Progress 09/01/20 to 08/31/23
Outputs
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. As explained above, in-person outreach workshops and field days have been replaced by virtual means of information dissemination. What opportunities for training and professional development has the project provided?During the three years of this funded project, we have provided training and professional development opportunities for 4 undergraduate students and 2 college graduated junior specialists. Two undergraduate students have been trained with all the knowledge and skills related to insect rearing and citrus planting. The other 2 undergraduate students were trained in basic molecular skills, such as polymerase chain reaction (PCR) and DNA or RNA extraction from single psyllids. The training activities were provided through one-on-one work with the PD, postdoctoral researchers, and/or junior specialists. In addition to trainings for undergraduate students, this project also provided more advanced training for junior specialists. The junior specialists working for this project are generally assigned to studies and small projects for our overall goals. One of the projects is the full genome assembly and analyses of different populations of D. citri. To work on the needed bioinformatics analyses, the junior specialist has been able to do bioinformatic analyses through Amazon Web Services computing servers paid by this project. At the end of the second year, we hired a new junior specialist and we have been training her in all psyllid-related knowledge and skills needed for this project. This project also provided trainings and professional development for 4 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. We attended and joined conferences, workshops, and training activities. 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. How have the results been disseminated to communities of interest?Due to COVID-19 restrictions during the first 2 years of this project, classical methods of extension outreach through in-person workshops and grower meetings were not possible. As a substitute, approaches were used that focused on the website and webinars. 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. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
For this funded project, we have 1) published detailed and high-quality chromosome-scale genome sequence analyses for the California (CA), Taiwan (TW), and Uruguay (UY) D. citri populations, 2) evaluated the effects of D. citri-specific virus(es), on D. citri biology and CLas acquisition and transmission, 3) established a system with color-coated psyllids for different treatments in assays so that all the treated psyllids could be placed on the same CLas-infected or -unindected citrus plants, 4)identified a novel bacterial endosymbiont originally from Hawaiian D. citri population, 5) tested more selected candidate microRNAs in D. citri for potential negative effects against CLas infection and/or D. citri, 6)established cricket paralysis virus into a mild strain viral vector. The progress we accomplished for this project leads us to achieve our goals of 1) engineering D. citri viral endosymbionts to induce desirable RNAi effects in the targeted D. citri populations and 2) deepening the understanding of this interconnected network to allow for manipulating symbionts as beneficial tools in the battle against CLas and D. citri. Objectives 1. Engineer insect-specific viruses for use in D. citri Diaphorina citri picorna-like virus (DcPLV)-- Due to the toxicity caused by the full DcPLV genome sequence in Escherichia coli, the DcPLV genome sequence was divided into three fragments, and cloned into the pMT vector separately. Thus, to avoid involving E. coli during cloning, we have been using a yeast-based cloning system to obtain full-lenth viral genome clones. This full-length DcPLV clone will then be used for in vitro transcription and the transcripts will be used to test the DcPLV infectivity in different insect cell lines and in D. citri directly. The alignment of the 2 full-length viral genome sequences, assembled from next generation sequencing and partially confirmed by Sanger sequencing, showed that there are 3 variants in the UY-D. citri population and only one variant/isolate in the TW-D. citri. All 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 in different D. citri populations, DcPLV isolates/variants from Uruguay population struggled to establish infections in D. citri Taiwan population. By comparing the DcPLV viral genome sequences from UY variants and TW isolate, we identified 109 nucleotide sequence in 5'UTR region of UY-DcPLV variants was truncated compared to the TW and BR (Brazilian) isolates. Diaphoria citri reovirus (DcRV)-- For our preliminary tests, we used T7 RNA polymerase to drive the transciption of the DcRV genome segments in the Drosophila S2 cells in attempts to recover DcRV virions. A plasmid expressing T7 RNA polymerase was also co-transfected with DcRV genome constructs into the S2 cells to drive the RNA transcription in the cells. However, no viral proteins were expressed from those constructs in the S2 cells when detected using Western blot analysis. We also test the viral gene expression using pAc5.1 plasmid vector that drives ORF expression by the Drosophila actin promoter in the S2 cells. 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. Taken together, our 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. Diaphoria citri densovirus (DcDV)-- As described in the previous report, 4,865 nt of full-length DcDV (5,071nt, ssDNA) genome sequence was cloned into the vector pBR322. Attempts at completing the inverted terminal repeats (ITR) of the DcDV insert have resulted in truncated inserts. The repetitive sequences and the secondary structure of the viral ITR were suspected to cause homologous recombination and consequently cause the truncated clones. As the only DNA virus identified in D. citri that replicates in nucleus, DcDV is important for artificial miRNA delivery. Therefore, we will use different cloning strategies to complete the viral genome construction and find the right conditions for delivering the potential infectious clone. 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. After 96 h, insects were moved to C. macrophylla plants and DcDV titre was tested in five individual insects every other day for 12days by qPCR. Our results suggested that DcDV is not horizontally transmitted via oral acquisition. 