Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 215 - Biological Control of Pests Affecting Plants | 4030 - Viruses | 1040 - Molecular biology | 40% |
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 3110 - Insects | 1100 - Bacteriology | 30% |
| 212 - Pathogens and Nematodes Affecting Plants | 4010 - Bacteria | 1101 - Virology | 30% |
Citrus is one of the most important crops in the United States, but it is currently threatened by a devastating disease: citrus greening/Huanglongbing (HLB). There are currently no effective strategies to manage HLB and there is increasing concern that using conventional insect control measures such as insecticides, could lead to resistance in Diaphorina citri (the Asian citrus psyllid) the insect vector of Candidatus Liberibacter asiaticus (CLas), causal agent of HLB, and indiscriminately target beneficial insects such as those used for biological control or pollination. We have identified several D. citri-specific viruses from different geographic D. citri populations. These are non-pathogenic insect-specific viruses (ISVs) and are natural components of the D. citri endosymbiont community. This research addresses the NIFAECDRE program focus areas in multiple ways. First, this project is aimed to provide a delivery system in D. citri for RNA interference therapeutics solutions for HLB disease control/management. The studies proposed here will also provide a better understanding of the HLB/vector/citrus pathosystem. In this project, we propose to (1) engineer one or more D. citri-specific viruses previously identified by our group, to deliver novel RNAi effectors targeting D. citri (the Asian citrus psyllid), other endosymbionts, and/or CLas as strategies to help manage HLB; (2) elucidate intricacies of the biology of the D. citri-specific viruses and their interactions between D. citri and CLas; (3) provide high efficient RNAi targets; (4) apply our developed tools and approaches to interfere with CLas infection and/or transmission by D. citri. In sum, this project could not only offer approaches for D. citri suppression, reduced CLas transmission, or behavior modification (attack and/or kill), but strategies for CLas titer reduction, competition, or acquisition /transmission by D. citri.
Our primary intent is to use D. citri-specific viruses to produce siRNAs and/or amiRNAs (artificial miRNAs) within D. citri and affect small RNA communications critical to the viral or bacterial endosymbionts in D. citri, or to target D. citri mRNAs important to CLas multiplication in, or transmission by D. citri. We have in culture three D. citri populations (CA, TW and UY), and D. citri-specific viruses from different geographic D. citri populations: 1) DcFLV, identified in D. citri from Florida but now a DcFLV infected CA-D. citri colony maintained in the Biosafety 3 facility at UC Davis, 2) DcRV, identified in D. citri from Florida, China and Hawaii, 3) DcPLV, identified in D. citri form Uruguay, Taiwan and Brazil, and 4) DcDV, identified in D. citri from Taiwan. Each of the four different D. citri viruses offers potential benefits for our efforts. DcRV is not spread horizontally among D. citri and thus offers opportunities for more contained spread in a population. DcFLV is widespread in some locations and could disseminate in D. citri populations. DcPLV appears to have certain D. citri host range constraints and our surveys for different populations suggested different transmission pathways and/or infection mechanisms in different geographical populations, thus might offer opportunities to target specific D. citri biotypes. Finally, DcFLV, DcRV and DcPLV are RNA viruses and will generate siRNA effectors, but DcDV is a DNA nuclear replicating virus and could be useful for delivering specific amiRNAs.The specific Objectives for this project include:
