Progress 09/15/23 to 09/14/24
Outputs
Target Audience: The target audience includes citrus growers throughout the United States (Florida, Texas, and California), the agro-chemical industry focused on citrus production, and consumers of citrus products. We engaged with the target audience through extension and outreach presentations at industry meetings and development of an informational website. Changes/Problems:We requested a one year extension for the project because the completion of objectives within the designated timeframe was delayed due to a combination of unforeseen challenges and external factors that significantly impacted our progress. These challenges included delays in onboarding two postdoctoral scientists in year 1, which led to setbacks in critical phases of the project, particularly in objectives 2 and 4. Additionally, we encountered technical issues in the implementation phase, which required additional time for troubleshooting and resolution of cell line growth (objective 4). Shortages in materials and contamination of primaryWolbachiacultures, as well as a population crash inD. citricolonies during winter 2023-24, further impacted the overall timeline for completing objectives (2 and 3) as within the original timeline. What opportunities for training and professional development has the project provided?Postdoctoral scientists attended international meetings to share our research work and communicate with other researchers. How have the results been disseminated to communities of interest? This work was selected for oral presentations at the International Research Conference on Huanglongbing (IRCHLB VII) at Riverside, California. This work will be formed into a manuscript to be submitted to a peer-review journal We integrate a diversity of methods to meet the objectives of this extension program: We regularly visit citrus growers to discuss their individual citrus pest management problems and how to implement solutions. During these visits, we often discuss current and ongoing research. We regularly provide education by fielding phone calls and readily welcome walk-in visits to discuss citrus pest management needs of growers. We have disseminated new information about citrus pest management using UF's Electronic Delivery Source (EDIS) system. This includes information on citrus pest management practices, citrus production practices, and the biology and identification of pests in Florida citrus, as well as other agricultural crops. In addition to using EDIS, we have consistently provided up-to-date information to clientele and county faculty through publications in popular citrus industry magazines. We co-maintain the website Science for Citrus Health (http://ucanr.edu/sites/scienceforcitrushealth/). I also regularly develop new extension articles for this website. This website serves a teaching tool to educate growers on the research and methods being conducted and developed to protect citrus from the causative agent of citrus greening. With respect to presenting information on GE alternative tools for citrus pest management and insecticide resistance in Florida, Stelinski is one of the leading entomologists to deliver presentations at grower seminars, workshops, and field days on these topics. We regularly disseminate new information by way of presenting PowerPoint presentations (lecture-style grower conferences and workshops), poster presentations with handouts (field days), and brief written reports (evening small group grower meetings). What do you plan to do during the next reporting period to accomplish the goals?Objective 2. Finish remaining acquisition and inoculation assays to identify effective combinations of GBPs for reducing Clas transmission. This information will be used to inform management strategies and select additional candidates for transformation in objective 4. Objective 3. Synthesizing dsRNAs targeting the nine gut surface proteins needed for subsequent experiments.Complete remaining acquisition and inoculation assays to identify effective combinations of GBPs for reducing Clas transmission. Additional assays will be conducted to evaluate whether dsRNA targets reduce feeding or effect ingestion/honeydew excretion. This information will be used to inform management strategies and select additional candidates for transformation in objective 4. Objective 4. Optimize expression and excretion of GBPs from Wolbachia and improve efficiency of transformant selection.Wolbachiacells lines producing GBPs will be introduced in ACP following optimization of peptide expression. In addition, we will draft manuscripts for submissionto peer-reviewed journals (Objectives 2, 3, 4) during the next year.
Impacts
What was accomplished under these goals?
