Progress 02/01/23 to 01/31/24
Outputs
Target Audience:2A. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE WORK YOU ARE DOING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we are using in silico design and experiment selection to identify peptides that selectively bind the HLB associated pathogen (CLas). Besides being used in our work - which will involve using them to construct peptides that specifically and effectively kill CLas - such peptides can also be used by other scientists in a variety of ways. For example, they could be used to (i) create CLas detection assays or (ii) to immuno-capture viable CLas to obtain in vitro cultivation inoculum. 2B. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE TOOLS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we are using several different methods to design and select for peptides that selectively bind to the HLB associated pathogen (CLas). Other scientists could learn from the methods that we are using to create such peptides. These methods include (i) in silico design, (ii) peptide aptamer selection and, (iii) Nanobody selection. In addition, other scientists will also be able to use the most up-to-date, effective, and rapid in planta method for testing anti-CLas molecules - which is the hairy root method created by Kranthi Mandadi. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is to construct an integrated metabolic model of the HLB pathosystem - citrus, CLas, APC - and their molecular interactions. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. This will be accomplish by, for example, analyzing omics datasets in the context of the model. This type of analysis greatly extends such datasets. For example, if an RNAseq dataset from infected citrus is analyzed in this way, the output is increased from a list of RNA transcripts to a dataset that includes all of the other reactions and pathways that are turned on or off in CLas and citrus under the conditions when that sample was collected - along with all of the interactions among the pathosystem organisms. 2C. STAKEHOLDERS THAT COULD BENEFIT FROM THE SOLUTIONS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS This objective is designed to benefit the citrus industry in the near-, intermediate-, and long-term by creating both prophylactic and curative HLB management strategies. This is because this objective is to create highly effective peptides that will specifically kill the HLB associated pathogen. We will deliver these peptides to CLas' habitat (citrus phloem) in several ways: (i) using CTV which is a near-term solution, (ii) using other vehicles that are not completely developed such as the novel virus-like RNA that Anne Simon is developing - which is an intermediate-term solution and, (iii) engineering citrus which is a longer-term solution. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is designed to benefit the citrus industry as we envision this model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This objective is designed to benefit the citrus industry by ensuring that the solutions we create are commercially viable, and to steer us away from solutions that are not commercially viable. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN This objective is designed to benefit the citrus industry in several ways. First, since we have included members of the industry in the development of this project, and as members of this project, we are confident that we are taking the necessary steps to make sure the solutions created by this project are commercially viable in terms of things like licensing and economic feasibility. Second, our outreach team is and will maintain communications to members of the industry concerning the progress we are making and the solutions that get developed. Third, we have a public outreach component that is to broadly educate the general public about HLB. We believe that if the general public was more aware of the HLB situation, they would be more inclined to contact their local, state and federal representatives and ask them to do what is needed to solve this problem. 2D. STUDENTS FROM YOUR FORMAL TEACHING AND OTHER EDUCATION ACTIVITIES Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate level formal teaching. For example, James Borneman teaches at least one course every year: MCBL 126, an undergraduate course in microbiomes. See Other Products or Outputs for more detailed information on project relevant teaching. OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN Changes/Problems:A Brief History of this Project in Relation to the COVID-19 Pandemic. This project has been considerably impacted by the COVID-19 pandemic. This project started on February 1 of 2019. The COVID-19 lock-down started in March of 2020. The lock down - including limited work schedules due to physical distancing requirements - lasted for over a year at the different project locations. For example, at the lead campus for this project, the University of California Riverside, the lock-downs and/or limited work schedules lasted until June of 2021. Therefore, only two of the four project-periods have not been impacted by the pandemic - the first year (02/01/2019 - 01/31/2020) and the last year (02/01/2022 - 01/31/2023). What opportunities for training and professional development has the project provided?Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used, including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate-level formal teaching. For example, James Borneman teaches at least one course every year: MCBL 126, an undergraduate course in microbiomes. See Other Products or Outputs for more detailed information on project-relevant teaching. How have the results been disseminated to communities of interest?Project updates and results were regularly disseminated among the team members. Project findings was also periodically shared with the scientific community, students, citrus growers and stakeholders in TX, CA and FL at various grower and extension meetings/conferences (see Other Products section). The broader scientific community and the citrus stakeholder community were also reached out through publications, newsletters (e.g., Citrograph), and the project's website (modelinghlb.weebly.com) which is under construction. What do you plan to do during the next reporting period to accomplish the goals?OBJECTIVE 1: Design, Construct, And Test Peptides That Specifically Target And Kill CLas To Create Highly Effective Prophylactic And Curative HLB Treatments We will continue to move all of the research described above forward as follows. The aforementioned new targeted antimicrobial peptides are being tested in greenhouse and field experiments. We will continue to design optimized CLas-targeted peptides. We will continue to test newly designed CLas-targeted antimicrobial peptides using in planta screening with the novel hairy root methodology. We will continue to test newly designed CLas-targeted peptides and CLas-targeted antimicrobial peptides using in vitro methods. We will continue to translate these findings into highly effective HLB treatments that can be used by citrus growers using the following activities: 1. Patent. Intellectual property protection is critical for creating products that growers can use. We will work with our industrial partners to both move our provisional patent into a utility patent and to work on product development. 2. Stakeholder and Advisory Committee Meetings. Team members will continue to meet and work with stakeholders and our advisory committee to discuss our findings and to seek their input to ensure the effective translation of these findings into products for HLB treatments. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM We will publish a paper describing the first metabolic model of citrus, which is for Citrus clementina. The model contains 2,522 genes, 8,659 metabolites, and 10,719 reactions - representing the largest genome-scale model build to day for any organism. To our knowledge, this network is the most comprehensive metabolic model reported to date (see The BIGG Database). We have recently submitted a manuscript describing this model. We will initiate the work to construct the first metabolic model of the Asian citrus psyllid (ACP). We will continue the work to mechanistically understand the Survivor Tree Phenotype, and translate that knowledge into effective HLB management strategies. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE We are preparing to evaluate the aforementioned approaches that seek to cure or prophylactically treat HLB in citrus groves. Once these strategies are identified, we will parameterize their cost and yield effects and complete the analysis in which we simulate costs and returns from adopting the strategies and compare them with those from current conventional cultural practices to determine the potential gains from the derived strategies relative to present best practices for mitigating HLB. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN. We will continue our outreach efforts by disseminating the findings of this project to the scientific community, students, citrus growers, and stakeholders in TX, CA and FL via various grower and extension meetings/conferences, publications, and newsletters (e.g., Citrograph). We will also make our website (modelinghlb.weebly.com) more complete and more useful. Here, we will aim to promote communication and networking between researchers and growers via an interactive web-based platform created for this project (managed by Georgios Vidalakis). Here, we will also endeavor to stimulate research initiatives and collaborations by crowd-sourcing our data and research results. Our website will inform the stakeholders and the general public about the progress and outcomes of our research. In addition, this website will serve as a hub for existing HLB online resources (Industry, californiacitrusthreat.org/; State, freshfromflorida.com; USDA, saveourcitrus.org; fruitmentorTM, www.fruitmentor.com/; etc.) to inform the public about the enormous threat HLB poses to the citrus industry, how they can identify HLB disease systems and ACP in their neighborhoods, and how they can communicate this information to the appropriate officials. We will incorporate a blog to disseminate updated information about our deliverable solutions so that end users, such as growers, can provide feedback to facilitate the development of products that will actually be useful for them. The blog will also allow us to communicate information about our research with the citrus community in an interactive manner. We will use Google Analytics to determine who our blog/website audience is and what blog posts/web pages they are most interested in. Our website will also provide free access to protocols for the HLB solutions that are developed by this project, along with the metabolic models and multi-omics data. It will also provide information about citrus events and access to our presentations.
Impacts
What was accomplished under these goals?