The breadth of our D. citri genetic resources, D. citri viruses in culture, and the depth of data generated so far make us uniquely able to integrate the diversity of multiple D. citri populations with various viral endosymbionts and CLas, but also as tools for biocontrol strategies against HLB. We are building from extensive infrastructure and expertise and will do what is proposed here in two years.The specific Objectives for this project include:Objective 1. Engineer one or more Diaphorina citri-specific viruses to deliver novel RNAi effectors targeting D. citri (the Asian citrus psyllid) and/or CLas as strategies to help manage HLB We will utilize different strategies for each virus depending on the different features and nature of each. Since the selected D. citri viruses each have different types of genomes, they present different obstacles for developing infectious clones, and we are tailoring cloning strategies for each. As noted above, each virus has potential advantages and with our team, we can do what is proposed here.Objective 2. Elucidate and manipulate the intricacies of the biology of the D. citri-specific viruses and their interactions between D. citri and CLas Thorough studies of the biology of ISVs and the interactions between the ISVs, D. citri and CLas are necessary not only for any translational applications, but also to gain a fundamental understanding of this complex system to ensure that we use optimal approaches. To elucidate and manupulate the intricacies of the biology of the D. citri-specific viruses and the interactions between D. citri and CLas, we will: i. Evaluate/compare CLas acquisition/transmission efficiency of CA-D. citri, TW-D. citri, UY-D. citri, which are infected with different ISVs ii. Identify transmission mechanism/pathways of D. citri-specific viruses in each D. citri population iii. Analyze small RNAs originating from each ISV from CA-, TW-, and/or UY-D. citri and from D. citri infected with or without CLas and compare the RNAi effects caused by the ISVs and CLas in D. citri populationsObjective 3. Perform more comprehensive gene annotations based on our advanced D. citri genome assembly results of CA-, TW-, and UY-D. citri We will cross examine the small RNA candidates found to be related to CLas infections in D. citri with the results of our new annotation data based on our high-quality chromosome-scale D. citri genome assemblies and select more promising RNAi targets for HLB control strategies. It is very likely that the infections of CLas and other symbionts in D. citri could trigger or affect D. citri responses, changing gene expression profiles and hence those genes could be ideal targets of our RNAi strategy. Our improved gene annotation studies will greatly enhance the accuracy of the predicted gene functions and help us choose the best desired targets for strongest effects.The engineered D. citri-specific virus(es) will be delivered into D. citri through oral feeding or microinjection. Because those viruses are ISVs, they (the viruses and RNAi effects) will persistently infect and remain not only in the tested psyllids, but also will vertically transmit to the progeny, spreading within psyllid populations. To confirm that the engineered virus can be vertically transferred, the modified virus will be tracked in D. citri progeny and evaluated for retention of introduced sequences. D. citri will be assessed for intended target effects and biology. We will critically assess the effects on 1) D. citri mortality, 2) CLas multiplication (titration) in D. citri by qPCR analyses, and 3) CLas acquisition/transmission efficiency. The CLas transmission assays (long- and/or short-term assays) will be done following the whole plant protocols described above.In this project, we will (1) develop engineered D. citri ISVs to interfere with CLas acquisition/infection/transmission, which could set a milestone not only for HLB control management, but in utilizing ISVs as applicable tools for plant applications (Objective 1); (2) investigate the biology of/between different D. citri populations, CLas, and the ISVs (Objective 2); and (3) utilize D. citri genome data with the intent of manipulating these interactions for developing efficacious, environmentally sound biological-based practices to help manage HLB (Objective 2-iii and 3); (4) assess best target candidates in large scale greenhouse trial assays for HLB controls (Objective 1, 2 and 3).