Objective 1. Completed in 2023. Objective 2: Several gut-binding peptides (GBPs) were evaluated for their efficiency in interfering withCandidatus Liberibacter asiaticus(CLas) acquisition by the Asian citrus psyllid (ACP): Specific gut-binding peptides (GBPs), notably P2 and NP3, effectively reduce the relative abundance of CLas in both ACP adults and nymphs in acquisition experiments. ACP fed on GBPs (NP2 and P2) exhibited a significantly reduced CLas infection rate. GBPs demonstrated no observable adverse effect on ACP fitness, suggesting that their application may not negatively impact ACP physiology and development. In healthy plants exposed to treatment with P2, the CLas inoculation rate was noticeably reduced, indicating potential transmission-blocking effects. ACP adults experienced a slight increase in mortality after feeding on several GBPs, particularly P2, though overall fitness was not compromised. Cumulatively, this suggests that P2 and NP3 could be promising candidates in managing CLas in citrus by reducing vector competence. Additional work could focus on optimizing dosage and assessing long-term impacts on ACP populations and CLas transmission. Objective 3: dsRNAs targeting nine of most abundant gut surface proteins in ACP have been synthesize and are stable for the entire feeding timeframe without causing significant mortality. All synthesized dsRNAs can knockdown the target transcript in ACP adults and nymphs with the downregulation ranged from 20 to 51% at 72 h, and from 41 to 63% at 120 h, in adults. In nymphs the knockdown efficiencies were 28-50% at 72 h, and 30-81% at 120 h. Nine target RNAi treatmentswere selected and tested to assess their effectiveness in reducing CLas acquisition in both ACP adults and nymphs. After24-hour and 48-hour acquisition access periods (AAPs), all dsRNA treatments significantly reduced the relative abundance of CLas in ACP adults compared to the control group. This suggests that RNAi treatments may be highly effective in suppressing CLas acquisition by ACP vectors. The fitness assays showed that nearly all dsRNA treatments had anegative effect on ACP fitness, indicating that RNAi treatments not only impact CLas acquisition but may also detrimentally affect ACP physiology and development. Objective 4 A wDi cell culture line was established using bacteria isolated fromD. citriadults and commercial S2Drosophilacells. One candidate GBP (DcNy2) and one negative control GBP (NegP) were selected forWolbachiatransformation. These GBPs were constructed into plasmid vectors for HDR donor construction. Extension/Outreach:Based on pre-tests and post-tests applied during some education events, I have quantified the knowledge, attitude, and skills gained during this training. More than 46% of my audience learned new knowledge about how the CLas pathogen is transmitted from tree to tree by Asian citrus psyllid. One hundred % of my audience learned how blocking movement of the CLas bacterium through the gut barrier could distupt transmission. More than 35% of my audience indicated that they would be willing to incorporate technologies they learned about in my educational programs into their current management programs. More than 40% of my audience acknowledged that current use of insecticides alone in their groves is insufficient for effective suppression of pest populations and huanglongbing
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Xie, N., K. S. Pelz-Stelinksi, and B. C. Bonning (2024). Silencing of the most abundant gut surface proteins of Asian citrus psyllid, International Research Conference on Huanglongbing (IRCHLB VII), Riverside, California.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Stelinski, L.L. 2023. Managing psyllids and other citrus pests. Citrus Expo. 16-17 August. (Several hundred Attendees). Florida State Fairgrounds, Tampa, FL
|
Progress 09/15/22 to 09/14/23
Outputs
Target Audience:The target audience includes citrus growers throughout the United States (Florida, Texas, and California), the agro-chemical industry focused on citrus production, and consumers of citrus products. We engaged with the target audience through extension and outreach presentations at industry meetings and development of an informational website. Changes/Problems:T THis year, we received a 12month no cost extension to the agreement. This extension was necessary due to unexpected delays that affected our ability to hire a postdoctoral researcher for our project. As a result, we have been unable to complete the proposed research objectives within the original timeline.OThe project required a postdoctoral researcher with expertise in pathogen transmission and transformation of cell lines to carry out the experimental work and data analysis. Despite our best efforts, we encountered unexpected visa delays and rejections that prevented us from hiring a qualified postdoctoral researcher for the project. We subsequently hired a suitable candidate for the position, and are now on track to complete the work in 2024. What opportunities for training and professional development has the project provided? Graduate student (PhD) Clebson Tavares presented a paper this work as part of his doctoral thesis "Midgut Surface Proteome of Diaphorina citri and Toxicity of Gut-Targeting Insecticidal Proteins for Use in Citrus Greening Management" Presentations "Midgut Surface Proteome of Diaphorina citri and Toxicity of Gut-Targeting Insecticidal Proteins for Use in Citrus Greening Management". Clebson S. Tavares, February 2023. How have the results been disseminated to communities of interest?SummaryBoth written and oral updates of the new technology being developed by this project has been delivered to the Florida citrus industry. In addition, we continue to provide outreach using The Science for Citrus Health (SCH) website. The SCH team led by P. Lemaux, L.L. Stelinski, and E. Stover is comprised of outreach specialists, postdocs and graduate students. The group produces outreach/extension documents and organizes/delivers events. To date, we have updated an online factsheet related to this project and continue to work on the rest of the proposed tasks as described below.We developed a factsheet and extension article on the project that is available. Stakeholders have been engaged at major annual meetings of citrus growers, the juice processing industry, and during a recent extension workshop designed to present new research taking place within IFAS. Written extension article Pelz-Stelinski, K.S., and L.L. Stelinski. 