies will likely require that nuPsyllid colonies be housed in a controlled/quarantine facility. Potential sites in each state were identified. An estimated population size for a nuPsyllid required for testing cannot be provided until the driver mechanism is selected. The effector mechanism may have associated fitness costs, as well, and these will have to be figured into rearing effort estimates. The initial plan is to piggyback nuPsyllid rearing efforts onto that of the existing parasitic wasp programs (Tamarixia) for initial testing with care to control for Tamarixia contamination. Modeling efforts to include better climate suitability GIS layer for ACP are in progress. An initial economic model was developed and captures the longer-term effects of HLB on the citrus supply response and the unique complexity associated with new planting decisions for this type of perennial crop. Details of the nuPsyllid project have been made available for public consumption through inclusion in trade journal articles, through the nuPsyllid web page and through other mechanisms, including newsletters of the CRDF. What do you plan to do during the next reporting period to accomplish the goals?At the Annual Team meeting the entire group of participants and stakeholders reviewed budget performance against milestones and adjusted spending plans towards the priority objectives for the remaining term of the grant. The entire group discussed revisions to focus the current technical plan based on the progress achieved, as well to address budget to plan variances. These contract modifications are now being implemented. All of the budget modifications are under the proposed scope of work. The administrative team has in several cases been able to propose redirecting some cash flow from within the program toward these more recently focused objectives. The priorities in the coming year are: accelerate development of a viral vector based on DCPLV or any other of the candidate viral vectors because this is likely to be the first tool for genetic manipulations and would be immediately useful for effector prioritization; analyze the phenotypes of non-native Wolbachia introduced into ACP; develop ACP transformation capacity at any level of efficiency because of the impact of success with this bottleneck on the ability to create the desired nuPsyllid colony; engage the grower community in a broad educational outreach to raise awareness of the alternatives for genetic technologies in the management of HLB.

Impacts
What was accomplished under these goals? The purpose of this NIFA-CAPS is to create options for management of HLB by replacing the wild type ACP with a population that is unable to transmit the bacterial causative agent CLas. Achieving this outcome will require progress in the following three areas of emphasis - An Effector Mechanism, A Driver System, and Diffusion. At the end of the fourth year of NIFA support significant progress has been accomplished in each of these areas as summarized below. Effector Mechanism - Initial assessments have not identified the required variation in CLas transmission to occur naturally in ACP populations. However the prospects for engineering a mechanism to achieve the desired phenotype are under active investigation. The effector is the content of the phenotypic change we aim to introduce. Candidate effectors are being identified through multiple parallel methods of investigation. Extensive transcriptome data sets (Transcriptome Computational Workbench; TCW) have been determined for whole adults, adult salivary glands, adult guts, and nymphs, infected or uninfected with CLas or CLso, and published datasets are made available to the research community, at www.sohomoptera.org/ACPPoP. The TCW is being updated to allow for protein sequence and spectral count data, in addition to the transcript and expression count data to allow for better identification of RNAi targets from protein data. Data from ongoing yeast two-hybrid, specific bait-prey co-transformations, co-immunoprecipitation, in silico transcriptional and proteomic profiling experiments support findings from previous TEM/SEM studies, and strongly suggest an "invasion model" in which CLas/CLso transforms the endocytic/exocytic host pathways to facilitate internalization, infection, and circulation in the psyllid host and vector. Based on proteome and transcriptome expression profiling, and/or yeast-two hybrid analyses in conjunction with a literature review of other pathosystems, 31 genes were selected as candidates for analysis. RNAi of 3 of these genes have caused significant psyllid mortality, compared to untreated controls whereas 12 genes have shown some reduced CLso transmission. All have predicted functions that corroborate the invasion model. In order to find additional targets for RNAi analysis, 10 putative phage-encoded effectors are being analyzed by expression profiling in psyllid adults and nymphs and an RNASeq study was conducted using CLas-infected and -uninfected ACP instars and adults. A system for screening molecules for binding to psyllid digestive tract epithelium has identified several candidate hexameric peptide ligands that bind specifically at submicromolar concentrations with different binding kinetics and also a potent bactericidal peptide. These sequences are ideal for the design of single gene products that could be used to block the ACPs ability to acquire/transmit CLas when expressed either within citrus or ACP. CLas acquisition transmission studies demonstrate that CLas multiplies in both nymphs and adults of ACP, but attains much higher levels in a shorter period of time post-acquisition when acquired by nymphs than when acquired by adults, and that adults may require longer access to infected plants compared to nymphs for CLas to reach higher levels in the psyllid. A comprehensive analysis of the ultrastructure of the Asian citrus psyllid stylet sheaths, salivary glands and alimentary canal was completed. Single chain antibody fragments (ScFvs) encoding genes have been isolated and are expressed in transgenic citrus and are being evaluated for their effects on CLas acquisition and transmission. Driver System - A new trait will not spread efficiently upon release within an existing population without a genetic bias of some kind. The driver is the medium of spread of the introduced phenotype--lack of CLas transmission. The drivers under investigation are viral, endosymbiont and chromosomal. An ACP picorna-like virus (DcPLV) was discovered in a worldwide search of ACP collections and is a leading candidate vector that might be of use for a paratransgenesis delivery system. Efforts continue to clone the complete genome as cDNA. Other ACP viruses being assessed as potential vehicles for transgenesis include ACP densovirus (DcDNV) and Diaphorina citri flavi-like virus (DcFLV). Unlike for DcPLV, there is evidence that these three viruses are in some, but not all, U.S. ACP populations. Experiments are also in progress to determine to determine if infection of insect cell lines, as opposed to whole insects, may offer a rapid approach for evaluating the efficacy of various