2. Identify novel endosymbiont RNAi targets in geographically distinct D. citri populations Three high quality chromosome-scale genome assemblies of CA-, TW-, and UY-D. citri were published in June 2022 by our group. These genomic resources are being used to map our small RNA libraries from CLas infected and uninfected samples. We are continuing analyzing small RNA mapping to the HH CLas genome, to identify putative direct targets. The target list will be used to test for phenotypes using synthesized dsRNA. 3. Use D. citri viruses to deliver specific small RNAs that interfere with D. citri, its endosymbiont microbiome, and/or CLas Our data generated from the NGS analyses of CLas-infected and CLas-uninfected CA-D. citri indicated that specific miRNAs in different dissected organs are affected by CLas infection in CA-D. citri. Based on the results of our analyses, we synthesized small RNAs to specifically target the miRNAs selected from the deep sequencing analyses and injected them into D. citri to screen for effects. The injected D. citri were placed on CLas-infected citrus and tested the CLas titer and/or other effects in D. citri. While seeing potential effects of those synthesized small RNA sequences, we are optimizing the methods used to verify the biological effects. We have established a color-coated psyllid approach suggested by Brazilian researchers to test different small RNA treated D. citri onto the same plant leaves to minimize the variations caused by the plant and psyllid stages and by individual plants. During this funding period, we predicted 2 D. citri primary miRNAs: pri-miRNA 317 and pri-miRNA 277 using RNAseq, small RNA deep sequencing and degradome sequencing technology. We also predicted the target gene transcripts of these 2 miRNAs from our bioinformatics analyses. We then expressed both the pri-miRNAs and the predicted sequences in Drosophila S2 cells and confirmed that most of the predictions were correct and the miRNAs could target the predicted target sequences and successfully down regulate the GFP expression with the expected target sequences. We also successfully used the pri-miRNAs as backbones for artificial miRNA expression and target to the expected target sequences in S2 cells. The 2 pri-miRNAs can be used in the viral vectors for future applications. Overall, we accomplished most of the proposed objectives. Although we couldn't resolve all the difficult aspects of this project, we produced significant and important progress that will benefit future HLB related studies and move closer to an innovative approach for HLB management.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
E. Henry, C.R. Carlson, and Y.-W. Kuo (2023) Candidatus Kirkpatrella diaphorinas gen. nov., sp. nov., an uncultured endosymbiont identified in a population of Diaphorina citri from Hawaii. International Journal of Systematic and Evolutionary Microbiology. DOI: 10.1099/ijsem.0.006111
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Y.-W. Kuo, B.W. Falk (2022) Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine-rich preference for virus- and non-virus-based expression vectors in plants. Plant Biotechnology Journal. 20, 1069 1084. DOI: 10.1111/pbi.13786
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
D.M. Galdeano, T. Rawat, W. Ingram, C.R. Carlson, G.R. Alves, and Y.-W. Kuo (2023) Plant pathogenic bacterial transmission competence and biology of its insect vector Diaphorina citri modulated by insect specific virus (In prep.)
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
C.R. Carlson, A.M. ter Horst, J.S. Johnston, E. Henry, B.W. Falk, and Y.-W. Kuo (2022) High quality, chromosome-scale genome assemblies: comparisons of three Diaphorina citri (Asian citrus psyllid) geographic populations. DNA Research. DOI: 10.1093/dnares/dsac027
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
E.E. Matsumura, J.C. Nigg, E.M. Henry, and B.W. Falk (2020) Development of a cricket paralysis virus-based system for inducing RNA interference-mediated gene silencing in Diaphorina citri. bioRxiv. DOI: https://doi.org/10.1101/2020.11.15.383588
- Type:
Websites
Status:
Published
Year Published:
2023
Citation:
Y.-W. Kuo, D.M. Galdeano, B.W. Falk, E. Stover, and P.G. Lemaux (2023) Virus-induced gene silencing (VIGS) using insect-specific viruses to manipulate psyllids as a strategy to help control citrus greening/HLB. Research Snapshots - Science for Citrus Health
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
D.M. Galdeano, C.R. Carlson, T. Rawat, W. Ingram, G.R. Alves, B.W. Falk, and Y.-W. Kuo (2023) Influence of Diaphorina citri flavi-like virus (DcFLV) on the biological aspects of Diaphorina citri, vector the causal agent of Huanlongbing. W28-4
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Y.-W. Kuo, D.M. Galdeano, C.R. Carlson, T. Rawat, and B.W. Falk (2023) Infection incidence and genome analyses of Diaphorina citri picorna-like virus isolates in Asian citrus psyllid, Diaphorina citri. W28-5
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
B.W. Falk. Plant Pathology, Everywhere Every Day. (2021) Plant Health 2021, Phytopathologist of Distinction Talk
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
D.M. Galdeano, C.R. Carlson, Y.-W. Kuo, and B.W. Falk (2022) Interactions of Diaphorina citri flavi-like virus (DcFLV) in Diaphorina citri, vector the causal agents of Huanlongbing. American Society for Virology Annual Meeting. P18-23
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
C.R. Carlson, A.M. ter Horst, J.S. Johnston, E. Henry, B.W. Falk, and Y.-W. Kuo (2022) The endogenous viral element landscapes of Diaphorina citri (Asian citrus psyllid) identified in new, chromosome-scale genome assemblies. American Society for Virology Annual Meeting. P26-2
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
Accelerating implementation of HLB-tolerant hybrids as new commercial cultivars for fresh and processed citrus Dr. Jinhe Bai, USDA-ARS, Fort Pierce, FL., Dr. Peggy G. Lemaux, University of California Berkeley, Berkeley, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Development of antimicrobial peptides from citrus to kill the CLas bacterium causing HLB Dr. Ed Stover, USDA-ARS, Fort Pierce, FL., Dr. Peggy G. Lemaux, University of California Berkeley, Berkeley, CA.