2022. Changing psyllids to make them incapable of spreading HLB. Citrus Industry Magazine. 103(11): 18-20. We developed an extension article describing this project forCitrus Industry Magazine. The article presents a description of paratransgenesis ofWolbachiato produce a nontransmitting Asian citrus psyllid (ACP) delivered to the Florida citrus industry. The article delivers a lay introduction to paratransgenesis for pest control and discusses where we are in the process of delivering potential tools for practical implementation. We discuss how modified endosymbionts can produce new proteins to modify the vector to inhibit pathogen transmission. Outreach via presentations and workshops. Stelinski, L.L. 2023. Managing psyllids and other citrus pests. Citrus Expo. 16-17 August. (Several hundred Attendees). Florida State Fairgrounds, Tampa, FL. A factsheet was developed as an education tool regarding our research and this project. This described our progress, challenges, and future applied opportunities. This was distributed as a hard-copy booklet at the meeting. Stelinski, L.L. et al. 2023. Asian citrus psyllid research and management. Citrus Insect, mite, and nematode workshop. 19January. UF/IFAS Citrus Research and Education Center, Lake Alfred, FL. This was a workshop included hands-on demonstrations in addition to a series of presentations during which time there was a longer period of stakeholder engagement. An 80-page educational binder was produced for all attendees that included copies of all presentations given. Stelinski, L.L. et al. 2022. Getting the most bang out of Asian citrus psyllid management under endemic greening. Citrus Expo. 17-18August. (Several hundred Attendees). Lee Civic Center, Fort Myers, FL. Stelinski presented a seminar discussingWolbachiaas a delivery system for CLas-blocking peptides or silencing RNAs at the 2022 Citrus and Specialty Crop Expo in Fort Myers FL (17-18 Aug). The seminar updated growers on our objective, discussed our progress to date, and challenges/opportunities for the future. Stelinski, L.L. et al. 2022. Harnessing biotechnology to manage greening: Exploring Bt proteins, RNAi, and paratransgenesis for vector control. International Citrus and Beverage Conference. 20-23 September. Clearwater Beach, Florida. There was significant interest from attendees. There was concern about when it would be available. Online outreach We have continued to use the SCH website (https://ucanr.edu/sites/scienceforcitrushealth/) as our main web-based medium for disseminating new information related to HLB. The SCH team continues to meet bi-monthly to discuss the next tasks relevant to the goals of the group.The Science for Citrus Health (SCH) team (current lead members: P. Lemaux, L.L. Stelinski, and Ed Stover) is comprised of outreach specialists, postdocs and graduate students. The group produces outreach/extension documents and organizes/delivers events. To date, we have completed the webinar portion of the objective and continue to work on the rest of the proposed tasks as described below. The website is currently operated and updated by the Stelinski lab. During the last quarter we published an information flier about the Science for Citrus health website on the California Research Board E-News mailer and the Florida Citrus Industry magazine newsletter to increase grower traffic to the site. We also published several new snapshots on the website site. What do you plan to do during the next reporting period to accomplish the goals?In the upcoming year, we plan to employ UV-crosslinking to identify the binding partners of the two peptides that were confirmed to bind to ACP nymph guts during this reporting period. This work will further enhance our understanding of ACP gut binding mechanisms and support the development of targeted control measures. In the coming year, our focus will be on completing the measurement of dsRNA efficiency in ACP nymphs. The impact of downregulating putativeCLas binding proteins in the ACP gut on the psyllid's ability to vectorCLas will be continued to be assessed in 2024. This ongoing research is critical for assessing the potential of RNAi as a method to reduce CLas transmission by targeting the most abundant gut surface proteins. Nymph feeding bioassays nymphs indicate the potential for effective dsRNA-mediated gene silencing. The assays are ongoing and will continue through 2024. Our findings also suggest that certain GBPs, particularly GBP2, have the potential to impede CLas acquisition in ACP adults. Further investigation and refinement of these peptides are needed to enhance their effectiveness.Inoculation assays started in August of 2023, and data is currently being processed. Results from these experiments will be included in the following report.We will continue our efforts to evaluate the impact of GBPs on nymph ACP and their ability to reduce CLas transmission throughout 2024.