OBJECTIVE 1: Design, Construct, and Test Peptides that Specifically Target and Kill CLas to Create Highly Effective Prophylactic and Curative HLB Treatments We have created much more effective anti-CLas peptides - which is a major step toward creating highly effective HLB treatments. Given that our newly designed CLas-targeted antimicrobial peptides are able to kill L. crescens at concentrations where non-targeted antimicrobial peptides (AMPs) have little effect - this suggests that delivery of these new targeted antimicrobial peptides (STAMPs) into citrus phloem using the Citrus tristeza virus (CTV) will transform an AMP-based treatment - which has been shown to be moderately effective in citrus field trials - into one that is highly effective in the field. To translate these findings into highly effective HLB treatments that can be used by citrus growers, we have implemented the following activities: Provisional Patent. Intellectual property protection is critical for creating products that growers can use. We obtained the following provisional patent: Mandadi, K.K., Borneman, J., Fernando, S., Irigoyen, S.C., Padilla, M., Ramasamy, M., Mallawarachchi, K.S. (2023). Development of specifically targeted binders and antimicrobial peptides (STAMPs) to control Candidatus Liberibacter spp. U.S. Provisional Patent, 63/497,753. Publication. Mallawarachchi, S., Haoqi W., Nirmitee M., Irigoyen, S., Padilla, C., Mandadi, K., Borneman, J., and Fernando, S. 2024. Specifically targeting antimicrobial peptides for inhibition of Candidatus Liberibacter asiaticus. J. App. Microbiol. 135, no. 4, lxae061. Publication. Mandadi, Kranthi. 2024. Advanced testing and commercialization of novel defensin peptides and therapies for HLB control. UCANR Science for Citrus Health. https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/https___ucanr.edu_sites_scienceforcitrushealth_Research_Snapshots_NIFA_I_938_/Mandadi1/ 2. Stakeholder and Advisory Committee Interactions. Team members have participated in meetings with stakeholders (e.g., Citrus Research Board, Texas Citrus Pest and Disease Management Corporation, U.S. Citrus) and our advisors (e.g., Southern Gardens Citrus). These meetings have enabled us to inform them of our promising results and plans as well as seek their input to ensure the effective translation of our findings into products for HLB treatments. OTHER RESULTS FROM THIS PAST YEAR ARE DESCRIBED BELOW DESIGNING & EVALUATING CLAS TARGETED PEPTIDES & STAMPS In total, we completed efficacy testing of ~6 different binders in various combinations and orientations of STAMPs against three culturable surrogates of CLas, as well as CLas-citrus hairy root (HR) assays. The results are promising and show that some binders and STAMPs had enhanced efficacy against the targets. The STAMPs were also moved to CTV cloning and testing which will be continued for the remainder of the project duration. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM BENEFITS TO THE COMMUNITY. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. In this project period, we finalized the genome-scale metabolic model for Citrus clementina. The model contains 2,522 genes, 8,659 metabolites, and 10,719 reactions - representing the largest genome-scale model build to day for any organism. To our knowledge, this network is the most comprehensive metabolic model reported to date (see The BIGG Database). We have recently submitted a manuscript describing this model. Another project leveraged from this work involves using the citrus metabolic model to accelerate the citrus engineering process by increasing the growth rates of citrus at the various steps in this process. This project is funded by the California Department of Food and Agriculture (CDFA). Another project leveraged from this work involves using the citrus metabolic model to understand the survivor tree phenotype. Citrus Growers in Florida observed a phenomenon where some trees - Survivor Trees - were not succumbing to the normal decline and death due to citrus huanglongbing disease (HLB) (Wang and Gmitter. 2014. Citrus Industry July:16). To verify such a potentially valuable phenomenon, in 2016 researchers at UC Riverside initiated a multiyear study to characterize these trees and to identify their microbes using funding from the Citrus Research Board, USDA ECDRE (2017-70016-26053), and CDFA (21-0001-051-SF). Orchards with putative Survivor Trees were identified via outreach to growers - led by Mike Irey (Southern Gardens Citrus) and Gary England (UF/IFAS Extension). The same trees in orchards from different regions in Florida were annually assessed for their HLB disease severity and composition of microbes. This work identified two commercial orchards with trees exhibiting little to no decline in their health, providing evidence that the Survivor Tree Phenotype is a real and reproducible phenomenon. Numerous microbes were associated with disease severity (Ginnan et al. 2020. Phytobiomes https://doi.org/10.1094/PBIOMES-04-20-0027-R), suggesting that host-microbe interactions were causing this phenotype. In 2022, our trip to Florida focused on the most promising orchard. While many trees in this orchard have died or are dying, other immediately adjacent trees have had their HLB symptoms completely reversed. This is both remarkable and potentially very important. Most recently, we are using the citrus metabolic model to perform a series of studies to mechanistically understand the Survivor Tree Phenotype. Thus far, all of our results point toward one microbe and the enzymes it makes as being the driver of the Survivor Tree Phenotype - where HLB disease severity is completely reversed. We are currently drafting a disclosure to obtain a patent for these findings, and the HLB management strategies that we envision will be created from these findings. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE We published four papers modeling the potential impact of Huanglongbing in California - see Products section. This involved revising a dynamic bioeconomic model to simulate citrus production when HLB is present or a threat, surveying stakeholders, and obtaining data from the scientific literature, USDA NASS, CDFA, UCANR, UF IFAS, DATOC, and other such sources. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN. See Section 5 C below.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Mallawarachchi, S., Haoqi W., Nirmitee M., Irigoyen, S., Padilla, C., Mandadi, K., Borneman, J., and Fernando, S. 2024. Specifically targeting antimicrobial peptides for inhibition of Candidatus Liberibacter asiaticus. J. App. Microbiol. 135, no. 4, lxae061.