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
Developing novel biological delivery methods for therapeutic agents and other biomolecules to enhance production of citrus Dr. Robert Shatters, USDA Horticultural Research Laboratory, Fort Pierce, FL., Dr. Peggy G. Lemaux, University of California Berkeley, Berkeley, CA.
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
Two-pronged approach to suppress the Asian citrus psyllid vector of HLB
Dr. Bryony Bonning, University of Florida., Dr. Peggy G. Lemaux, University of California Berkeley, Berkeley, CA.
|
Progress 09/01/21 to 08/31/22
Outputs
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 and 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 ~9000 visits and ~3500 downloads. During this period, 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 49 Research Snapshots (four during this period), broken into five categories, Disease Management, Early Detection Techniques, Established Orchards, Psyllid Management, and General Tools. Additions to the Citrus Series Podcast, Beyond the Bench (https://ucanr.edu/sites/scienceforcitrushealth/podcast/) were also made. In these pieces, 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 (three during this period). 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. Portions were translated into Spanish during this period. Another effective outreach method for our target audiences is the use of webinars. During this period, three webinars were held, two in English. "Management Program for Nipaecoccus viridis (hibiscus mealybug)", had 150 attendees, 143 from the U.S. and the rest from six other countries. "Citrus Thrips Biology, Monitoring, and Management", had 194 attendees with 178 from CA and the rest from other states, Mexico and Peru. A third webinar in Spanish, "Biología y Manejo del Psílido Asiático de Cítricos (PAC)", attracted over 100 attendees from the U.S., Mexico, Spain, Ecuador, Argentina, Venezuela and Costa Rica. Webinars provide ~30-40 min talks by experts. Webinars are advertised in California, Texas and Florida; as appropriate, CEU (pesticide license) credits were offered. Changes/Problems:Because our personnel change, we will not only continue our previous efforts for all the objectives, but include more biology, feeding, acquisition, and transmission assays of different symbiont-infected populations and study host-microbe and microbe-microbe interactions in D. citri. As explained above, in-person outreach workshops and field days have been replaced by virtual means of information dissemination. What opportunities for training and professional development has the project provided?During the second year of this funded project, we have provided training and professional development opportunities for 2 undergraduate students and 2 college graduated junior specialists. One undergraduate student has been trained with all the knowledge and skills related to insect rearing and citrus planting. The other undergraduate student was trained in basic molecular skills, such as polymerase chain reaction (PCR) and DNA or RNA extraction from single psyllids. The training activities were provided through one-on-one work with the project scientist, postdoctoral researchers, or junior specialists. In addition to trainings for undergraduate students, this project also provided more advanced training for junior specialists. The junior specialists working for this project are generally assigned to studies and small projects for our overall goals. One of the projects is the full genome assembly and analyses of different populations of D. citri. To work on the needed bioinformatics analyses, the junior specialist has been able to do bioinformatic analyses through Amazon Web Services computing servers paid by this project. At the end of the second year, we hired a new junior specialist and we have been training her in all psyllid-related knowledge and skills needed for this project. 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 third/final year of this project as more COVID restrictions are lifted. 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. How have the results been disseminated to communities of interest?Due to COVID-19 restrictions, classical methods of extension outreach through in-person workshops and grower meetings were not possible. As a substitute, approaches were used that focused on the website and webinars. 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. What do you plan to do during the next reporting period to accomplish the goals?We have been making good progress in the first and second year of this project. 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. Objectives 1. Engineer insect-specific viruses for use in D. citri Because of the different features and natures of each insect-specific viruses found in D. citri, we will use different approaches to finish the constructs of full viral genome clones mentioned previously. We will also test if there are other viral proteins or enzymes needed for viral replication initiation in D. citri. Due to the lack of any D. citri cell lines, it makes it very hard to test any expression clones or D. citri-specific viral infectious clones for D. citri. We will continue testing the best strategies and plasmid vectors for delivering full-genome viral clones of the D. citri-specific viruses directly in psyllids and other insect cells, such as mosquito cell line- c6/36 and hemiptera cell line- sf9. 2. Identify novel endosymbiont RNAi targets in geographically distinct D. citri populations We have done small RNA and transcriptome deep sequencing of CA- D. citri and DcRV-infected CA-D. citri during the first year of the project. During this reported period, (second year) we have now generated small RNA and RNAseq sequencing data of TW- D. citri and UY-D. citri. We will identify geographically distinct RNAi targets and potential RNAi targets across different populations based on bioinformatic analyses. Specifically, we will be looking for candidates that are highly changed in insects without exposure to CLas and after exposure to CLas. We will first map the small RNAs with different individual viral and bacterial endosymbionts and look for profile changes between CLas-positive and -negative D. citri. Identified candidates will also be assessed for potential usefulness in virus-induced gene silencing (VIGS) driven RNAi based on the genes they target and microbial or host origin. At the end of these filtering steps, we will have a list of candidate miRNAs, virus-induced siRNAs, small RNA target regions in symbionts, and target genes for each D. citri population. 3. Use D. citri viruses to deliver specific small RNAs that interfere with D. citri, its endosymbiont microbiome, and/or CLas Although all the viruses we identified in D. citri are insect borne and specific for D. citri, those viruses could transmit differently through generations, and could even be limited to specific population(s)/haplotype. Therefore, while developing insect-specific viral vectors for RNAi delivery, in addition to DcFLV-positive D. citri, we will also study the biology, feeding, acquisition, and transmission mechanisms of each selected virus-infected populations. While we continue testing more candidate miRNA targets, we will also test additional RNAi targets against D. citri endosymbionts and/or CLas using synthesized dsRNA inducers based on our bioinformatic analyses results. We will then construct the selected RNAi inducers into viral infectious clones developed in Objective 1 for stronger RNAi effects in D. citri. By using the RNA (DcFLV, DcPLV, and DcRV) and DNA (DcDV) viruses here we can deliver different types of small RNAs in D. citri. We will use the cytoplasmically replicating RNA virus(es) DcFLV, DcRV, or DcPLV to generate siRNAs. The amiRNAs will be cloned into miRNA backbones (primary miRNAs) of miR317 or miR277 of D. citri. DcDV, a nuclear replicating virus, will be used to generate artificial microRNAs (amiRNAs), miRNA mimics, or sequences that target specific miRNAs. Sequences of candidate target genes of endosymbionts or of D. citri predicted in Objective 2 will be cloned into the RNA viral vectors to induce desired siRNAs and RNAi effects in D. citri. These combined experiments will allow us to engineer D. citri viral endosymbionts to induce desirable RNAi effects in the targeted D. citri populations and will deepen the understanding of this interconnected network and allow for manipulating symbionts be as beneficial tools in the battle against CLas and D. citri. Outreach efforts will continue along the same trajectory as those described in this report.