Impacts
What was accomplished under these goals?
Objective 1: Characterize ACP Gut Binding Peptides Identifying Additional ACP Gut Binding Peptides We conducted screenings using a unique kimwipe feeding method with ACP nymphs, and this led to the enrichment of numerous peptides. We selected the top nine most enriched peptides based on specific criteria, ensuring their stability and hydrophilicity. In Vivo Binding Confirmation and Comparative Proteomics We fused these selected peptides with a fluorescent protein (mCherry) and confirmed their binding to ACP nymphs' guts in living organisms. Additionally, we identified the binding partners of two GBPs, providing insights into how these peptides interact with the ACP gut. Identification and Silencing of ACP Gut Surface Proteins In our ongoing research to reduce CLas transmission by silencing essential gut surface proteins in ACP, we've made significant progress: We identified nine candidate proteins for silencing through RNA interference (RNAi) after comparing ACP adult and nymph gut surface proteins. We successfully synthesized double-stranded RNA (dsRNA) targeting these nine proteins, with precision in design. We tested the efficacy of dsRNAs on adult ACP through membrane feeding bioassays, observing notable reductions in protein levels, albeit with variations among the proteins. Nymph feeding bioassays are currently underway, showing promising results with substantial knockdown of specific proteins. Our work is contributing to understanding how RNAi can potentially disrupt CLas transmission through the manipulation of ACP gut proteins. Objective 2.Test the ability of GBP to interfere withCLas attachment to the ACP gut epithelium. We used feeding assays to assess the potential of GBPs to compete with CLas within the ACP gut, with a particular emphasis on examining the impact of these peptides on CLas acquisition and transmission. To evaluate the effect of GBPs on CLas attachment to the gut epithelial cells of ACP, we conducted transmission feeding assays with uninfected ACP adults (1-2 days old). GBPs (GBP1-P5) and a non-gut binding peptide (GBP6, negative control) were synthesized and dissolved in either nuclease-free water (hydrophilic peptides) or dimethyl sulfoxide (DMSO, hydrophobic peptides). Additionally, control groups included a sucrose control and sucrose + DMSO treatments. Inoculation assays were conducted to confirm the efficacy of GBPs in reducing CLas transmission to susceptible plants. GBP-exposed ACP were released onto uninfected leaves following acquisition feeding. For the adult acquisition feeding assay, a total of 5 replications were conducted. CLas acquisition was observed to be higher in ACP adults fed on GBP1 and GBP6 after 48 hours of exposure to CLas-infected plants. This was followed by GBP4, GBP5, GBP3, sucrose, and sucrose + DMSO. No significant differences were found among these treatments. Notably, GBP2 demonstrated an 83% reduction in CLas acquisition compared to GBP6 (non-gut binding peptide). The CLas infection rate was highest among adults fed on sucrose, sucrose + DMSO, and GBP1, GBP3, GBP4, GBP5, and GBP6 (54-75% CLas-positive) compared to GBP2 (39% CLas-positive). For the adult acquisition feeding assay, a total of 4 replications were performed for this experiment. The highestCLas acquisition was observed on ACP nymphs fed with peptide 6, followed by peptide 5, sucrose, sucrose + DMSO, and peptide 1. However, the lowestCLas acquisition was observed on ACP nymphs fed with peptide 2; followed by peptides 3 and 4. Nevertheless, peptide 4 was not statistically different than the rest of the treatments.CLas infection rate was significantly higher among nymphs that were fed on peptide 6, followed by peptide 5, 1, sucrose, and sucrose + DMSO (50-80%CLas-positive) compared to peptide 2, 3, and 4 (30-35%CLas-positive). An additional replication will be performed, and results will be added to the following report. Based on the results of these experiments and objective 1, we selected three GBPs that are being evaluated under objective 4. 