Target Audience
?Our outreach and extension efforts are aimed toward the citrus industry, growers, media and the general public. These efforts are communicated through several outlets and are conducted by a team of volunteers, which include AES faculty, Cooperative Extension specialists, postdocs and graduate students from the University of California (Berkeley, Davis, Riverside, Division of Agriculture and Natural Resources), University of Florida and Texas A&M. Meetings of the team are held virtually to discuss emerging citrus issues, new citrus research projects for potential Research Snapshots (see below), Podcasts (see below), ideas and feedback from webinars (see below) and other issues related to the citrus industry and HLB. Among these efforts is the Science for Citrus Health (SCH) website (https://ucanr.edu/sites/scienceforcitrushealth/). This resource provides information about the ACP/HLB situation, including materials to help growers and the media understand approaches being used to combat the disease. Created in May 2015, and since updated, the SCH site has to date had ~12,000 visits and ~5700 downloads. Additionally, we converted some site information into Spanish (https://ucanr.edu/sites/scienceforcitrushealth/Home_Page_in_Spanish/) - important for the Hispanic population that has close working relationships with the citrus industry and growers. This year, we presented our results of how an Diaphorina citri specific virus affected CLas transmission efficiency at the IRCHLB conference for citrus growers. One SCH section, Research Snapshots (https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/) involves working with funded citrus project researchers to create informational pieces that describe their approaches and accomplishments related to ACP/HLB. To insure they are reader-friendly, they are written in a language understandable to growers, the media and the general public. Currently, there are 47 Research Snapshots, broken into five categories, General Topics, Disease Management, Early Detection Techniques, Psyllid Management, and General Tools. We added a new category, Ongoing NIFA Projects, in which we list the ongoing efforts of researchers funded by NIFA grants. There are currently 16 Snapshots on this topic, including one by Kuo, "SP: Virus-induced gene silencing (VIGS) using insect-specific viruses to manipulate psyllids as a strategy to help control citrus greening/HLB". There is also a section, Citrus Series Podcast, Beyond the Bench (https://ucanr.edu/sites/scienceforcitrushealth/podcast/), where researchers are asked about their science, how they got involved in their work, the impact of what they do, and what their job is like. Nine such podcasts are now featured. An extensive collection of PP slides (https://ucanr.edu/sites/scienceforcitrushealth/Outreach_Resources/PowerPoint/) is also available, providing explanations of genetic engineering and editing and how they are used to modify citrus and psyllids to address HLB. Portions were translated into Spanish. Another effective outreach method for our target audiences is the use of webinars. Since February 2021, at the start of the pandemic, SCH sponsored these webinars, many in collaboration with the UC Integrated Pest Management program. We have had seven webinars on a variety of topics of interest to citrus growers and researchers, one presented in Spanish. Webinars provide ~30-40 min talks by experts and were attended by participants worldwide, from U.S., Mexico, Spain, Peru, Ecuador, Argentina, Venezuela and Costa Rica. Each webinar attracted 150-250 attendees and post-event surveys indicated that participants planned to implement some of the information learned. As appropriate, CEU (pesticide license) credits were offered. Topics included: Emerging Technologies to Manage HLB and ACP; ACP and HLB Management in the Field; Use of Particle Films to Manage HLB; Biology and Management of ACP (in Spanish); Citrus Thrips Biology; Developing Management Tactics for Mealy Bugs; and Research Update on Asian Citrus Psyllid Development.
Changes / Problems
Because our personnel change, we included more biology, feeding, acquisition, and transmission assays of different symbiont-infected populations and study host-microbe and microbe-microbe interactions in D. citri. In addition, we added tests for functional promoter(s) in D. citri because the current available insect promoters didn't work in the D. citri cells. We expect to start testing our viral clones when we identify a working promoter for D. citri cells.
Training & Professional Development
During this report period, we have provided training and professional development opportunities for one graduate student, two college graduated Junior Specialist, and two Postdoctoral Researchers. The undergraduate student has been trained with all the knowledge and skills related to insect rearing, sample collection, citrus planting, DNA and RNA extraction from single psyllid, and polymerase chain reaction (PCR). The training activities were provided through one-on-one work with the project director, postdoctoral researchers, or junior specialists. In addition to trainings for undergraduate students, this project also provided more advanced training for junior specialists. The junior specialist working for this project was assigned to studies and small projects for our overall goals. The projects include the vertical and horizontal transmission of DcPLV, and the effects of DcPLV infection to CLas infection in D. citri. These projects also provided trainings and professional development for postdoctoral researchers and the project scientist working on this project. This project provides funding for postdocs, the project scientist and other personnel to attend conferences and workshops where they can present the results of the studies, learn state-of-the-art techniques, experience the latest progress in science, and communicate with other scientists who also work in this field. The project has provided opportunities for training activities and professional development in the second year of this project. The outreach efforts provide training for graduate students and postdocs in public science communication. During this period, four graduate students from UC Davis, UC Riverside and the University of Florida participated and one postdoctoral fellow from UC Riverside. This involved participation in writing Research Snapshots, as well as producing podcasts and video segments.