effectors and if a well characterized ssRNA nodavirus (Flock House Virus) may be useful for this purpose. The genetic diversity of Wolbachia in ACP, was compared across selected gene sequences. Two wDi strains were detected among the samples tested. Some populations were co-infected with multiple wDi strains. These findings suggest that Wolbachia-induced cytoplasmic incompatibility may exist in ACP. ACP was successfully transfected with a supergroup A Wolbachia of Drosophila melanogaster by microinjection. Due to the low number of individuals produced, establishing co-infected isolines for use in experiments is ongoing. Proof of concept has been established for several chromosomal-based gene drive systems for population replacement in the psyllid. DNA vectors for a preferred system, engineered translocations, have been constructed and implemented in a model system predicted to yield a relatively high threshold system that will feature genetic containment and likely public acceptance advantages. This system shows great potential for ACP-HLB control because it should be readily transferrable once ACP can be transformed and is robust to mutations anticipated to inactivate drive while genes of interest can be easily linked to the translocation breakpoint. Use of this technology in ACP requires a transgenesis system of gene constructs and transformation of the psyllid germline. This priority is being pursued with both embryo injection and injection of adult males and females using a variety of transfection reagents and methods including the gene gun and electromagnetic rail gun. A number of constructs that carry different promoters, a reporter gene and a source of transposase have been generated and injected into nymphs and adults. There is evidence for transient expression in somatic tissues, but no evidence for germline transmission yet. Additional efforts include attempts to modify the psyllid genome using Cas9-based direct integration of foreign DNA at DNA breaks created by direct injection of a Cas9- and guide RNA-encoding plasmid. Diffusion - Once a nuPsyllid population is developed, its successful use will depend on series of factors based on the overall phenotype and fitness of the population in the environment and most importantly, will depend on human adoption, including the behavior of regulatory agencies, growers and consumers. All of these attributes must be modeled accurately for a nuPsyllid release to be used effectively. As for any other innovation, diffusion is the rate of change. Several aspects of the technical and communication plan for diffusion of this proposed HLB solution can be addressed most effectively only when an actual candidate nuPsyllid is available for release. The ability to rear, release and monitor psyllids has been initiated and is of immediate use in HLB disease management applications outside of this proposal. USDA-APHIS field response personnel in Southern California were interviewed to capture factors affecting the success of regulatory programs and to transfer this information to the nuPsyllid modeling team. Super-stimulant attractants based on methyl-salicylate and methyl-jasmonate are under development to improve trapping efficiencies to monitor psyllid populations.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fisher, T.W., and Brown, J.K., 2014. Transcriptomic and proteomic analysis of Candidatus Liberibacter asiaticus and solanacearum during psyllid infection and vector-mediated transmission. APS-CPS Joint Meeting, Minneapolis, MN. August 9-13, 2014.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Cicero, J. M., Fisher, T.W., and Brown, J.K. 2016. Localization of Ca. Liberibacter solanacearum and evidence for surface appendages in the potato psyllid vector. Phytopathology 106:142-154.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Russell, C.W. and K.S. Pelz-Stelinski. 2015. Development of an artificial diet and feeding system for juvenile stages of the Asian citrus psyllid, Diaphorina citri. Entomologia Experimentalis et Applicata 154:171-176
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hoffmann M., M.R. Coy, H.N. Kingdom-Gibbard, and K.S. Pelz-Stelinski. 2014. Wolbachia infection density in populations of the Asian citrus psyllid (Hemiptera: Liviidae). Environmental Entomology 43: 1215-1222.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Pelz-Stelinski, K.S. 2015. Battling the Asian citrus psyllid and citrus greening in Florida. 8th International Integrated Pest Management Symposium. 23-26 March, Salt Lake City, UT
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Hoffmann, M., C. Russell, M. Coy, and K.S. Pelz-Stelinski. 2015. Endosymbiotic control of the Asian citrus psyllid, Diaphorina citri) (Hemiptera: Liviidae): Diversity and Ecology of Wolbachia in Florida ACP Populations. The 4th International Research Conference on Huanglongbing, 8-13 February, Orlando, Florida.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Hoffmann, M., M. Coy, C.W. Russell, and K.S. Pelz-Stelinski. 2014. Molecular characterization and ecological survey of Wolbachia in Florida Diaphorina citri (Hemiptera: Liviidae) populations. The 62nd Annual Meeting of the Entomological Society of America, 16-19 November, Portland, Oregon.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Pelz-Stelinski, K.S. 2015. Incapacitating psyllids to host the greening bacteria or transmit the disease. International Research Conference on Huanglongbing Grower Day. Florida State College, Avon Park, FL. South 7 April (370 Attendants).
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Pelz-Stelinski, K.S. 2015. Developing new solutions for HLB management. Florida Citrus Growers Institute. Citrus Research and Education Center, Lake Alfred, FL. 12 March (309 Attendants).
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Fisher, T.W., M. Vyas, R. He, W. Nelson, J.M. Cicero, M. Willer, R. Kim, R. Kramer, G.A. May, J.A. Crow, C.A. Soderlund, D.R. Gang, and J.K. Brown. 2014. Comparison of potato and Asian citrus psyllid adult and nymph transcriptomes identified vector transcripts with potential involvement in circulative, propagative Liberibacter transmission. Pathogens 3: 875-907; doi: 10.3390/pathogens3040875.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Vyas, M., Fisher, T., He, R., Nelson, W.H., Yin, G., Cicero, J.M., Willer, M., Kim, R., Kramer, R., May, G.A., Crow, J.A., Soderlund, C.A., Gang, D.R., and Brown, J.K. 2015. Asian citrus psyllid expression profiles suggest Candidatus Liberibacter asiaticus-mediated alteration of adult nutrition and metabolism, and of nymphal development and immunity. PLoS One 10(6), e0130328.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Cicero, J.M., Fisher, T.W., Qureshi, J.A., Stansly, P.A., and Brown, J.K. 2016. Colonization and intrusive invasion of potato psyllid by Ca. Liberibacter solanacearum. Phytopathology First look: doi: http://dx.doi.org/10.1094/PHYTO-03-16-0149-R.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ammar, E.D., Ramos, J.E., Hall, D.G., Dawson, W.O. and Shatters Jr, R.G., 2016. 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), p.e0159594.)