2.2.3 Binding of GBPs may affect the function of ACP gut surface proteins, many of which are digestive enzymes and transporters. To address the potential impact of GBP exposure, the effect of GBPs on ACP life history was evaluated in a series of fitness studies that assessed fecundity and survival.Fecundity assays were started in August of 2023. The experiment is ongoing, and data is currently being processed. Results from these experiments will be included in the following report. A total of 4 replications were performed for this experiment. ACP adults (1-2 days old) were assessed to determine whether gut-binding peptides influenced the survival of males and females among treatments. After being fed with a solution containing peptides 1, 2, and 4, males ACP had significantly lower survival than male adults fed a sucrose solution. Nevertheless, survival was not different among males ACP fed with the remaining treatments compared to sucrose. After being fed with a solution containing peptides 2 and 3, females ACP had significantly lower survival than female adults fed with a sucrose solution. However, survival was not significantly different among females ACP fed with the remaining treatments compared to sucrose. Objective 3.Silence expression of putative ACP gut proteins bound by CLas. This objective focuses on the evaluation of synthesized dsRNAs' effectiveness and potential impacts on the Asian citrus psyllid (ACP), aiming to better understand their utility for controlling this vector of citrus greening disease (CLas).To assess the effects of synthesized dsRNAs on ACP, we conducted membrane feeding bioassays targeting both adult (4-5 days old) and nymph (4th or 5th instar) psyllids. The results of our experiments provide valuable insights into the efficacy and impact of dsRNAs on ACP. For adult ACP, the transcript levels of APN, CroqM, and CAD at 72 h after dsRNA exposure were significantly reduced by 51%, 40%, and 45%, respectively, when compared to dsGFP. However, this downregulation did not improve at 120 h. BSG transcript levels were significantly decreased by dsRNA but only by 26% at 72 h relative to dsGFP. ALP, Gamma-glutamyltranspeptidase 1, monocarboxylate transporter, V-ATPase-A, and calcium-transporting ATPase transcript levels were lowest at 120 h, with reductions of 52%, 57%, 63%, 41%, and 41% relative to GFP, respectively. For ACP nymphs, our ongoing nymph feeding bioassays have shown promising results for all nine dsRNAs. In two replicates for dsV-ATPase-A and dsAPN, the transcript levels of these two proteins were knocked down by 40% and 70%, respectively, at 120 h. Our progress in evaluating dsRNA efficacy against ACP gut surface proteins has yielded significant findings, particularly in adult ACP where several proteins showed substantial knockdown. Ongoing experiments with nymphs indicate the potential for effective dsRNA-mediated gene silencing. Objective 4.DevelopWolbachiaas a delivery system for CLas-blocking peptides or silencing RNAs.We are currently employing an existing Wolbachia transformation system. We are modifying the ACP Wolbachia (wDi) using CRISPR-Cas9 coupled with homology directed recombination (HDR) to express previously described peptides that disrupt the successful establishment of CLas in ACP.We are currently transforming peptide genes into Wolbachia cells. Previous workhas optimized the CRISPR-Cas9 guide RNA and cut site in the wDi genome.