Impacts
What was accomplished under these goals?
With the urgent needs of solving the problems caused by the disease Huanglongbing (HLB), different approaches are being investigated to help manage the disease including targeting Diaphorina citri (the Asian citrus psyllid), the insect vector of Candidatus Liberibacter asiaticus (CLas), causal agent of this devastating citrus disease. However, there is increasing concern that conventional insect control measures such as insecticides could lead to resistance in D. citri and indiscriminately target beneficial insects. This underscores the importance of studying the biology of D. citri, so that alternative control measures can be devised. Insect endosymbionts, which are vertically transmitted to insect progeny or sometimes horizontally transmitted to other individuals, play key roles in insect-plant and/or insect-plant-pathogen interactions and influence different aspects of insect biology. In addition to developing viral infectious vectors of the D. citri-specific viruses identified by our group, it is also important to study the biology effects of each insect-specific virus on D. citri and CLas. During the second year of this funded project, we have 1) published detailed and high-quality chromosome-scale genome sequence analyses for the California (CA), Taiwan (TW), and Uruguay (UY) D. citri populations, 2) evaluated the effects of D. citri-specific virus(es), on D. citri biology and CLas acquisition and transmission, 3) established a system with color-coated psyllids for different treatments in assays so that all the treated psyllids could be placed on the same CLas-infected or -unindected citrus plants, 4)identified a novel bacterial endosymbiont originally from Hawaiian D. citri population, 5) tested more selected candidate microRNAs in D. citri for potential negative effects against CLas infection and/or D. citri. We are making good progress to achieve the goals of this project. The progress we accomplished during the first year of this project leads us to achieve our goals of 1) engineering D. citri viral endosymbionts to induce desirable RNAi effects in the targeted D. citri populations and 2) deepening the understanding of this interconnected network to allow for manipulating symbionts as beneficial tools in the battle against CLas and D. citri. Objectives 1. Engineer insect-specific viruses for use in D. citri Diaphorina citri picorna-like virus (DcPLV)-- Due to the toxicity caused by the full DcPLV genome sequence in Escherichia coli, the DcPLV genome sequence was divided into three fragments, and cloned into the pMT vector separately. Thus, to avoid involving E. coli during cloning, we have been using a yeast-based cloning system to obtain full-lenth viral genome clones. We attempt to transform E. coli cells with the yeast purified plasmid as well as with lysates of the transformed yeast-cells. Although E. coli transformation was successful, Sanger sequencing results showed additional sequence of 1000 bp inserts, which later was found to be fragments from E. coli, and multiple nucleotide sequence errors in the DcPLV clones. This full-length DcPLV clone will then be used for in vitro transcription and the transcripts will be used to test the DcPLV infectivity in different insect cell lines and in D. citri directly. Diaphoria citri reovirus (DcRV)-- For our preliminary tests, we used T7 RNA polymerase to drive the transciption of the DcRV genome segments in the Drosophila S2 cells in attempts to recover DcRV virions. The S2 cells were transfected with plasmids containing all the ten DcRV genome segments that were driven by the T7 promoter. A plasmid expressing T7 RNA polymerase was also co-transfected with DcRV genome constructs into the S2 cells to drive the RNA transcription in the cells. However, no viral proteins were expressed from those constructs in the S2 cells when detected using Western blot analysis. We also test the viral gene expression using pAc5.1 plasmid vector that drives ORF expression by the Drosophila actin promoter in the S2 cells. We are now also testing to express the viral genes in plants in attempts to obtain the DcRV viral virions in plant cells. Diaphoria citri densovirus (DcDV)-- As described in the previous report, 4,865 nt of full-length DcDV (5,071nt, ssDNA) genome sequence was cloned into the vector pBR322. Attempts at completing the inverted terminal repeats (ITR) of the DcDV insert have resulted in truncated inserts. The repetitive sequences and the secondary structure of the viral ITR were suspected to cause homologous recombination and consequently cause the truncated clones. We are now testing other cloning strategies to include the viral ITRs into the full-genome clones into the pAc5.1 plasmid vector. As the only DNA virus identified in D. citri that replicates in nucleus, DcDV is important for artificial miRNA delivery. Therefore, we will use different cloning strategies to complete the viral genome construction and find the right conditions for delivering the potential infectious clone. 