Dissemination Streams
Approaches were used that focused on the website and webinars. During this report period, we submitted a manuscript about our findings of how DcFLV affected the development of D. citri and CLas transmission. We also published a Research Snapshot describing our ongoing NIFA project for the general readers during the last report period. Based on tracking methods, these approaches appeared effective in reaching intended audiences and providing current information on research efforts to combat HLB. The Science for Citurs Health website also provides links to podcasts, videos and webinars.
Next Reporting Steps
We have been making good progress during this report period, identifying the transmission mechanisms of D. citri-specific viruses, identifying different isolates of DcPLV, the infectivity of DcPLV isolates in different D. citri populations, complete DcPLV full genome sequence of UY and TW isolate/variants, and revealing the effects caused by DcFLV infection in D. citri. For the next reporting period, we will continue our efforts to achieve our goals of this project. The following are some of our key focus areas for the next and final year of this project. Objective 1. Engineer one or more Diaphorina citri-specific viruses to deliver novel RNAi effectors targeting D. citri (the Asian citrus psyllid) and/or CLas as strategies to help manage HLB Dr. Bryony Bonning kindly sent us 2 stable FL-D. citri cell lines, Dici1 and Dici3 for us to test our viral clones. We started testing the methods establishing other D. citri cell line(s) from the D. ctir populations we maintain in the Contained Research Facility at UC Davis following the methods published last year by Wu et. al. (Bonning group, Florida). We have established an approach to inoculate the D. citri cell line, Dici3 with DcRV purified virions during this report period. We also cloned all 10 segments of DcRV genome into different expression vectors. We are currently testing for functional promoters for D. citri cell lines, which is crucial for us to express RNAs or proteins and express infectious clones in D. citri cell lines. In addition to DcRV, we will also develop protocols for purifying DcDV and DcPLV viral particles and transfect the available D. citri cell lines with different virions to study the cellular response to the infections of different D. citri-specific viruses. We are also testing protocols for DNA and RNA transfection. Our full viral genome clones of the RNA viruses will be in vitro transcribed and transfected into D. citri cell lines before we identify any functional promoters for D. citri cells. We will also test DcDV DNA genome using the established transfection protocol as well. Objective 2. Elucidate and manipulate the intricacies of the biology of the D. citri-specific viruses and their interactions between D. citri and CLas We are currently testing the effects of DcPLV infection for CLas transmission assays. We have established a TW-D. citri population that is 100% infected by TW-DcPLV isolate. We will use this population and the DcPLV-free TW-D. citri for CLas transmission assays using the same approach we used for CLas transmission of DcFLV-infected and -uninfected CA-D. citri. Furthermore, to further confirm our preliminary data, we will continue analyzing our data from the crossing assays with (1) DcPLV-infected male TW-D. citri and DcPLV-uninfected female TW-D. citri; and (2) DcPLV-uninfected male TW-D. citri and DcPLV-infected female TW-D. citri to verify if the virus is transmitted to the progenies by transovarial transmission. We will also initiate the identification of factors affecting CLas transmission by comparing the DcFLV-infected and -uninfected D. citri. Initially, we will transfect DcFLV into available D. citri cell lines and study cellular responses of DcFLV infection. If our CA, TW, and UY D. citri cell lines will be ready in time, we will also test the DcFLV infection in the cells developed from different D. citri lineages. Objective 3. Perform more comprehensive gene annotations based on our advanced D. citri genome assembly results of CA-, TW-, and UY-D. citri Previously, we completed deep sequencing analyses for small RNAs from CA, TW, UY D. citri populations infected or un-infected by CLas. The analyzed psyllids were synchronized to be at the same stage (age) and the sex was separated. To gain more information and cross examine the signals affected by DcFLV and CLas infection, we will use the previous obtained deep sequencing data with the new results we will acquire from the cellular responses to DcFLV infection and predict more potential factors involved in CLas transmission. Furthermore, we will use our previous deep sequencing data of RNAs with our D. citri genome assembly data to further perform more comprehensive gene annotations as well.