Publications
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Pelz-Stelinski, K.S., and L.L. Stelinski. 2022. Changing psyllids to make them incapable of spreading HLB. Citrus Industry Magazine. 103(11): 18-20.
|
Progress 09/15/21 to 09/14/22
Outputs
Target Audience:The target audience includes citrus growers throughout the United States (Florida, Texas, and California), the agro-chemical industry focused on citrus production, and consumers of citrus products. We engaged with the target audience through extension and outreach presentations at industry meetings and development of an informational website. Changes/Problems:Wolbachiaculture growth slowed during summer 2022 due to incubator failure, required re-establishing a new line from cryogenic stocks. This resulted in slowed progress on Objective 4. Colonies recovered after the issue was resolved, allowing progress on this objective to continue. Processing of visa applications resulted in the delayed hiring of postdoctoral scientists in the Bonning and Pelz-Stelinski labs. This delayed progress onobjectives 2 and 3 for several months past the intended start. What opportunities for training and professional development has the project provided?Graduate student (PhD) Clebson Tavares presented a paper and two posters for this work: Presentations Contributed paper: "Midgut surface proteome of the Asian citrus psyllid, Diaphorina citri". Clebson S. Tavares, Ruchir Mishra, Bryony C. Bonning. Annual meeting of Entomological Society of America, Oct 31-Nov 1, 2021 Poster presentations? Florida Entomological Society annual meeting, June 20-22, Gainesville, FL CREC Posters and Pastries 2022, April 22 Relative abundance of gut surface proteins in nymphs and adults of the Asian citrus psyllid, Diaphorina citri Clebson S. Tavares, Ruchir Mishra, Pierre N. Ghobrial, Bryony C. Bonning How have the results been disseminated to communities of interest?Accomplishments Summary Both written and oral updates of the new technology being developed by this project has been delivered to the Florida citrus industry. In addition, we continue to provide outreach using the Science for Citrus Health (SCH) website (https://ucanr.edu/sites/scienceforcitrushealth/). The SCH team (P.G. Lemaux, L.L. Stelinski, M. Setamou, and others) is comprised of outreach specialists, postdocs and graduate students. The group produces outreach/extension documents and organizes/delivers events. To date, we have updated an online factsheet related to this project and continue to work on the rest of the proposed tasks as described below. Written extension article We developed an extension article describing this project for Citrus Industry Magazine. This will be the first published description of paratransgenesis of Wolbachia to produce a non-transmitting Asian citrus psyllid (ACP) delivered to the Florida citrus industry. The article delivers a lay introduction to paratransgenesis for pest control and discusses where we are in the process of delivering potential tools for practical implementation. We discuss how modified endosymbionts can produce new proteins to modify the vector to inhibit pathogen transmission. Conventional outreach during industry meeting Stelinski presented a seminar discussing Wolbachia as a delivery system for CLas-blocking peptides or silencing RNAs at the 2022 Citrus and Specialty Crop Expo in Fort Myers FL (17-18 Aug). The seminar updated growers on our objective, discussed our progress to date, and challenges/opportunities for the future. Online outreach We have continued to use the SCH website as our main web-based medium for disseminating new information related to HLB to growers, researchers, media and the general public. The SCH team continues to meet bi-monthly to discuss the next tasks relevant to the goals of the group. The SCH team encountered a significant roadblock in the past six months brought on by staffing shortages at UC, which slowed are ability to update and post information to our website. Also, the tracking function was not working so we were unable to quantify visits and downloads. Work is in progress to make Stelinski's laboratory at UF the main administrator of the website, which we envision will get us back on track. Angel Hoyte in Stelinski's lab will serve as the website administrator. We anticipate that this problem should be resolved within the next month. We have held five webinars with international audiences since February, including a focus on use of new genetic technologies (biotechnology) and pest and disease issues. These webinars offered continued education units to growers in both California and Florida. This webinar series is being developed to address a breadth of management approaches in citrus and has featured speakers across several disciplines, including entomology, plant pathology, and horticulture. Although specific technologies have been the focus of individual presentation, the series is being developed to serve as a systems-based catalogue of potential tools for integration when approaching HLB management. Future webinars and a podcast are under development. Future plans: Stelinski submitted abstract entitled: "Bt pesticidal protein-based strategies for suppression of Asian citrus psyllid for HLB management: Challenges and Opportunities" to International Citrus Juice and Beverage Conference in Tampa Bay in March. Stelinski presented an overview of our goals and an update of our progress to the Juice Processing sector of the citrus industry during this meeting. What do you plan to do during the next reporting period to accomplish the goals?Objective 1.Confirmation of in vivo gut binding by fluorescence microscopy is planned for the coming year. Objective 2.Results from transmission assays to screen GBP blocking will be reported in the coming year. Assays to detection CLas in ACP following exposure to GBPs are alsoplanned for the coming year. Objective 3.Gene silencing RNAs that downregulate CLas receptor proteins, thereby impeding entry of CLas into the psyllid vector will be evaluated in the coming year. Bioasssays of dsRNAs with actin and alpha-tubulin as reference genes are underway. Objective 4. Wolbachia-based system for delivery of the most effective gut binding peptides and / or silencing RNAs to ACP (will start during the next reporting period.Analysis of GBP for disruption of CLas to plantsis planned for the coming year.