- Type:
Other
Status:
Published
Year Published:
2024
Citation:
Mandadi, Kranthi. 2024. Advanced testing and commercialization of novel defensin peptides and therapies for HLB control. UCANR Science for Citrus Health. https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/https___ucanr.edu_sites_scienceforcitrushealth_Research_Snapshots_NIFA_I_938_/Mandadi1/
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Kwak, Y., & Hansen, A. K. 2023. Unveiling metabolic integration in psyllids and their nutritional endosymbionts through comparative transcriptomics analysis. Iscience, 26(10).
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Kaplan, J., Johnston, E., Singh, A. 2023. Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: Results for Valencia orange production. Research Note 2023-4, September 2023. https://www.csus.edu/faculty/k/kaplanj/researchnotes/2023-04-valencias_rn.pdf.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Kaplan, J., Johnston, E., Singh, A. 2023. Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: Results for Navel orange production. Research Note 2023-3, August 2023. https://www.csus.edu/faculty/k/kaplanj/researchnotes/2023-03-navels_rn.pdf.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Kaplan, J., Johnston, E., Singh, A. 2023. Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: Results for tangerine & mandarin production. Research Note 2023-2, August 2023. https://www.csus.edu/faculty/k/kaplanj/researchnotes/2023-02-tangerine_mandarin_rn1.pdf.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Kaplan, J., Johnston, E., Singh, A. 2023. Potential economic consequences from Huanglongbing (aka citrus greening disease) in California commercial citrus: Results for lemon production. Research Note 2023-1, July 2023. https://www.csus.edu/faculty/k/kaplanj/researchnotes/2023_01_lemons_rn.pdf.
|
Progress 02/01/22 to 01/31/23
Outputs
Target Audience:2A. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE WORK YOU ARE DOING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we are in silico designing and experimentally selecting for peptides that will selectively bind to the HLB associated pathogen (CLas). Besides being used in our work - which will involve using them to construct peptides that specifically and effectively kill CLas - such peptides could also be used by other scientists in a variety of ways. For example, they could be used to (i) create CLas detection assays or (ii) to immuno-capture viable CLas for in vitro cultivation inoculum. 2B. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE TOOLS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we are using several different methods to design and select for peptides that will selectively bind to the HLB associated pathogen (CLas). Other scientists could learn from the methods that we are using to create such peptides. These methods include (i) in silico design, (ii) peptide aptamer selection and, (iii) Nanobody selection. In addition, other scientists will also be able to use the most effective and rapid in planta method for analyzing the effect of molecules on CLas. This is the hairy root method created by Kranthi Mandadi. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is to construct an integrated metabolic model of the HLB pathosystem - citrus, CLas, APC - and their molecular interactions. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. This will be accomplish by, for example, analyzing omics datasets in the context of the model. This type of analysis greatly extends such datasets. For example, if an RNAseq dataset from infected citrus is analyzed in this way, the output is increased from a list of RNA transcripts to a dataset that includes all of the other reactions and pathways that are turned on or off in CLas and citrus under the conditions when that sample was collected - along with all of the interactions among the pathosystem organisms. 2C. STAKEHOLDERS THAT COULD BENEFIT FROM THE SOLUTIONS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS This objective is designed to benefit the citrus industry in the near-, intermediate-, and long-term by creating both prophylactic and curative HLB management strategies. This is because this objective is to create highly effective peptides that will specifically kill the HLB associated pathogen. We will deliver these peptides to CLas' habitat (citrus phloem) in several ways: (i) using CTV which is a near-term solution, (ii) using other vehicles that are not completely developed such as the novel virus-like RNA that Anne Simon is developing - this is an intermediate-term solution and, (iii) engineering citrus which is a longer-term solution. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is designed to benefit the citrus industry as we envision this model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This objective is designed to benefit the citrus industry by ensuring that the solutions we create are commercially viable, and to steer us away from solutions that are not commercially viable. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN This objective is designed to benefit the citrus industry in several ways. First, since we have included members of the industry in the development of this project, and as members of this project, we are confident that we are taking the necessary steps to make sure the solutions created by this project are commercially viable in terms of things like licensing and economic feasibility. Second, our outreach team is and will maintain communications to members of the industry concerning the progress we are making and the solutions that get developed. Third, we have a public outreach component that is to broadly educate the general public about HLB. We believe that if the general public was more aware of the HLB situation, they would be more inclined to contact their local, state and federal representatives and ask them to do what is needed to solve this problem. We are doing this through a variety of means including creating an App that will explain the HLB pathosystem, why it is a problem, and what researchers and the citrus industry are doing to solve this problem. 2D. STUDENTS FROM YOUR FORMAL TEACHING AND OTHER EDUCATION ACTIVITIES Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate level formal teaching. For example, James Borneman teaches at least one course every year: MCBL 126, an undergraduate course in microbiomes. See Other Products or Outputs for more detailed information on project relevant teaching. OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN Changes/Problems:A Brief History of this Project in Relation to the COVID-19 Pandemic. This project has been considerably impacted by the COVID-19 pandemic. This project started on February 1 of 2019. The COVID-19 lock-down started in March of 2020. The lock down - including limited work schedules due to physical distancing requirements - lasted for over a year at the different project locations. For example, at the lead campus for this project, the University of California Riverside, the lock-downs and/or limited work schedules lasted until June of 2021. Therefore, only two of the four project-periods have not been impacted by the pandemic - the first year (02/01/2019 - 01/31/2020) and the last year (02/01/2022 - 01/31/2023). What opportunities for training and professional development has the project provided?Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used, including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate-level formal teaching. For example, James Borneman teaches at least one course every year: MCBL 126, an undergraduate course in microbiomes. See Other Products or Outputs for more detailed information on project-relevant teaching. How have the results been disseminated to communities of interest?Project updates and results were regularly disseminated among the team members. Project findings was also periodically shared with the scientific community, students, citrus growers and stakeholders in TX, CA and FL at various grower and extension meetings/conferences (see Other Products section). The broader scientific community and the citrus stakeholder community were also reached out through publications, newsletters (e.g., Citrograph), and the project's website (modelinghlb.weebly.com) which is under construction. What do you plan to do during the next reporting period to accomplish the goals?OBJECTIVE 1: Design, Construct, And Test Peptides That Specifically Target And Kill CLas To Create Highly Effective Prophylactic And Curative HLB Treatments We will continue to move all of the research described above forward as follows. The aforementioned new targeted antimicrobial peptides will be put into CTV and tested in greenhouse and field experiments. We will continue to design optimized CLas-targeted peptides. We will continue to test newly designed CLas-targeted antimicrobial peptides using in planta screening with the novel hairy root methodology. We will continue to test newly designed CLas-targeted peptides and CLas-targeted antimicrobial peptides using in vitro methods. We will continue to translate these findings into highly effective HLB treatments that can be used by citrus growers using the following activities: 1. Patent. Intellectual property protection is critical for creating products that growers can use. We will work with our industrial partners to both move our provisional patent into a utility patent and to work on product development. 2. Stakeholder and Advisory Committee Meetings. Team members will continue to meet and work with stakeholders and our advisory committee to discuss our findings and to seek their input to ensure the effective translation of these findings into products for HLB treatments. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM We will write a manuscript that will present the first metabolic model of citrus, which is for Citrus clementina. The model currently contains 10,493 reactions, 6,771 metabolites, and 3,245 genes. To our knowledge, this network is the most comprehensive metabolic model reported to date (see The BIGG Database). We will initiate the work to construct the first metabolic model of the Asian citrus psyllid (ACP). We will continue the work to mechanistically understand the Survivor Tree Phenotype, and translate that knowledge into effective HLB management strategies. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE We are preparing to evaluate the aforementioned approaches that seek to cure or prophylactically treat HLB in citrus groves. Once these strategies are identified, we will parameterize their cost and yield effects and complete the analysis in which we simulate costs and returns from adopting the strategies and compare them with those from current conventional cultural practices to determine the potential gains from the derived strategies relative to present best practices for mitigating HLB and ACP. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN. We will continue our outreach efforts by disseminating the findings of this project to the scientific community, students, citrus growers, and stakeholders in TX, CA and FL via various grower and extension meetings/conferences, publications, and newsletters (e.g., Citrograph). We will also make our website (modelinghlb.weebly.com) more complete and more useful. Here, we will aim to promote communication and networking between researchers and growers via an interactive web-based platform created for this project (managed by Georgios Vidalakis). Here, we will also endeavor to stimulate research initiatives and collaborations by crowd-sourcing our data and research results. Our website will inform the stakeholders and the general public about the progress and outcomes of our research. In addition, this website will serve as a hub for existing HLB online resources (Industry, californiacitrusthreat.org/; State, freshfromflorida.com; USDA, saveourcitrus.org; fruitmentorTM, www.fruitmentor.com/; etc.) to inform the public about the enormous threat HLB poses to the citrus industry, how they can identify HLB disease systems and ACP in their neighborhoods, and how they can communicate this information to the appropriate officials. We will incorporate a blog to disseminate updated information about our deliverable solutions so that end users, such as growers, can provide feedback to facilitate the development of products that will actually be useful for them. The blog will also allow us to communicate information about our research with the citrus community in an interactive manner. We will use Google Analytics to determine who our blog/website audience is and what blog posts/web pages they are most interested in. Our website will also provide free access to protocols for the HLB solutions that are developed by this project, along with the metabolic models and multi-omics data. It will also provide information about citrus events and access to our presentations.
Impacts
What was accomplished under these goals?
OBJECTIVE 1: Design, Construct, and Test Peptides that Specifically Target and Kill CLas to Create Highly Effective Prophylactic and Curative HLB Treatments We have created much more effective anti-CLas peptides - which is a major step toward creating highly effective HLB treatments. Given that our newly designed CLas-targeted antimicrobial peptides are able to kill L. crescens at concentrations where non-targeted antimicrobial peptides (AMPs) have little effect - this suggests that delivery of these new targeted antimicrobial peptides (STAMPs) into citrus phloem using the Citrus tristeza virus (CTV) will transform an AMP-based treatment - which has been shown to be moderately effective in citrus field trials - into one that is highly effective in the field. To translate these findings into highly effective HLB treatments that can be used by citrus growers, we have implemented the following activities: 1. Patent Disclosure. Intellectual property protection is critical for creating products that growers can use. An application for a provisional patent was submitted on April 24 (2023) -- U.S. Patent Office Serial No. 63/497,753. Mandadi, K., Borneman, J., Fernando, S., Irigoyen, S., Padilla, C., Ramasamy, M., Mallawarachchi, S. (2022). Development of specifically targeted binders and antimicrobial peptides (STAMPs) to control Candidatus Liberibacter spp. 2. Stakeholder and Advisory Committee Interactions. Team members have participated in meetings with stakeholders (e.g., Citrus Research Board, Texas Citrus Pest and Disease Management Corporation, U.S. Citrus) and our advisors (e.g., Southern Gardens Citrus). These meetings have enabled us to inform them of our promising results and plans as well as seek their input to ensure the effective translation of our findings into products for HLB treatments. OTHER RESULTS FROM THIS PAST YEAR ARE DESCRIBED BELOW DESIGNING CLAS TARGETED PEPTIDES This year we tested various combinations of CLas-targeting peptides, linkers, and antimicrobial peptides. To date, there are no effective strategies to predict optimal combinations of these three components, so this has to be done empirically. EVALUATING CLAS TARGETED ANTIMICROBIAL PEPTIDES A. Testing Newly Designed Targeted Peptides for their Ability to Bind to CLas and Surrogates. To date, we have tested 19 putative targeted peptides using in vitro binding assays. Of them, 4 showed good binding to three CLas surrogates and CLas from enriched plant extracts. B. Efficacy Evaluations of the CLas Targeted Antimicrobial Peptides (STAMPS). To date, we have tested the efficacy of 6 different STAMPs against three culturable surrogates of CLas and in vitro CLas-citrus hairy root (HR) assays and a few more tests are underway. The results are promising and show that some STAMPs had enhanced efficacy against the targets when compared to the AMPs alone. We will continue the efficacy evaluations in the next reporting period. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM BENEFITS TO THE COMMUNITY. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. In this project period, we have completed the work that has biologically validated the first in silico metabolic model of citrus, which is for Citrus clementina. The model currently contains 10,493 reactions, 6,771 metabolites, and 3,245 genes. To our knowledge, this network is the most comprehensive metabolic model reported to date (see The BIGG Database). We have started the process of writing a manuscript that will present this new model and all of its features. Another project leveraged from this work involves using the citrus metabolic model to accelerate the citrus engineering process by increasing the growth rates of citrus at the various steps in this process. This project is funded by the California Department of Food and Agriculture (CDFA). Another project leveraged from this work involves using the citrus metabolic model to understand the survivor tree phenotype. Citrus Growers in Florida observed a phenomenon where some trees - Survivor Trees - were not succumbing to the normal decline and death due to citrus huanglongbing disease (HLB) (Wang and Gmitter. 2014. Citrus Industry July:16). To verify such a potentially valuable phenomenon, in 2016 researchers at UC Riverside initiated a multiyear study to characterize these trees and to identify their microbes using funding from the Citrus Research Board, USDA ECDRE (2017-70016-26053), and CDFA (21-0001-051-SF). Orchards with putative Survivor Trees were identified via outreach to growers - led by Mike Irey (Southern Gardens Citrus) and Gary England (UF/IFAS Extension). The same trees in orchards from different regions in Florida were annually assessed for their HLB disease severity and composition of microbes. This work identified two commercial orchards with trees exhibiting little to no decline in their health, providing evidence that the Survivor Tree Phenotype is a real and reproducible phenomenon. Numerous microbes were associated with disease severity (Ginnan et al. 2020. Phytobiomes https://doi.org/10.1094/PBIOMES-04-20-0027-R), suggesting that host-microbe interactions were causing this phenotype. In 2022, our trip to Florida focused on the most promising orchard. While many trees in this orchard have died or are dying, other immediately adjacent trees have had their HLB symptoms completely reversed. This is both remarkable and potentially very important. Most recently, we are using the citrus metabolic model to perform a series of studies to mechanistically understand the Survivor Tree Phenotype. Thus far, all of our results point toward one microbe and the enzymes it makes as being the driver of the Survivor Tree Phenotype - where HLB disease severity is completely reversed. We are currently drafting a disclosure to obtain a patent for these findings, and the HLB management strategies that we envision will be created from these findings. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE Our work on this project began in the spring of 2021 with revising a dynamic bioeconomic model to be used to simulate citrus production when HLB is present or a threat. During this time, we also surveyed growers, industry representatives, farm advisors, research collaborators, and others to guide the adaptation of the model strategies to combat HLB and ACP. Additional data for the simulation model was been collected from the scientific literature, USDA NASS, CDFA, UCANR, UF IFAS, DATOC, and other such sources. We are preparing to evaluate the aforementioned approaches that seek to cure or prophylactically treat HLB in citrus groves. Once these strategies are identified, we will parameterize their cost and yield effects and complete the analysis in which we simulate costs and returns from adopting the strategies and compare them with those from current conventional cultural practices to determine the potential gains from the derived strategies relative to present best practices for mitigating HLB and ACP. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN. See Section 5 C below.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Wang, H.; Mulgaonkar, N.; Mallawarachchi, S.; Ramasamy, M.; Padilla, C.S.; Irigoyen, S.; Coaker, G.; Mandadi, K.K.; Fernando, S. Evaluation of Candidatus Liberibacter asiaticus Efflux Pump Inhibition by Antimicrobial Peptides. Molecules 2022, 27, 8729. https://doi.org/10.3390/molecules27248729
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Younghwan Kwak, Jacob Argondona, Patrick H. Degnan, Allison K. Hansen. 2023. Chromosomal-level assembly of Bactericera cockerelli reveals rampant gene family expansions impacting genome structure, function and insect-microbe-plant-interactions. Molecular Ecology Resources. Mol Ecol Resour. 2023 Jan;23(1):233-252. doi: 10.1111/1755-0998.13693. Epub 2022 Aug 16. PMID: 35925827.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Allison K. Hansen, Ariana Sanchez, Younghwan Kwak. 2022. Divergent host-microbe interaction and pathogenesis proteins detected in recently identified Liberibacter species. Microbiology Spectrum. Aug 31;10(4):e0209122. doi: 10.1128/spectrum.02091-22. Epub 2022 Jul 28. PMID: 35900091; PMCID: PMC9430466.