Target Audience
Outreach and extension efforts are aimed toward the citrus industry, growers, media and the general public. These efforts are communicated through several outlets and are conducted by a team of volunteers, which include AES faculty, Cooperative Extension specialists, postdocs and graduate students from the University of California (Berkeley, Davis, Riverside, Division of Agriculture and Natural Resources), University of Florida and Texas A&M. Meetings of the team are held virtually to discuss emerging citrus issues, new citrus research projects for potential Research Snapshots (see below), Podcasts (see below), ideas and feedback from webinars (see below) and other issues related to the citrus industry and HLB. Among these efforts is the Science for Citrus Health (SCH) website (https://ucanr.edu/sites/scienceforcitrushealth/). This resource provides information about the ACP/HLB situation, including materials to help growers and the media understand approaches being used to combat the disease. Created in May 2015, and since updated, the SCH site has to date had ~12,000 visits and ~5700 downloads. Additionally, we converted some site information into Spanish (https://ucanr.edu/sites/scienceforcitrushealth/Home_Page_in_Spanish/) - important for the Hispanic population that has close working relationships with the citrus industry and growers. One SCH section, Research Snapshots (https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/) involves working with funded citrus project researchers to create informational pieces that describe their approaches and accomplishments related to ACP/HLB. To insure they are reader-friendly, they are written in a language understandable to growers, the media and the general public. Currently, there are 47 Research Snapshots, broken into five categories, General Topics, Disease Management, Early Detection Techniques, Psyllid Management, and General Tools. In the past year we added a new category, Ongoing NIFA Projects, in which we list the ongoing efforts of researchers funded by NIFA grants. There are currently 16 Snapshots on this topic, including one by Kuo, "SP: Virus-induced gene silencing (VIGS) using insect-specific viruses to manipulate psyllids as a strategy to help control citrus greening/HLB". There is also a section, Citrus Series Podcast, Beyond the Bench (https://ucanr.edu/sites/scienceforcitrushealth/podcast/), where researchers are asked about their science, how they got involved in their work, the impact of what they do, and what their job is like. Nine such podcasts are now featured. An extensive collection of PP slides (https://ucanr.edu/sites/scienceforcitrushealth/Outreach_Resources/PowerPoint/) is also available, providing explanations of genetic engineering and editing and how they are used to modify citrus and psyllids to address HLB. Portions were translated into Spanish. Another effective outreach method for our target audiences is the use of webinars. Since February 2021, at the start of the pandemic, SCH sponsored these webinars, many in collaboration with the UC Integrated Pest Management program. To date we have had seven webinars on a variety of topics of interest to citrus growers and researchers, one presented in Spanish. Webinars provide ~30-40 min talks by experts and were attended by participants worldwide, from U.S., Mexico, Spain, Peru, Ecuador, Argentina, Venezuela and Costa Rica.. Each webinar attracted 150-250 attendees and post-event surveys indicated that participants planned to implement some of the information learned. As appropriate, CEU (pesticide license) credits were offered. Topics included: Emerging Technologies to Manage HLB and ACP; ACP and HLB Management in the Field; Use of Particle Films to Manage HLB; Biology and Management of ACP (in Spanish); Citrus Thrips Biology; Developing Management Tactics for Mealy Bugs; and Research Update on Asian Citrus Psyllid Development.
Changes / Problems
Because our personnel change, we included more biology, feeding, acquisition, and transmission assays of different symbiont-infected populations and study host-microbe and microbe-microbe interactions in D. citri.