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ammar, E.D., Hall, D.G. and Shatters, R.G., 2016. 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; http://www.sciencedirect.com/science/article/pii/S2213879X16000067
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ammar, E.D., Hall, D.G. and Shatters Jr, R.G., 2015. Ultrastructure and development of the new stylets inside pre-molting first instar nymphs of the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). Florida Entomologist, 98(1), pp.373-376.)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nouri, S., Salem, N., Nigg, J. C., Falk, B. W. 2016. Diverse array of new viral sequences identified in worldwide populations of the Asian citrus psyllid (Diaphorina citri) using viral metagenomics. J. Virology 90: 2434  2445).
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Nigg, J.C., Nouri, S., and Falk, B.W. 2016. Complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri. Genome Announcements, In Press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Nouri, S., Salem, N., Falk, B.W. 2016. Complete genome sequence of Diaphorina citri Associated C virus (DcACV), a novel putative RNA virus of the Asian citrus psyllid. Genome Announcements, In Press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Matsumura, E. E., Nerva, L., Nigg, J. C., Falk, B. W., Nouri, S. Complete genome sequence of the largest known flavi-like virus, Diaphorina citri Flavi-Like Virus, a novel virus of the Asian citrus psyllid, Diaphorina citri. American Society for Microbiology; Genome Announcements. In Press. Volume 4 Issue 5, Sept/Oct 2016
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Nouri, S. Diverse array of new viral sequences identified in worldwide populations of the Asian citrus psyllid (Diaphorina citri) using viral metagenomics. Society for Invertebrate Pathology, July 27, 2016, Tours, France.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Chu, C., M. Hoffman p, and Pelz-Stelinski, K.S. 2016. Inter-population variability of endosymbiont densities in the Asian citrus psyllid, Diaphorina citri. Microbial Ecology. 71: 999-1007.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Hay, B. Scheduled presentation at International Congress of Entomology, 2016
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Patt, J.M. Annual Meeting of the Entomological Society of America, Minneapolis, MN, November 2015
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Mitchell, P.D., McRoberts, N., Brown, Z. (submitted). Economic and social factors in deploying gene drive technology. Journal of Responsible Diffusion.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Patt, J.M. Annual Florida Citrus Show, Fort Pierce, FL, January 2016
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Patt, J.M. Annual Meeting of the American Chemical Society, Philadelphia, PA, August 2016
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Patt, J.M. International Congress of Entomology, Orlando, FL, September 2016
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Citrograph: Teiken, C., P. Lemaux, B. Grafton-Cardwell, and N. McRoberts. 2015. Genetic Engineering to protect citrus from HLB Citrograph 6(1): 24-31.

Progress 09/01/14 to 08/31/15

Outputs
Target Audience:Target audiences include the primary benefactors of the research, the U.S. citrus growers. In addition, the target is the scientific community who is engaged in developmental research that has allowed this research project to be envisioned, and on whose progress we will continue to move forward. The general public is a target of our outreach. As consumers, they are interested and concerned about how research solutions are implemented to solve practical problems, and have shown interest in the foundations of this research project. Finally, policy-makers who often are involved in funding research for Florida citrus, need to be appraised of the project, its goals, and expectations that come from progress. The Outreach Team has determined that 1) because the effector and driver systems are all progressing equally it will not be possible to eliminate one or more from the outreach efforts and 2) an educational program should focus on the context of genetic technologies in general so that the nuPsyllid option for disease management is contrasted for example with a genetically modified citrus host and other technology options. Together with molecular biologist and extension expert Peggy Lemaux, Powerpoint presentation materials have been drafted and are being reviewed with the extension group. Changes/Problems:Like many new projects of this magnitude communication and reporting between the 15 institutions involved in this project is not simple. The project meetings and progress reports indicate that, despite this complexity, the project is on target with its timeline of objectives. There are no major changes to the project in terms of approach and none are anticipated. However, in this continuation year 3 planning we have spent significant administrative effort in cash flow management to direct funds to the most important current objectives within the original proposed scope of work. CRDF, as the primary on this project, requires quarterly written progress reports on its funded projects, and we have included this term in all nuPsyllid project subcontracts, although it is not required under the NIFA terms and conditions. Consequently, we are receiving and posting these brief progress reports that are generated by each participant and coordinated through the team leaders up to a collective quarterly progress report submitted by the Project Director. We feel this keeps the team members focused on the goals and allows us to communicate regularly on progress. What opportunities for training and professional development has the project provided?Elements of this project are being conducted in University and USDA laboratories in a number of states. These sub-projects are providing considerable professional training to undergraduate and graduate students through direct involvement in the nuPsyllid project. In addition, the project employs a number of Post-Doctoral trainees in the labs, whose contributions to the research objectives serve also to provide them additional professional training. All involved in this project are being exposed to the approaches and mechanics of team research on a large scale, and team meetings involve shared experience on how component research objectives fit into the larger picture. How have the results been disseminated to communities of interest?A large annual Team meeting was held on Feb. 8th, 2015 in advance of the HLB International Conference in Orlando, FL including stakeholders, administrative management and advisors. This was a great opportunity for the Driver and Effector teams, who are primarily involved in attempting to isolate or build a nuPsyllid colony to present results to those responsible for the ultimate downstream release and adoption of this technology. Presentations on the project goals and objectives, as well as progress to date have occurred at meetings of the citrus growers in California, Texas and Florida numerous times during the year. There is a substantial effort to rear and release any type of nuPsyllid under development: Florida, Texas, and California will each develop and maintain its own colony to provide nuPsyllids, if available, for initial greenhouse studies and pilot field releases within its borders. The decision as to where to house nuPsyllid colonies within each state will be likely have to be made at several administrative levels. Regulatory agencies will likely require that nuPsyllid colonies be housed in a controlled/quarantine facility. Potential sites in each state were identified. An estimated population size for a nuPsyllid required for testing cannot be provided until the driver mechanism is selected. The effector mechanism may have associated fitness costs, as well, and these will have to be figured into rearing effort estimates. The initial plan is to piggyback nuPsyllid rearing efforts onto that of the existing parasitic wasp programs (Tamarixia) for initial testing with care to control for Tamarixia contamination. Modeling efforts to include better climate suitability GIS layer for ACP are in progress. An initial economic model was developed captures the longer-term effects of HLB on the citrus supply response and the unique complexity associated with new planting decisions for this type of perennial crop. An abstract of the work was submitted to the Agriculture and Applied Economics Association meeting in San Francisco, July 26-28, 2015. Details of the nuPsyllid project have been made available for public consumption through inclusion in trade journal articles, through the nuPsyllid web page and through other mechanisms, including newsletters of the CRDF. What do you plan to do during the next reporting period to accomplish the goals?At the Annual Team meeting the entire group of participants and stakeholders used a risk matrix methodology to force rank whether they perceived any of the three driver systems to be most likely implemented. Because there was no compelling winner (or loser) from this analysis, we are continuing with the concurrent work plan originally proposed with respect to the Driver and Effector teams. The entire group discussed revisions to focus the current technical plan based on the progress achieved, as well as budget to plan variances. The administrative team has in several cases been able to propose redirecting some cash flow from within the program toward these more recently focused objectives. The priorities in the coming year are: accelerate development of a viral vector based on DCPLV because this is likely to be the first tool for genetic manipulations and would be immediately useful for effector prioritization; analyze the phenotypes of non-native Wolbachia introduced into ACP; develop ACP transformation capacity at any level of efficiency because of the impact of success with this bottleneck on the ability to create the desired nuPsyllid colony; engage the grower community in a broad educational outreach to raise awareness of the alternatives for genetic technologies in the management of HLB.