2. Identify novel endosymbiont RNAi targets in geographically distinct D. citri populations Three high quality chromosome-scale genome assemblies of CA-, TW-, and UY-D. citri were published in June 2022 by our group. These genomic resources are being used to map our small RNA libraries from CLas infected and uninfected samples. We are continuing analyzing small RNA mapping to the HH CLas genome, to identify putative direct targets. The target list will be used to test for phenotypes using synthesized dsRNA. The whole-genome sequencing of our four D. citri populations also resulted in the discovery of a novel and heritable bacterial endosymbiont in one of the D. citri populations. The novel bacterial endosymbiont appears to have a reduced genome in comparison to its closest relative. A descriptive manuscript is currently in prep to publish these results. We also tested the biology, feeding behavior, acquisition, and transmission of CLas differences of DcFLV-positive and -negative D. citri. The results showed that 1) the duration of egg, nymphal and developmental were significantly longer in DcFLV-positive D. citri; 2) the feeding behavior was similar in both DcFLV-positive and -negative D. citri; 3) DcFLV-negative showed higher acquisition rate while showing lower CLas titers in the CLas-positive psyllids compared with DcFLV-positive psyllids; 4) DcFLV-positive D. citri showed significant higher CLas transmission rate and the transmitted citrus plants showed significantly higher CLas titers. in our transmission assays. 3. Use D. citri viruses to deliver specific small RNAs that interfere with D. citri, its endosymbiont microbiome, and/or CLas Our data generated from the NGS analyses of CLas-infected and CLas-uninfected CA-D. citri indicated that specific miRNAs in different dissected organs are affected by CLas infection in CA-D. citri. Based on the results of our analyses, we synthesized small RNAs to specifically target the miRNAs selected from the deep sequencing analyses and injected them into D. citri to screen for effects. The injected D. citri were placed on CLas-infected citrus and tested the CLas titer and/or other effects in D. citri. While seeing potential effects of those synthesized small RNA sequences, we are optimizing the methods used to verify the biological effects. Due to the variability of CLas distribution and titer in citrus plants and through the progression of infection, it could be hard to conclude the biological effects of CLas infection in D. citri. However, now we have established a color-coated psyllid approach suggested by Brazilian researchers to test different small RNA treated D. citri onto the same plant leaves to minimize the variations caused by the plant and psyllid stages and by individual plants. We will continue testing different miRNA mimics and/or anti-miRNAs and develop optimal method(s) for clear biological effects.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Y.-W. Kuo, B.W. Falk (2022) Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine-rich preference for virus- and non-virus-based expression vectors in plants. Plant Biotechnology Journal. 20, pp. 1069-1084; DOI: 10.1111/pbi.13786
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
C.R. Carlson, A.M. ter Horst, J.S. Johnston, E. Henry, B.W. Falk, and Y.-W. Kuo (2022) High quality, chromosome-scale genome assemblies: comparisons of three Diaphorina citri (Asian citrus psyllid) geographic populations. DNA Research. DOI: 10.1093/dnares/dsac027
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
E. Henry, C.R. Carlson, and Y.-W. Kuo (2022) Candidatus Kirkpatrella diaphorinas gen. nov., sp. nov., an uncultured endosymbiont identified in a population of Diaphorina citri from Hawaii. International Journal of Systematic and Evolutionary microbiology (In prep.)
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
E.E. Matsumura, S. Vu, C.R. Carlson, T. Rawat, Y.-W. Kuo, E. Henry, J.C. Jared, and B.W. Falk (2022) Development of a cricket paralysis virus-based system for inducing RNA interference-mediated gene silencing in Diaphorina citri. International Journal of Molecular Sciences (In prep.)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
D.M. Galdeano, C.R. Carlson, Y.-W. Kuo, and B.W. Falk (2022) Interactions of Diaphorina citri flavi-like virus (DcFLV) in Diaphorina citri, vector the causal agents of Huanlongbing. American Society for Virology Annual Meeting. P18-23
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
C.R. Carlson, A.M. ter Horst, J.S. Johnston, E. Henry, B.W. Falk, and Y.-W. Kuo (2022) The endogenous viral element landscapes of Diaphorina citri (Asian citrus psyllid) identified in new, chromosome-scale genome assemblies. American Society for Virology Annual Meeting. P26-2
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
Accelerating implementation of HLB-tolerant hybrids as new commercial cultivars for fresh and processed citrus Dr. Jinhe Bai, USDA-ARS, Fort Pierce, FL., Dr. Peggy G. Lemaux, University of California Berkeley, Berkeley, CA.
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
Development of antimicrobial peptides from citrus to kill the CLas bacterium causing HLB Dr. Ed Stover, USDA-ARS, Fort Pierce, FL., Dr. Peggy G. Lemaux, University of California Berkeley, Berkeley, CA.
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
Developing novel biological delivery methods for therapeutic agents and other biomolecules to enhance production of citrus Dr. Robert Shatters, USDA Horticultural Research Laboratory, Fort Pierce, FL., Dr. Peggy G. Lemaux, University of California Berkeley, Berkeley, CA.