Training & Professional Development
During this report period, we have provided training and professional development opportunities for one graduate student, one college graduated Junior Specialist, and two Postdoctoral Researchers. The undergraduate student has been trained with all the knowledge and skills related to insect rearing, sample collection, citrus planting, DNA and RNA extraction from single psyllid, and polymerase chain reaction (PCR). The training activities were provided through one-on-one work with the project director, postdoctoral researchers, or junior specialists. In addition to trainings for undergraduate students, this project also provided more advanced training for junior specialists. The junior specialist working for this project was assigned to studies and small projects for our overall goals. One of the projects is the full genome assembly and analyses of DcPLV. This project also provided trainings and professional development for postdoctoral researchers and the project scientist working on this project. This project provides funding for postdocs, the project scientist and other personnel to attend conferences and workshops where they can present the results of the studies, learn state-of-the-art techniques, experience the latest progress in science, and communicate with other scientists who also work in this field. The project has provided opportunities for training activities and professional development in the second year of this project. We will aim to attend and join more conferences, workshops, and training activities in the second year of this project. The outreach efforts provide training for graduate students and postdocs in public science communication. During this period, four graduate students from UC Davis, UC Riverside and the University of Florida participated and one postdoctoral fellow from UC Riverside. This involved participation in writing Research Snapshots, as well as producing podcasts and video segments.
Dissemination Streams
Approaches were used that focused on the website and webinars. During this report period, we published a Research Snapshot describing our ongoing NIFA project for the general readers. Based on tracking methods, these approaches appeared effective in reaching intended audiences and providing current information on research efforts to combat HLB. The website also provides links to podcasts, videos and webinars.
Next Reporting Steps
We have been making good progress in the first year of this project, identifying the transmission mechanisms of D. citri-specific viruses, identifying different isolates of DcPLV, the infectivity of DcPLV isolates in different D. citri populations, complete DcPLV full genome sequence of UY and TW isolate/variants, and revealing the effects caused by DcFLV infection in D. citri. For the next reporting period, we will continue our efforts to achieve our goals of this project. The following are some of our key focus areas for the next and final year of this project. Objective 1. Engineer one or more Diaphorina citri-specific viruses to deliver novel RNAi effectors targeting D. citri (the Asian citrus psyllid) and/or CLas as strategies to help manage HLB To test our D. citri-specific viral clones in D. citri cells, we started testing the methods establishing D. citri cell line(s) following the methods published this year by Wu et. al. (Bonning group, Florida). Previously, we have cloned all 10 segments of DcRV genome into different expression vectors. We will first try to inoculate the D. citri primary cell lines with DcRV purified virions. We will then test our DcRV clones in the D. citri primary cells and test express the required viral proteins, using baculovirus system or plasmid vectors containing insect promotors, to initiate infections from the clones. Objective 2. Elucidate and manipulate the intricacies of the biology of the D. citri-specific viruses and their interactions between D. citri and CLas We are currently preparing plants and DcPLV-infected and uninfected TW-D. citri for CLas transmission assays. We have established a TW-D. citri population that is 100% infected by TW-DcPLV isolate. We will use this population and the DcPLV-free TW-D. citri for CLas transmission assays using the same approach we used for CLas transmission of DcFLV-infected and -uninfected CA-D. citri. Furthermore, to further confirm our preliminary data, we will perform crossing assays with (1) DcPLV-infected male TW-D. citri