Impacts
What was accomplished under these goals? The purpose of this NIFA-CAPS is to create options for management of HLB by replacing the wild type insect vector (ACP) with a population that is unable to transmit the bacterial causative agent Candidatus Liberibacter asiaticus (CLas). Achieving this outcome will require progress in the following three areas of emphasis - An Effector Mechanism, A Driver System, and Diffusion. At the end of the third year of NIFA support significant progress has been accomplished in each of these areas as summarized below. Effector Mechanism - Initial assessments have not identified the required variation in CLas transmission to occur naturally in ACP populations. However the prospects for engineering a mechanism to achieve the desired phenotype are under active investigation. The effector is the content of the phenotypic change we aim to introduce. Candidate effectors are being identified through multiple parallel methods of investigation. There is a promising list in various stages of validation from in silico-bioinformatics (proteomic and transcriptomic), genetic (yeast two-hybrid), physical methods (immunoprecipitation-pull down and mass spectrometry identification of proteins in the complex). In order to use an Effector for insect replacement, we need to disrupt these interactions while maintaining psyllid fitness. An extensive transcriptome database (the Transcriptome Computational Workbench) has been constructed and annotated for whole adults and nymphs, and from dissected salivary glands and guts of insects infected or not infected with CLas. They have been made available to the research community at www.sohomopter.org/ACPPOP and the manuscripts titled "Asian citrus psyllid expression profiles suggest Candidatus Liberibacter asiaticus-mediated alteration of adult nutrition and metabolism, and of nymphal development and immunity" and "Comparison of potato and Asian citrus psyllid adult and nymph transcriptomes identified vector transcripts with potential involvement in circulative, propagative Liberibacter transmission" were published in PLOS ONE and Pathogens, respectively. Using the yeast two-hybrid method, we have completed ACP gut and salivary gland library matings (35), CLas library matings (37) and Y2H specific "Bait" to "Prey" matings (reciprocal confirming mating, 30), and recently, a novel batch screen was conducted between a CLas bait-ACP prey library mating, to reinforce the potential role of several complexes and individual proteins identified using results obtained from the in silico approach. Co-Immunoprecipitation (CoIP or pulldowns) yield complexes of proteins from psyllid extracts reacted with the various over-expressed candidate proteins that are identified by mass spectrometry analysis. Using PCR, RT-PCR from cDNA and CoIP, the expression of three prophage gene products expressed in the psyllid has been demonstrated. All three implicate interactions in the invasion process. To date CoIP has been achieved and repeated multiple times with two ACP proteins, two CLas proteins and one prophage protein. RNAi knockdowns studies for 21 candidates are completed or are in progress; at least 6 targets have shown significantly reduced Liberibacter transmission in a functional bioassay using the a single-gene RNAi approach. These results have allowed us to construct a model of invasion showing that both prophage and CLas effectors may co-opt the clathrin coated pit endocytic pathway into a pathogen mediated phagocytosis involving 'membrane ruffling', a mode of entry/exit ascribed to several well known human pathogenic bacteria. This insight has greatly enhanced our ablity to prioritize new cadidates for experimentation, such that putative effectors that can be shown to act in concert can be targetted collectively, and further abate ACP-Liberibacter interactions essential for adhesion and multiplication in the gut, exiting the gut, and entering the salivary glands. A system for screening 4-amino acid peptide libraries for binding to psyllid digestive tract epithelium has identified at least 8 candidate ligands that bind specifically at submicromolar concentrations with different binding kinetics. Competition studies with unbiotinylated versions for one have shown competitive binding indicating the biotin is not required for gut binding. Similar competition studies are underway for the other peptides. Peptide binding has been shown to be stable in adults that acquired the peptide during feeding as nymphs. Proving this stability was crucial to functional use of the peptides in the acquisition/transmission inhibitory bioassays. Single chain antibody fragments (ScFvs) encoding genes have been isolated and are expressed in transgenic citrus. There are 18 separate transgenic lines each producing the ScFvs recognizing separate epitopes from two different surface antigens of CLas. These plants are at various stages of development with some ready to be used in CLas transmission assays to determine if any scFV's, when expressed in a plant, block acquisition of CLas by the psyllid. Driver System - A new trait will not spread efficiently upon release within an existing population without a genetic bias of some kind. The driver is the medium of spread of the introduced phenotype--lack of CLas transmission. The drivers under investigation are viral, endosymbiont and chromosomal. Viruses infecting psyllids were found by deep sequencing and bioinformatic analysis of small RNA libraries from over 30 diverse ACP collections from 18 locations around the world and the Diaphorina citri picorna-like virus (DCPLV) was selected for development into a recombinant vector system to be implemented for reverse genetics and to understand some of the biology of DCPLV. A collection of 6 non-native Wolbachia isolines is currently being screened for efficacy in reducing Las transmission. The diversity and distribution of native Wolbachia strains in existing Florida psyllid populations has been identified and phylogentically characterized. One minor candidate stain appears to be an excellent candidate for population replacement because it is negatively correlated with the presence of other psyllid endosymbionts. High throughput methods for establishing and screening new Wolbachia cell cultures from psyllid samples have been developed, yielding approximately 30 new native ACP Wolbachia cell cultures. Proof of concept has been established for several chromosomal-based gene drive systems for population replacement in the psyllid. DNA vectors for a preferred system, engineered translocations, have been constructed and implemented in a model system predicted to yield a relatively high threshold system that will feature genetic containment and likely public acceptance advantages. In order to generate translocations by design, site-specific cleavages to promote recombination are engineered with both the cas9/guide RNA system and homing endonucleases. This system shows great potential for ACP-HLB control because it should be readily transferrable once ACP can be transformed and is robust to mutations anticipated to inactivate drive while genes of interest can be easily linked to the translocation breakpoint. Use of this technology in D. citri requires a transgenesis system of gene constructs and transformation of the psyllid germline. This priority is being pursued with both embryo injection and injection of adult males and females using a variety of transfection reagents and methods including the gene gun and electromagnetic rail gun. Several aspects of the technical and communication plan for diffusion of this proposed HLB solution can be addressed most effectively only when an actual candidate nuPsyllid is available for release. The ability to rear, release and monitor psyllids has been initiated and is of immediate use in HLB disease management applications outside of this proposal.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: NOTE: include J Brown Nov 2014 Publication - see page 24 of pdf file--CRDF funding acknowledged