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
Two-pronged approach to suppress the Asian citrus psyllid vector of HLB
Dr. Bryony Bonning, University of Florida., Dr. Peggy G. Lemaux, University of California Berkeley, Berkeley, CA.
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Outreach and extension efforts provide information in English to the citrus industry, growers, media and the general public. The website, Science for Citrus Health (https://ucanr.edu/sites/scienceforcitrushealth/), covers the ACP/HLB situation and provides resources for growers and others to better understand techniques and approaches being developed to battle the disease. Created in May 2015 and recently updated, the SCH site has to date had ~7300 visits and ~2700 downloads, >500 during this reporting period. Currently, we are converting site information to Spanish (https://ucanr.edu/sites/scienceforcitrushealth/Home_Page_in_Spanish/ ), given the relationship of the Hispanic population to the citrus industry and growers. These outreach efforts are conducted by a team of AES faculty, extension specialists, grad students and postdocs from the University of California (Berkeley, Davis, Riverside), University of Florida and Texas A&M. Meetings of the team are held monthly to discuss emerging citrus issues, new citrus research projects for potential Research Snapshots and Podcasts (see below), feedback from completed webinars (see below), ideas for future webinars and other issues related to the citrus industry and HLB. For Research Snapshots (https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/), we worked with citrus project researchers (six during this period) to develop descriptions and images depicting their strategies and accomplishments related to HLB. These pieces were translated into language understandable by growers, the media and the general public. Currently, there are 36 Research Snapshots, broken into five categories, Disease Management, Early Detection Techniques, Established Orchards, Psyllid Management, and General Tools. Also, we initiated the Citrus Series Podcast, Beyond the Bench (https://ucanr.edu/sites/scienceforcitrushealth/podcast/), which asks researchers about their science and to explain how they got interested in their work, the impact of what they do, and what their job is like. Five such podcasts are now featured on the site. An extensive collection of PP slides is also available on the site that provides descriptions of genetic engineering and editing and how they might be used to modify citrus and psyllids. Portions are currently being translated into Spanish. Another outreach method, to reach our target audiences, webinars, was initiated this year. In February 2021, we conducted a webinar, "Emerging technologies to manage Asian citrus psyllid and Huanglongbing", which featured 20-minute talks by experts, including a PI on this grant, Bryce Falk, with a panel discussion at the end. The webinar was advertised in California, Texas and Florida; CEU (pesticide license) credits were offered. The webinar had 163 participants, including 16 international participants. Of the attendees, 90% agreed or strongly agreed that the webinar met their learning objectives and they gained new knowledge. A second webinar was held in July entitled, "Managing Asian citrus psyllid and citrus growth using particle films", with the same format as the preceding webinar. Two experts, one from UFL and one from UCR, gave a 30 min talk followed by an engaging discussion. There were 211 participants from 12 countries and 7 different states; 72 CA participants received CEU credits. Of attendees, 80% agreed or strongly agreed that the webinar was relevant to their work and 97% agreed or strongly agreed that they gained new knowledge. Changes/Problems:Although there are some delays due to the coronavirus pandemic, we believe we will fulfill the objectives proposed at the end of this project. What opportunities for training and professional development has the project provided?During the first year of this funded project, we have provided training and professional development opportunities for 2 undergraduate students and 2 college graduated junior specialists. One undergraduate student has been trained with all the knowledge and skills related to insect rearing and citrus planting. He will be trained for insect and plant sampling in the second year of this project. The other undergraduate student has been trained in basic molecular skills, such as polymerase chain reaction (PCR) and DNA or RNA extraction from single psyllids. The training activities were provided through one-on-one work with the project scientist, postdoctoral researchers, or junior specialists. In addition to trainings for undergraduate students, this project also provided more advanced training for junior specialists. The junior specialists working for this project are generally assigned to studies and small projects for our overall goals. One of the projects is the full genome assembly and analyses of different populations of D. citri. To work on the needed bioinformatics analyses, the junior specialist attended a 2 week bioinformatics training workshop and has been able to do bioinformatic analyses through Amazon Web Services computing servers paid by this project. 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 first year of this project. We will aim to attend and join more conferences, workshops, and training activities in the second/final year of this project as more COVID restrictions are lifted. How have the results been disseminated to communities of interest?Restrictions due to the COVID-19 pandemic have restricted classical methods of extension outreach through in-person workshops and grower meetings. Thus, in order to disseminate the above-described content, we used new approaches through the website and webinars, as described above. These approaches were effective in reaching our intended audiences and providing them with up-to-date information on the state of research efforts to combat HLB in citrus. In addition to efforts described above, the team prepared an article (https://citrusindustry.net/category/hlb-management/ ) for Citrus Industry News in October 2020, describing the informational resources available on the SCH website. A group of graduate students at UC Riverside created an Instagram account to highlight Research Snapshots and to reach a wider audience. We also created a YouTube channel (https://www.youtube.com/channel/UC2bdSHP_rsOJgNqx7HEOyOg) to provide opportunities for Research Snapshot authors to share videos related to their work. People have watched videos over 1000 times since the channel was launched. What do you plan to do during the next reporting period to accomplish the goals?We have been making good progress in the first year of this project. 