and DcPLV-uninfected female TW-D. citri; and (2) DcPLV-uninfected male TW-D. citri and DcPLV-infected female TW-D. citri to verify if the virus is transmitted to the progenies by transovarial transmission. We will also analyze the UY-DcPLV genome sequence in the infected F1 TW-D. citri and compare the viral genome sequence with the UY-DcPLV in UY-D. citri. This work will identify essential segments of the viral genome for viral adjustments to different D. citri populations. Objective 3. Perform more comprehensive gene annotations based on our advanced D. citri genome assembly results of CA-, TW-, and UY-D. citri We will cross examine our small RNA candidates found to be related to CLas infections in D. citri with the results of our new annotation data based on our high-quality chromosome-scale D. citri genome assemblies and select more promising RNAi targets for HLB control strategies. <br><br>
<br>What was accomplished under these goals? Our primary intent is to use D. citri-specific viruses to produce siRNAs and/or amiRNAs (artificial miRNAs) within D. citri and affect small RNA communications critical to the viral or bacterial endosymbionts in D. citri, or to target D. citri mRNAs important to CLas multiplication in, or transmission by D. citri. We have in culture three D. citri populations (CA, TW and UY), and D. citri-specific viruses from different geographic D. citri populations: 1) DcFLV, identified in D. citri from Florida but now a DcFLV infected CA-D. citri colony maintained in the Biosafety 3 facility at UC Davis, 2) DcRV, identified in D. citri from Florida, China and Hawaii, 3) DcPLV, identified in D. citri form Uruguay, Taiwan and Brazil, and 4) DcDV, identified in D. citri from Taiwan. Each of the four different D. citri viruses offers potential benefits for our efforts. DcRV is not spread horizontally among D. citri and thus offers opportunities for more contained spread in a population. DcFLV is widespread in some locations and could disseminate in D. citri populations. Objective 1. Engineer one or more Diaphorina citri-specific viruses to deliver novel RNAi effectors targeting D. citri (the Asian citrus psyllid) and/or CLas as strategies to help manage HLB We now have full genome clones of DcRV, DcPLV that are ready for infectivity tests. We also have done other analyses and assays for the D. citri-specific viruses we have in culture: Diaphorina citri reovirus (DcRV) To understand the manner of DcRV transmission, we assessed the DcRV incidence in our DcRV infected California Diaphorian citri (CA-D. citri) population. Results of our RT-quantitative PCR (RT-qPCR) assays showed 100% of D. citri insects were infected. To determine whether DcRV could be horizontally transmitted among D. citri insects, we next used oral and injection approaches in attempts to transmit DcRV. RT-qPCR analysis showed that none of the recipient D. citri insects acquired DcRV. RT-qPCR analysis showed that DcRV could be detected from the feeding zone of the leaf, but the mean viral genome copies was 103-fold lower than that in adults of infected D. citri insects. No DcRV could be detected in the distal parts of the same plants, suggesting that DcRV did not move within plant tissues from the location where it was deposited. Our results also showed that DcRV could not establish infection in CA-D. citri in the leaf-feeding assays. Taken together, these data strongly suggest that the CA-D. citri insects could not be infected by DcRV via oral acquisition. It was also revealed that DcRV could not infect the Citrus macrophylla plant, which was used in our assays. Diaphorina citri densovirus (DcDV) To determine whether DcDV is horizontally transmitted, we performed a "kissing model" as described in the DcRV assays. We were not able to detect DcDV above the qPCR detection threshold at any timepoint. To test whether DcDV can be orally acquired when supplied in artificially high concentration, we homogenized 400 adult TW-D. citri in liquid nitrogen and filtered the resuspended homogenate through a 0.22 uM filter. In parallel, 0.5 g of TW-D. citri was used for partial virion purification and the resulting virion prep was diluted to produce an artificial diet mixture. As with the diet-sharing experiment, we were not able to detect DcDV above the qPCR detection threshold at any timepoint. Our results suggested that DcDV is not horizontally transmitted via oral acquisition. Diaphorian citri picorna-like virus (DcPLV) Two