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: Target audiences include the primary benefactors of the research, the U.S. citrus growers. In addition, the target is the scientific community who is engaged in developmental research that has allowed this research project to be envisioned, and on whose progress we will continue to move forward. The general public is a target of our outreach. As consumers, they are interested and concerned about how research solutions are implemented to solve practical problems, and have shown interest in the foundations of this research project. Finally, policy-makers who often are involved in funding research for Florida citrus, need to be appraised of the project, its goals, and expectations that come from progress. Together with molecular biologist and extension expert, Peggy Lemaux, two grower seminars were developed and presented in Santa Paula, CA (June 27, 2014) and Exeter, CA (July 17, 2014) entitled “Food fights in the marketplace: Is there a way to use genetics to address HLB disease in citrus?” Based on the results of a questionnaire presented to this audience of over 100 growers, it appears that the California segment of the citrus industry represented by this sample, demonstrates strong acceptance of genetic solutions but remain concerned about public acceptance and government approvals. This audience was split on whether genetically modified trees or insects were preferable. Changes/Problems: Like many new projects of this magnitude communication and reporting between the 15 institutions involved in this project was not simple. The project meetings and progress reports indicate that, despite this complexity, the project is on target with its timeline of objectives. There are no major changes to the project in terms of approach and none are anticipated. CRDF, as the primary on this project, requires quarterly written progress reports on its funded projects, and we have included this term in all nuPsyllid project subcontracts, although it is not required under the NIFA terms and conditions. Consequently, we are receiving and posting these brief progress reports which are generated by each participant and coordinated through the team leaders up to a collective quarterly progress report which is submitted by the Project Director. We feel this keeps the team members focused on the goals and allows us to communicate regularly on progress. What opportunities for training and professional development has the project provided? Elements of this project are being conducted in University and USDA laboratories in a number of states. These sub-projects are providing considerable professional training to undergraduate and graduate students through direct involvement in the nuPsyllid project. In addition, the project employs a number of Post-Doctoral trainees in the labs, whose contributions to the research objectives serve also to provide them additional professional training. All involved in this project are being exposed to the approaches and mechanics of team research on a large scale, and team meetings involve shared experience on how component research objectives fit into the larger picture. How have the results been disseminated to communities of interest? Numerous team meetings of team leaders and various collaborators have occurred. Interim annual progress and project overviews were presented to the Scientific and Regulatory Advisors and to Tom Turpen, Project Director and Mary Lou Polek, Administrative Team Co-Director in a Team Leader Annual Meeting on May 12th and 13th in Dallas, TX. The team discussed revisions to the current technical plan based on the progress achieved, as well as budget to plan variances. Presentations on the project goals and objectives, as well as progress to date have occurred at meetings of the citrus growers in California, Texas and Florida numerous times during the year. Presentation and discussion of the nuPsyllid project has occurred during telephonic meetings as well, notably There is a substantial effort to rear and release any type of nuPsyllid under development. In July this group organized conference call and discussed the following key topics: Florida, Texas, and California will each develop and maintain its own colony to provide nuPsyllids for initial greenhouse studies and pilot field releases within its borders. The decision as to where to house nuPsyllid colonies within each state will be likely have to be made at several administrative levels. Regulatory agencies will likely require that nuPsyllid colonies be housed in a controlled/quarantine facility. Potential sites in each state were identified. An estimated population size for a nuPsyllid required for testing cannot be provided until the driver mechanism is selected. The effector mechanism may have associated fitness costs, as well, and these will have to be figured into rearing effort estimates. The design of release and monitoring studies will also need to be postponed until the driver and effector mechanisms are selected. Modeling studies that will help predict the spread of nuPsyllid have been developed and are being refined and validated with historical datasets on HLB spread. The Outreach Team met and determined that 1) because the effector and driver systems are all progressing equally it will not be possible to eliminate one or more from the outreach efforts and 2) an educational program should focus on the context of genetic technologies in general so that the nuPsyllid option for disease management is contrasted for example with a genetically modified citrus host. Details of the nuPsyllid project have been made available for public consumption through inclusion in trade journal articles, through the nuPsyllid web page and through other mechanisms, including newsletters of the CRDF. What do you plan to do during the next reporting period to accomplish the goals? The proposal plan will continue to be followed, focusing on parallel development of effectors and drivers. Additional attention to areas which can be advanced through complementary funding also are being evaluated within the nuPsyllid Team, and Advisors. A comprehensive Annual meeting that will include all researchers associated with the project has been announced for a day just in advance of the upcoming HLB International conference in Orlando, Feb. 9-13, 2015. The priorities in the coming year are: select and prioritize effectors; obtain antibody reagents for top effector candidates; use the bioassay platform based on artificial nymph diet for comparative testing of the phenotypes in ACP, maximizing transmission blockage and minimizing fitness loss; accelerate development of a viral vector because this is likely to be the first tool for genetic manipulations and would be immediately useful for effector prioritization; analyze the phenotypes of non-native Wolbachia introduced into ACP; determine if Wolbachia transformation is a feasible goal; develop ACP transformation capacity at any level of efficiency; continue to ready the engineered translocation constructs; begin to model the logistics of rearing and releasing nuPsyllid around hypothetical specifications and explicit assumptions; engage the grower community in a broad educational outreach to raise awareness of the alternatives for genetic technologies in the management of HLB.