Impacts
What was accomplished under these goals?
The impact of our project for the current year was the identification of nine key gut binding peptides (GBP) that compete with CLas for attachment to the Asian citrus psyllid (ACP) gut.These peptides are critical for identifying the pathway for, and ultimately blocking, CLas transmission by ACP. We will use these peptides in the remainder of this project to develop symbionts and dsRNA to interfere with CLas movement in ACP, as novel tools for management of the HLB pathosystem.This targeted, problem-solving effort will provide an innovative and sustainable approach for blocking CLas spread by the psyllid vector, toward a practical management tool for grower use. Obj. 1. Characterize ACP gut binding peptides Major activities completed / experiments conductedIdentify additional ACP gut binding peptidesThe PhD C7C phage display library was screened for identification of ACP nymph gut binding peptides. Candidate gut binding peptides were identified using an optimized bioinformatics pipeline. A total of 455 peptides were enriched with log2-fold change ≥ 5 in both rounds of biopanning. Following exclusion of false positives, the top 26 gut binding peptides predicted to stable and hydrophilic clustered into three groups on Gibbs Cluster Analysis, and nine peptides were selected for further analysis with representatives from each cluster.Constructs were generated for expression of peptide-linker-mCherry fusion proteins for five of the candidate peptides. These fusion proteins will be used to confirm peptide binding under in vivo conditions. Identify peptide binding partners.Binding partners for GBP3 and GBP4 were identified usingUV crosslinking pull down assay, western blot analysis of UV crosslinked proteins, and LC-MS/MS. As these peptides were shown to bind some of the most abundant proteins on the surface of the psyllid gut, they are ideal candidates for testing for competition with CLas for gut binding. Compare ACP adult and nymph gut surface proteins.We conducted proteomic analysis and compared the relative abundance of gut surface proteins between adults and nymphs. Brush border membrane vesicles (BBMV) were prepared from guts dissected from 6,000 nymphs and 10,000 adults, and samples run in triplicate on a timsTOF mass spectrometer. A total of 1516 and 1219 proteins were identified in BBMV from ACP adults and nymphs, respectively. Bioinformatics analysis software and manual curation predicted that 112 adult and 87 nymph proteins localize to the surface of the microvilli. After data normalization using the normalized spectrum abundance factor (dNSAF) method followed by Studentttest, we found that the relative abundance of 26 proteins significantly differed between nymphs and adults. Data collectedThe top 26 enriched peptides that were predicted to be stable and hydrophilic, passed SAROTUP suite analyses and have fold change ≥ 5 were subjected to Gibbs cluster analysis (Andreatta et al., 2013). The Gibbs algorithm clustered these peptides into three groups. The peptides thatwere predicted to be stable and hydrophilic, passed SAROTUP suite analyses and have log2-fold change ≥ 5 and belonged to different Gibbs cluster groups were prioritized for further analysis. To analyze millions of peptides identified from eluted phage, we used a bioinformatics pipeline to select the best candidate gut binding peptides (Mishra et al., 2021). In vivogut binding peptides from ACP (GBP3 and 4) were synthesized by Genemed Synthesis Inc with Biotin and p-benzyol-l-phenylalanine (Bpa) modifications at the N- and C-termini, respectively. Western blot analysis was used to confirm whether GBP3 and GBP4 peptides crosslinked to BBMV protein(s). The results from western blot suggest the presence of a unique or more intense protein band or bands (between 150 to 25 kDa; Figure 1) in UV exposed lane. Summary statistics and discussion of resultsThe top 3 binding partners of GBP3 are aminopeptidase-N like, neprilysin-2-like and facilitated trehalose transporter Tret1 based on sequest HT score. Further, GBP4 bound to aminopeptidase-N like, facilitated trehalose transporter Tret1 and alkaline phosphatase-like proteins. Aminopeptidase N (APN) and alkaline phosphatase (ALP) are known Cry pesticidal protein receptor. APN and ALP are the top two most abundant proteins of ACP BBMV (Tavares et al., 2022). These results suggest that GBP4 and GBP3 are ideal peptides to test for competition with CLas for gut binding. Key outcomes or other accomplishments realizedThe top 26 gut binding peptides predicted to stable and hydrophilic clustered into three groups and nine peptides were selected for further analysis with representatives from each cluster.Constructs were generated for expression of peptide-linker-mCherry fusion proteins for five of the candidate peptides. These fusion proteins will be used to confirm peptide binding under in vivo conditions in ACP. Binding partners for GBP3 and GBP4 were identified. As these peptides were shown to bind some of the most abundant proteins on the surface of the psyllid gut, they are ideal candidates for testing for competition with CLas for gut binding and were selected for evaluation in ACP assays to evaluate transmission.We found that the relative abundance of 26 proteins significantly differed between nymphs and adults. Obj. 2. Test the ability of GBP to interfere with CLas attachment to the ACP gut epithelium. Major activities completed / experiments conductedTransmission assays to screen GBP blockingGBP 3, 4, and 15 peptides were synthesized, and bioassaysare underway. Data collected andSummary statistics and discussion of resultsData are currently being generated in ongoing experiments. Data and key outcpmes will be described in the next report. Obj. 3. Silence expression of putative ACP gut proteins bound by CLas Major activities completed / experiments conductedBased on the gut surface proteome data, primers were designed, and dsRNAs synthesizedwith the MEGAscript RNAi kitto targetDiaphorina citriaminopeptidase N and alkaline phosphatase. GFP dsRNAs were generated as a negative control treatment. Bioasssays with actin and alpha-tubulin as reference genes are underway. Data collected andSummary statistics and discussion of resultsData are currently being generated in ongoing experiments.ata and key outcpmes will be described in the next report. Obj. 4. DevelopWolbachiaas a delivery system for CLas-blocking peptides or silencing RNAs Major activities completed / experiments conductedA protein-based system was used to transform wDi using CRISPR/Cas9-mediated homology-directed recombination technology. Transgenes for a promotor and control sequence (Venus promotor + GFP) were integrated in the wDi genome. 500 ng of circular plasmid donor DNA (pDonor) containing the insert gene and both flanking homology arms were used per sample.Four biological replicates were conducted initially per sample, with 850,000 wDicells electroporated per sample. Data collected andSummary statistics and discussion of resultsFollowing introduction of the genetic material, successful integration of sequences was confirmed by PCR. Constructs were sequenced using an automated sequencing system (Applied Biosystems, Foster City, CA) and confirmed against the GenBank database. Insertion and transcription of gene sequences was evaluated by quantitativereverse transcription(qRT-PCR). Protein separation from isolated wDiLacI-VenuconfirmsVenusexpression.Going forward, the promotor-driven expression system will be usedtransformations of cells with GBPs and dsRNA. Key outcomes or other accomplishmentsConfirmation of promotor-driven expression of control gene in endosymbiont vector.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Tavares, C.S., Mishra, R., Ghobrial, P.N., Bonning, B.C. 2022. Composition and abundance of midgut surface proteins in the Asian citrus psyllid, Diaphorina citri. J. Proteomics 261:104580 doi: 10.1016/j.jprot.2022.104580
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Midgut surface proteome of the Asian citrus psyllid, Diaphorina citri . Clebson S. Tavares, Ruchir Mishra, Bryony C. Bonning. Annual meeting of Entomological Society of America, Oct 31-Nov 1, 2021
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Pelz-Stelinski K.S., and L. Stelinski. 2022. Changing psyllids to make them incapable of spreading HLB. Citrus Industry 103(11): 18-20.
|