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. Objectives 1. Engineer insect-specific viruses for use in D. citri Because of the different features and natures of each insect-specific viruses found in D. citri, we will use different approaches to finish the constructs of full viral genome clones mentioned previously. We will also test if there are other viral proteins or enzymes needed for viral replication initiation in D. citri. In addition, we will start to develop infectious clones of Diaphorina citri reovirus (DcRV). We will first clone all 10 segments of the viral genome into DNA vectors. We will then follow the successful reovirus reverse genetics strategies used for other reoviruses, such as bluetongue virus (BTV) and African horse sickness virus (AHSV), which are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family). Due to the lack of D. citri cell lines, we will first test the clones in the Drosophila S2 cells. We will also test the clones in D. citri once we establish a suitable promoter that can be used in D. citri. 2. Identify novel endosymbiont RNAi targets in geographically distinct D. citri populations To identify novel endosymbiont and/or CLas RNAi targets, we first used CA-D. citri and DcRV-infected CA-D. citri to develop a working pipeline. We now have done small RNA and transcriptome deep sequencing of CA- D. citri and DcRV-infected CA-D. citri and are currently analyzing the data and working on pipeline development. Next, we will prepare and generate sequencing data of TW- D. citri and UY-D. citri. We will identify geographically distinct RNAi targets and potential RNAi targets across different populations based on bioinformatic analyses. Specifically, we will be looking for candidates that are highly changed in insects without exposure to CLas and after exposure to CLas. Identified candidates will also be assessed for potential usefulness in virus-induced gene silencing (VIGS) driven RNAi based on the genes they target and microbial or host origin. At the end of these filtering steps, we will have a list of candidate miRNAs, virus-induced siRNAs, small RNA target regions in bacterial symbionts, and target genes for each D. citri population. 3. Use D. citri viruses to deliver specific small RNAs that interfere with D. citri, its endosymbiont microbiome, and/or CLas Although all the viruses we identified in D. citri are insect borne and specific for D. citri, those viruses could transmit differently through generations, and could even be limited to specific population(s)/haplotype. Therefore, while developing insect-specific viral vectors for RNAi delivery, we will also study the D. citri population range and transmission mechanisms of each selected virus. While we continue testing more candidate miRNA targets, we will also test additional RNAi targets against D. citri endosymbionts and/or CLas using synthesized dsRNA inducers based on our bioinformatic analyses results. We will then construct the selected RNAi inducers into viral infectious clones developed in Objective 1 for stronger RNAi effects in D. citri. By using the RNA (DcFLV, DcPLV, and DcRV) and DNA (DcDV) viruses here we can deliver different types of small RNAs in D. citri. We will use the cytoplasmically replicating RNA virus(es) DcFLV, DcRV, or DcPLV to generate siRNAs. The amiRNAs will be cloned into miRNA backbones (primary miRNAs) of miR317 or miR277 of D. citri. DcDV, a nuclear replicating virus, will be used to generate artificial microRNAs (amiRNAs), miRNA mimics, or sequences that target specific miRNAs. Sequences of candidate target genes of endosymbionts or of D. citri predicted in Objective 2 will be cloned into the RNA viral vectors to induce desired siRNAs and RNAi effects in D. citri. These combined experiments will allow us to engineer D. citri viral endosymbionts to induce desirable RNAi effects in the targeted D. citri populations and will deepen the understanding of this interconnected network and allow for manipulating symbionts be as beneficial tools in the battle against CLas and D. citri.
Impacts
What was accomplished under these goals?
Different approaches are being investigated to help manage Huanglongbing (HLB) including targeting Diaphorina citri (the Asian citrus psyllid), the insect vector of Candidatus Liberibacter asiaticus (CLas), causal agent of this devastating citrus disease. However, there is increasing concern that conventional insect control measures such as insecticides could lead to resistance in D. citri and indiscriminately target beneficial insects. This underscores the importance of studying the biology of D. citri, so that alternative control measures can be devised. Insect endosymbionts, which are vertically transmitted to insect progeny or sometimes horizontally transmitted to other individuals, play key roles in insect-plant and/or insect-plant-pathogen interactions and influence different aspects of insect biology. During the first year of this funded project, we have 1) completed detailed and high-quality genome sequence analyses for the California (CA), Taiwan (TW), and Uruguay (UY) D. citri populations, 2) confirmed the D. citri true genome size, 3) completed full genomes of D. citri endosymbionts, 4) analyzed small RNAs derived from bacterial endosymbionts, and 5) tested several selected candidate microRNAs in D. citri for potential negative effects against CLas infection and/or D. citri. We are making good progress to achieve the goals of this project. The progress we accomplished during the first year of this project leads us to achieve our goals of 1) engineering D. citri viral endosymbionts to induce desirable RNAi effects in the targeted D. citri populations and 2) deepening the understanding of this interconnected network to allow for manipulating symbionts as beneficial tools in the battle against CLas and D. citri. Objectives 1. Engineer insect-specific viruses for use in D. citri Diaphorina citri picorna-like virus (DcPLV)-- Prior to the start of this project, we had already cloned cDNAs derived from DcPLV genome in a plasmid backbone driven by the Drosophila-derived metallothionein gene promoter (pMT). However, due to the toxicity caused by the full DcPLV genome sequence in Escherichia coli, the DcPLV genome sequence was divided into three fragments, 3.1, 3.5 and 3.7 Kb in length, and cloned into