isolates of DcPLV identified from Uruguay (UY) and Taiwan (TW) showed 100% and 30-40% virus infection incidence in the populations, respectively. To test whether the virus or the D. citri populations affected the different incidences, we injected virus crude extract/filtrate of UY- or TW-D. citri into psyllids from a DcPLV-free TW-D. citri population. The results showed 100% of the injected psyllids were positive for DcPLV infection, whereas the F1 progenies (nymphs) from the UY-DcPLV and TW-DcPLV injected psyllids showed ~33% and 100% infection rates, respectively. The variants/isolates shared over 94% nucleotide identity and over 99% amino acid sequence identity and similarity. The results from our assays suggested even with different isolates that are over 94% and 99% identities of nucleotide and amino acid sequences, DcPLV isolates/variants from Uruguay population struggled to establish infections in D. citri Taiwan population. A clone, pCC1-T7p-DcPLV-T7t, is established and now available for testing in insect cells line. This will be digested for use in in vitro transcription using a T7 polymerase, and different insect cell lines will be transfected with the transcripts to test DcPLV infectivity. Objective 2. Elucidate and manipulate the intricacies of the biology of the D. citri-specific viruses and their interactions between D. citri and CLas Among the D. citri-specific viruses identified previously, Diaphorina citri flavi-like virus (DcFLV) was found in the D. citri populations in Florida and China. The role of DcFLV in D. citri biology and in the transmission efficiency of CLas by D. citri could be critical and yet are still unknown. Our results showed the time of oviposition, number of eggs laid for each female and the total eggs laid by DcFLV-negative CA-D. citri females were higher than for DcFLV-positive D. citri females. However, the time of development of eggs to nymph and nymphs to adults were faster in DcFLV-negative D. citri compared to DcFLV-positive D. citri. Also, DcFLV-negative D. citri produced more honeydew than DcFLV-positive D. citri. In sum, our results suggested DcFLV delayed the time of female D. citri laying eggs and development from eggs to adults but didn't affect the viability of the eggs, nymphs, and adults and the life span of the CA-D. citri. We also conducted CLas transmission assays for DcFLV-positive and -negative CA-D. citri. Our results showed that the CLas transmission by the DcFLV-positive D. citri was 20% higher than the transmission by the DcFLV-negative CA-D. citri. Our study provides insight into how DcFLV affects D. citri biology and how DcFLV influences CLas transmission by D. citri, thus this knowledge can provide new strategies to HLB control. Objective 3. Perform more comprehensive gene annotations based on our advanced D. citri genome assembly results of CA-, TW-, and UY-D. citri In total, repeat masking of CA-, TW-, and UY-D. citri assemblies resulted in 38.26% (108,248,856bp), 37.91% (107,246, 725bp), and 36.87% (98,311,742bp) being identified as repeat regions, respectively. Among major classes of repeat ele- ments, LTRs and DNA transposon elements were the most abun- dant, at 4.05% and 4.03% of CRF-CA, 3.4% and 3.97% of CRF- TW, and 4.2% and 2.89% of CRF-UY, respectively. Approximately 44-47% of all repeat regions identified in each assembly remain un- classified. BRAKER2 predicted 20,184 protein-coding gene models in the CA assembly, 20,357 gene models in the TW assembly, and 19,083 gene models in the UY assembly. BUSCO analysis of predicted proteins using the Hemipteran_odb10 dataset showed high completeness with scores of 95.3% (CA), 95.4% (TW), and 96.1% (UY) complete. Similar BUSCO scores were observed using the Insecta_odb10 and Arthropoda_odb10 datasets. From our predicted gene models, we used EnTAP to successfully annotate 16,047 gene models in CA, 15,985 gene models in TW, and 15,198 gene models in UY. 895,968 GO terms were assigned to 12,462 CA genes, 870,081 GO terms were assigned to 12,462 TW genes, and 844,334 GO terms were assigned to 11,909 UY genes. A total of 4,508, 4,417, and 4,223 genes from CA, TW, and UY, respectively, were successfully assigned KEGG orthology (KO) terms and accordingly mapped to 309, 312, and 309 biological pathways. Predicted proteins clustered into a total of 15,598 orthologous groups, with 14,554, 14,501, and 14332 orthologous groups belonging to CA, TW, and UY, respectively. <br><br><b>Publications</b><br>