Impacts
What was accomplished under these goals? The purpose of this NIFA-CAPS is to create options for management of HLB by replacing the wild type insect vector (ACP) with a population that is unable to transmit the bacterial causative agent (CLas). Achieving this outcome will require progress in the following three areas of emphasis – An Effector Mechanism, A Driver System, and Diffusion. Effector Mechanism – Initial assessments have not identified the required variation in CLas transmission to occur naturally in ACP populations. However the prospects for engineering a mechanism to achieve the desired phenotype are under active investigation. The effector is the content of the phenotypic change we aim to introduce. Candidate effectors are being identified through multiple parallel methods of investigation including bioinformatics, proteomics, yeast two-hybrid (Y2H), peptide-ligand and scFV-ligand libraries. An Effector Workshop with the participation of Team Leaders Bob Shatters and Judy Brown, Project Directors and additional collaborating scientists was held in Ft. Pierce, FL in Dec. 2013. There is a growing list of candidate effectors generated from bioinformatics (proteomic and transcriptomic), genetic (yeast two-hybrid) and physical methods (Far-Westerns). In the case of the yeast two-hybrid method numerous candidates are being tested reciprocally to confirm results. This workflow of the Effector team has already generated more high quality targets than can be analyzed in bioassays. In many cases loss of gene expression through RNAi is highly toxic to psyllids. We have only conceived of two tools to use to disrupt the Effector Mechanism, RNAi and competitive protein ligand inhibitors. Secondary metabolites or RNA aptamers are potential additional options. In order to use an Effector for insect replacement, we need to disrupt these interactions while maintaining psyllid fitness. The extensive transcriptome data set (the Transcriptome Computational Workbench) has been created from whole adults and nymphs as well as dissected salivary glands and guts of insects infected or not infected with CLas is now available to the research community at www.sohomopter.org/ACPPOP. Using the yeast two-hybrid method, gut and salivary gland library matings (24/ea) have been performed and the results reinforce the likely role of several complexes and individual proteins identified in the level 2 biological processes of the Gene Ontology categories of Adhesion, Nutrition, Invasion and Immune functions. Using RNAi knockdowns at least 5 of these targets show a significant reduction in CLas transmission in a functional transmission bioassay. A system for screening peptide libraries for protein ligand inhibitors was established based on the elements listed below. These results led to the decision to use 4-amino acid peptide libraries for the screening program. Several peptides have been identified from multiple experiments that bind specifically to digestive tract membranes at submicromolar concentrations. These results were corroborated with live adult and nymphs fed on artificial diet or leaves containing the biotinylated test peptides. scFv antibody to CLas surface antigens, FlhA and OmpA have been purified in quantities that will enable tests for effects on CLas transmission in a functional bioassay. Citrus flush takes up peptides in xylem and phloem when leaf petioles are placed in solutions containing the peptides. About 20% of the psyllids (nymphs or adults) that have fed on these leaves take up these peptides and these peptides can be detected throughout the alimentary canal. Previous work on Asian citrus psyllid feeding has shown that about 20% of the time they feed on the xylem. Peptides introduced by this method are found primarily in the xylem. A new diet feeding strategy allows nymph development from early instars through to adult emergence. This will enable transmission blocking studies with a system that reflects the most efficient acquisition of CLas in the natural system. Driver System – A new trait will not spread efficiently upon release within an existing population without a genetic bias of some kind. The driver is the medium of spread of the introduced phenotype--lack of CLas transmission. The drivers under investigation are viral, endosymbiont and chromosomal. Three viruses of ACP were discovered by deep sequencing and bioinformatic analysis of small RNA libraries from diverse ACP collections from 18 locations around the world. These viruses include a Reovirus (dsRNA), two Iflavirus-like isolates (+sense, ssRNA) and a Densovirus (ssDNA). Because the Ifla-like viruses are most readily manipulated as a gene vector, efforts are now focused on obtaining the full genome sequence. To date, RNAs have been extracted from 33 different ACP populations collected from 9 locations around the world. The Ifla-like viruses discovered in this work may be members of a new genus because parts of the genome show highly significant similarity to known members of the genus Iflavirus whereas other regions are more similar in genome organization to the genus Dicistrovirus. Over 95% of the genome appears to be cloned and efforts are now focused on identifying and confirming the 5’ termini of each of the genomic RNAs to enable a recombinant vector system to be implemented for reverse genetics. There are significant regional differences in Wolbachia infection levels and sequence types among Florida psyllid populations assessed using multi locus sequence typing (MLST). All investigated D. citri populations harbor wDi from supergroup A. A qPCR assay was developed for ACP Wolbachia using a primer pair for the wsp gene. Artificial feeding and microinjection systems have been developed to introduce Wolbachia into ACP for the purpose of screening psyllids for vector and host competence. Inoculation of ACP with new Wolbachia infections must occur during the nymphal stage in order for the infection to establish and be maintained in the insect. Cell cultures of a range of Wolbachia strains have been established and novel Wolbachia strains have been introduced into ACP at the nymphal stage. Infected adults will be mated so that progeny can be evaluated for subsequent transmission, CI, and fitness assays. Using this system, foreign Wolbachia infections in ACP have been confirmed in adult ACP via PCR. Proof of concept has been established in a model system for chromosomal-based gene drive systems for population replacement in the psyllid. However, the challenge with single or two-locus underdominance is to fully rescue the siblings that should be protected by the antidote in an engineered toxin-antidote system. A third drive mechanism, engineered translocations has been constructed and implemented in a model system and future work will focus on this relatively high threshold system that will feature genetic containment and likely public acceptance advantages. This system shows great potential for ACP-HLB control because it should be readily transferrable once ACP can be transformed and is robust to mutations anticipated to inactivate drive while genes of interest can be easily linked to the translocation breakpoint. Diffusion – Once a nuPsyllid population is developed, its successful use will depend on series of factors based on the overall phenotype and fitness of the population in the environment and most importantly, will depend on human adoption, including the behavior of regulatory agencies, growers and consumers. All of these attributes must be modeled accurately for a nuPsyllid release to be used effectively. As for any other innovation, diffusion is the rate of change. Several aspects of the technical and communication plan can be addressed most effectively only when an actual candidate nuPsyllid is available for release. The ability to rear, release and monitor psyllids has been initiated and is of immediate use in HLB disease management applications outside of this proposal.