the pMT vector separately. Thus, to avoid involving E. coli during cloning, we are now testing a yeast-based cloning system. Next, we will co-transform the linear pYES1L vector and the DcPLV fragments into the MaV203 Competent Yeast Cells. This system takes advantage of homologous recombination in Saccharomyces cerevisae to join DNA fragments that share end terminal homology into a single molecule. This full-length DcPLV clone will then be used for in vitro transcription and the transcripts will be used to test the DcPLV infectivity in different insect cell lines and in D. citri directly. Diaphorina citri flavi-like virus (DcFLV)-- Cloning of DcFLV (27,724 nt) is nearly complete. The last three fragments and the middle three fragments have been combined and only one cloning reaction to combine all fragments remains. They will initially be cloned into a vector with a pMT promoter to test in Drosophila S2 cells. To successfully trigger in vivo transcription of our potential virus infectious DNA clones, we will first develop and test working promoter(s) for D. citri. Due to the lack of useful information and knowledge of D. citri endogenous promoter sequences, we are currently testing the Drosophila U6-2 promoter and a putative D. citri ubiquitin promoter with the reporter gene GFP and will determine the transcription start site. We will continue to test more putative D. citri promoters based on our genome data and transcriptome analyses. Diaphorian citri denso virus (DcDV)-- 4,865 nt of full-length DcDV (5,071nt, ssDNA) genome sequence was cloned into the vector pBR322. Attempts at completing the inverted terminal repeats (ITR) of the DcDV insert have resulted in truncated inserts. The repetitive sequences and the secondary structure of the viral ITR were suspected to cause homologous recombination and consequently cause the truncated clones. As the only DNA virus identified in D. citri that replicates in nucleus, DcDV is important for artificial miRNA delivery. Therefore, we will use different cloning strategies to complete the viral genome construction and find the right conditions for delivering the potential infectious clone. 2. Identify novel endosymbiont RNAi targets in geographically distinct D. citri populations During the first year of this funded project, we completed the genomes of all D. citri populations and their endosymbionts using a combination of short-read WGS, Hi-C sequencing, PacBio HiFi sequencing, and RNAseq. We completed and annotated three high-quality, chromosome-scale genome assemblies of D. citri isolates from California, Taiwan, and Uruguay. These assemblies represent both major D. citri haplotypes: the Southeast Asian haplotype (TW and UY) and the Southwest Asian haplotype (CA). Flow cytometry and K-mer analyses indicated the true genome size of D. citri to be approximately 280Mb, with 12 autosomes and a XX/X0 sex determination system. Chromosome 7 has been identified as the putative X chromosome through sequencing coverage analyses and interspecific synteny. BUSCO analyses showed high completeness and low duplication of single copy orthologs. These assemblies will provide invaluable genomic resources to researchers. A manuscript describing these new resources is in preparation. These genomic resources are being used to map our small RNA libraries from CLas infected and uninfected samples. Within the bacterial symbionts we have found a predominance of tRNA-derived small RNAs, especially in the symbiont Candidatus Profftella armatura. Additionally, we generated lists of predicted bacterial sRNAs based on highly mapped regions of the genome that also have hairpin secondary structure. Regions of interest are identified and extended to the full area of mapped reads, then analyzed to determine the folding prediction of each region, since bacterial small RNAs are usually 50-500nt in length. Targets identified in CLas infected samples will be compared to those identified in CLas uninfected samples. We are also analyzing small RNA mapping to the HH CLas genome, to identify putative direct targets. The target list will be used to test for phenotypes using synthesized dsRNA. The whole-genome sequencing of our four D. citri populations also resulted in the discovery of a novel and heritable bacterial endosymbiont in one of the D. citri populations. The novel bacterial endosymbiont appears to have a reduced genome in comparison to its closest relative, indicating it may have evolved to be host-dependent. A descriptive manuscript is currently in prep to publish these results. 3. Use D. citri viruses to deliver specific small RNAs that interfere with D. citri, its endosymbiont microbiome, and/or CLas Our preliminary data generated from the NGS analyses of CLas-infected and CLas-uninfected CA-D. citri indicated that specific miRNAs in different dissected organs are affected by CLas infection in CA-D. citri. Based on the results of our analyses, we synthesized small RNAs to specifically target the miRNAs selected from the deep sequencing analyses and injected them into D. citri to screen for effects. The injected D. citri were placed on CLas-infected citrus and tested the CLas titer and/or other effects in D. citri. While seeing potential effects of those synthesized small RNA sequences, we are optimizing the methods used to verify the biological effects. Due to the variability of CLas distribution and titer in citrus plants and through the progression of infection, it could be hard to conclude the biological effects of CLas infection in D. citri. We will continue to test different miRNA mimics and/or anti-miRNAs and develop optimal method(s) for clear biological effects.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Y.-W. Kuo and B.W. Falk. Artificial microRNA guide strand selection shows a purine-rich preference for virus- and non-virus-based expression vectors in plants. (2021) Plant Biotechnology Journal (under re-submission)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
B.W. Falk. Plant Pathology, Everywhere Every Day. (2021) Plant Health 2021, Phytopathologist of Distinction Talk
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
E. Henry, C.R. Carlson, and B.W. Falk (2021) Discovery and full genome assembly of a novel bacterial endosymbiont of Diaphorina citri. (In prep)
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
C.R. Carlson, E. Henry, J.S. Johnston, B.W. Falk, and Y.-W. Kuo (2021) High-quality, chromosome scale genome assemblies of Asian citrus psyllid (Diaphorina citri) geographic isolates. (In prep)
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