Publications

Progress 09/01/12 to 08/31/13

Outputs
Target Audience: Target audiences include the primary benefactors of the research, the U.S. citrus growers. In addition, the target is the scientific community who is engaged in developmental research that has allowed this research project to be envisioned, and on whose progress we will continue to move forward. The general public is a target of our outreach. As consumers, they are interested and concerned about how reseach solutions are implemented to solve practical problems, and have shown interest in the foundations of this research project. Finally, policy-makers who often are involved in funding research for Florida citrus, need to be appraised of the project, its goals, and expectations that come from progress. Changes/Problems: Like many new projects of this magnitude, the sub-contracting of the 15 institutions involved in this project was not simple and some delay in onset of project activities resulted. The project meetings and progress reports indicate that, despite this ramp-up period, the project is on target with its timeline of objectives. There are no major changes to the project in terms of approach and none are anticipated. CRDF, as the primary on this project, requires quarterly written progress reports on its funded projects, and we have included this term in allnuPsyllid project subcontracts, although it is not required under the NIFA terms and conditions. Consequently, we are receiving and posting these brief progress reports which are generated by each participant and coordinated through the team leaders up to acollective quarterly progress report which is submitted by the Project Director. We feel this keeps the team members focused on the goals and allows us to communicate regularly on progress. What opportunities for training and professional development has the project provided? Elements of this project are being conducted in University and USDA laboratories in a number of states. These sub-projects are providing considerable professional training to undergraduate and graduate students through direct involvement in the nuPsyllid project. In addition, the project employs a number of Post-Doctoral trainees in the labs, whose contributions to the research objectives serve also to provide them additional professional training. All involved in this project are being exposed to the approaches and mechanicsof team research on a large scale, and team meetings involve some level of training on how component research objectives fit into the larger picture. It is our goal in 2013-14 to incorporate structured project and program management training into the team meetings, and have encouraged the NIFA Program Leader for this project area to participate on behalf of NIFA in this activity. How have the results been disseminated to communities of interest? Numerous team meetings of team leaders and various collaborators have occurred. Progress and project overviews were presented to the nuPsyllid Stakeholder Advisory Group and to Mary Lou Polek, Administrative Team Co-Director in their Annual Meeting on August 26th, in Lake Alfred, FL by Team Leaders, Bob Shatters, Joe Patt and Tom Turpen, Project Director. Presentations on the project goals and objectives, as well as progress to date have occurred at meetings of the citrus growers in California, Texas andFlorida numerous times during the year. Presentation and discussion of the nuPsyllid project has occurredduring telephonic as well as formal conference meetings (Annual Forum)of the National Citrus Health Response Program (CHRP), which is jointly managed byUSDA, APHIS and the National Citrus Research Council, a citrus grower organization. Details of the nuPsyllid project have been made available for public consumption through inclusion in trade journal articles, through the nuPsyllid web page and through other mechanisms, including newsletters of the CRDF. What do you plan to do during the next reporting period to accomplish the goals? The proposal plan will continue to be followed, focusing on parallel development of effectors and drivers. Additional attention to areas which can be advanced through complementary funding also are being evaluated within the nuPsyllid Team, and among the advisors. Opportunities have arisen to add depth to the team through recruitment of additional scientists with parallel capabilities and with ongoing research that will contribute. In one example, all 8 nuPsyllid Team Leaders and all 4 Scientific and Regulatory Advisors met in person on April 11 and 12 in Riverside, CA at a first Annual Meeting together with MaryLou Polek CO-PD and Tom Turpen, PD. Based on the presentations and discussions during and subsequent to the Annual Meeting, CRDF approved a supplemental CRDF-funded budget and contract extension at their Board meeting on Aug. 27th that will enhance the work plan critical for psyllid transformation developed by Al Handler, with Team Leaders Bruce Hay, and Kirsten Pelz-Stelinski. Additionally, the team will continue to focus on improving our ability to identify the optimal nuPsyllid phenotype while we also begin to model the complex system of release and monitoring around hypothetical specifications and assumptions.

Impacts
What was accomplished under these goals? The purpose of this NIFA-CAPS is to create options for management of HLB by replacing the wild type insect vector (ACP) with a population that is unable to transmit the bacterial causative agent (CLas). Achieving this outcome will require progress in the following three areas of emphasis – An Effector Mechanism, A Driver System, and Diffusion. Effector Mechanism Initial assessments have not identified the required variation in CLas transmission to occur naturally in ACP populations. However the prospects for engineering a mechanism to achieve the desired phenotype are under active investigation. The effector is the content of the phenotypic change we aim to introduce. Candidate effectors are being identified through multiple parallel methods of investigation including bioinformatics, proteomics, yeast two-hybrid, peptide and scFV libraries. Two de novo assemblies of the ACP transcriptome were created from eight Illumina paired-end libraries which were constructed from pooled psyllids: 1) whole bodies, 2) guts, and 3) salivary glands of the uninfected adult ACP; 4) whole bodies, 5) guts, and 6) salivary glands from CLas-infected adult ACP, 7) uninfected ACP nymphs, and 8) CLas-infected ACP nymphs. The transcripts from the ACP transcriptome were combined with the transcripts from the similarly constructed potato psyllid transcriptome, annotated and compiled into an extensive multi-taxon transcriptome database www.sohomoptera.org/psyllid at which multiple tools for data mining have been developed and implemented. Yeast-2 hybrid studies were initiated to study protein-protein interactions important in psyllid-Liberibacter interactions involved in the circulative, propagative pathway. Three cDNA libraries: uninfected gut, uninfected salivary gland and infected bacteria were made. Also three Mate & Plate “prey” libraries (uninfected gut, uninfected salivary gland and infected bacteria) were made. To date 12 gut library matings and 12 salivary gland library matings have been performed. Data analysis has been completed for 20 of those experiments yielding roughly 44 ACP gene products for testing in the RNAi phase of the project. To date, good quality dsRNA has been synthesized using the MEGAscript RNAi kit for five psyllid genes predicted to be involved in cytoskeleton formation, defense response, vesicle transport, or transcytosis. Knock-down results for effects of one of the cytoskeleton-related genes show an 18% reduction in Liberibacter transmission. From a large expression library screen, a set of 5 single chain antibody fragments (scFv) were identified and shown to bind to specific surface antigens of CLas. Bulk purification of the scFv antibodies is in progress under conditions required to maintain scFV binding. Construction of a peptide library was initiated to identify candidate effectors in a binding inhibition assay using ACP alimentary canal membrane preparations. Peptides in the library contain 4-10 amino acids, a double glycine linker and a biotin label to enable use of streptavidin-fluorescent tags. CLas positive citrus was used to develop an enrichment protocol for obtaining extracts from citrus that are enriched with the CLas bacterium. It was observed that the petiole and proximal leaf midrib region where enriched for CLas. Based on this observation, a single leaf acquisition/transmission bioassay was developed by combining artificial diet and feeding of petiole extract. An initial experiment showed the detection of transmitted CLas in the excised leaves in less than 3 weeks. A plant-based screening protocol was developed for monitoring psyllid transmission that allows full ACP life-stage development in excised plant flush. dsRNAs are delivered to plants through use of a transient expression vector (CTV). After infected ACP populations are exposed to dsRNAs that target various ACP genes by feeding in planta, the survivors were found to be free of CLas. The adults that emerged from nymphs, that developed on these plants from eggs laid by the original CLas positive adults, also were devoid of CLas. In controls, 15% to 20% of these newly emerged psyllids typically tested positive for CLas. Driver System A new trait will not spread efficiently upon release within an existing population without a genetic bias of some kind. The driver is the medium of spread of the introduced phenotype---lack of CLas transmission. The drivers under investigation are viral, endosymbiont and chromosomal. Three viruses of ACP were discovered by deep sequencing and bioinformatic analysis of small RNA libraries from diverse ACP collections. These viruses include a Reovirus (dsRNA), an Iflavirus (+sense, ssRNA) and a Densovirus (ssDNA). RNA libraries include samples from China, Taiwan, Brazil, Pakistan and Florida and will include Texas and California. A virus-free ACP colony was established for biological assays to evaluate these ACP pathogens as potential drivers. A virus-free D. citri colony has been established at UC Davis. Wolbachia was found to be present in all collections of ACP from 20 different Florida sites with infection frequencies of 86-96%. This population diversity has been charactierized via multi locus sequence typing (MLST) and a quantitative real-time (qPCR) assay has been developed to assess Wolbachia infection rates. Two isofemale lines were confirmed to be free of Wolbachia. These lines remain unstable, probably due to population bottle-necking. Additionally, antibiotic treatments are being used to clear ACP nymphs and adults of Wolbachia infection. Two virulent strains of Wolbachia have been selected for introduction into Wolbachia-cleared psyllid cultures via direct hemolymph transfer or cell culture transfection by microinjection. Chromosomal drivers were investigated in the model genetic system Drosophila where an invasive system modeled on the naturally occurring Medea element has been established. A feature that may be required for release of transgenic insects for some applications is the ability to test the release in the field at a threshold below that required for population replacement. Transgenes would spread to fixation under high release threshold conditions and would be lost under low release threshold conditions. This concept was confirmed in a synthetic system targeting a known haplolethal gene. The focus now is on two-locus underdominance, a lower threshold system. In two-locus underdominance each of two nonhomologous chromosomes (A and B) carry toxin-antidote pairs in which the toxin present on one chromosome (Toxin 1) is linked to an antidote (Antidote 2) that represses Toxin 2. Toxin 2 is located on a nonhomologous chromosome, linked with Antidote 1, which represses Toxin 1. In such a system, organisms can only survive if they carry A and B chromosomes or only wild type (+) chromosomes. 2-locus underdominance gene drive systems show an introduction threshold of 26% even in the absence of other element-associated fitness costs. Once the frequency of A/B individuals surpasses a critical threshold, matings with other A/B individuals are common, while matings with wild type are rare. In this regime the underdominant chromosomes spread at the expense of wild types, resulting in the complete loss of wild type individuals from the population. Transgenes can be removed from the wild population, simply by diluting the replaced population with wild types such that the frequency of transgenics falls below ~26%. Diffusion Once a nuPsyllid population is developed, its successful use will depend on series of factors based on the overall phenotype and fitness of the population in the environment and most importantly, will depend on human adoption, including the behavior of regulatory agencies, growers and consumers. All of these attributes must be modeled accurately for a nuPsyllid release to be used effectively. As for any other innovation, diffusion is the rate of change.

Publications