- Type:
Other
Status:
Submitted
Year Published:
2022
Citation:
INVENTION DISCLOSURE: Mandadi, K.K., Borneman, J., Fernando, S., Irigoyen, S.C., Padilla, M., Ramasamy, M., Mallawarachchi, K.S. (2022). Development of specifically targeted binders and antimicrobial peptides (STAMPs) to control Candidatus Liberibacter spp. Texas A&M University System (TAMUS) invention disclosure. 6003AGLR22
|
Progress 02/01/21 to 01/31/22
Outputs
Target Audience:2A. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE WORK YOU ARE DOING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we are in silico designing and experimentally selecting for peptides that will selectively bind to the HLB associated pathogen (CLas). Besides being used in our work - which will involve using them to construct peptides that specifically and effectively kill CLas - such peptides could also be used by other scientists in a variety of ways. For example, they could be used to (i) create CLas detection assays or (ii) to immuno-capture viable CLas for in vitro cultivation inoculum. 2B. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE TOOLS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we are using several different methods to design and select for peptides that will selectively bind to the HLB associated pathogen (CLas). Other scientists could learn from the methods that we are using to create such peptides. These methods include (i) in silico design, (ii) peptide aptamer selection and, (iii) Nanobody selection. In addition, other scientists will also be able to use the most effective and rapid in planta method for analyzing the effect of molecules on CLas. This is the hairy root method created by Kranthi Mandadi. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is to construct an integrated metabolic model of the HLB pathosystem - citrus, CLas, APC - and their molecular interactions. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. This will be accomplish by, for example, analyzing omics datasets in the context of the model. This type of analysis greatly extends such datasets. For example, if an RNAseq dataset from infected citrus is analyzed in this way, the output is increased from a list of RNA transcripts to a dataset that includes all of the other reactions and pathways that are turned on or off in CLas and citrus under the conditions when that sample was collected - along with all of the interactions among the pathosystem organisms. 2C. STAKEHOLDERS THAT COULD BENEFIT FROM THE SOLUTIONS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS This objective is designed to benefit the citrus industry in the near-, intermediate-, and long-term by creating both prophylactic and curative HLB management strategies. This is because this objective is to create highly effective peptides that will specifically kill the HLB associated pathogen. We will deliver these peptides to CLas' habitat (citrus phloem) in several ways: (i) using CTV which is a near-term solution, (ii) using other vehicles that are not completely developed such as the novel virus-like RNA that Anne Simon is developing - this is an intermediate-term solution and, (iii) engineering citrus which is a longer-term solution. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is designed to benefit the citrus industry as we envision this model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This objective is designed to benefit the citrus industry by ensuring that the solutions we create are commercially viable, and to steer us away from solutions that are not commercially viable. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN This objective is designed to benefit the citrus industry in several ways. First, since we have included members of the industry in the development of this project, and as members of this project, we are confident that we are taking the necessary steps to make sure the solutions created by this project are commercially viable in terms of things like licensing and economic feasibility. Second, our outreach team is and will maintain communications to members of the industry concerning the progress we are making and the solutions that get developed. Third, we have a public outreach component that is to broadly educate the general public about HLB. We believe that if the general public was more aware of the HLB situation, they would be more inclined to contact their local, state and federal representatives and ask them to do what is needed to solve this problem. We are doing this through a variety of means including creating an App that will explain the HLB pathosystem, why it is a problem, and what researchers and the citrus industry are doing to solve this problem. 2D. STUDENTS FROM YOUR FORMAL TEACHING AND OTHER EDUCATION ACTIVITIES Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate level formal teaching. For example, James Borneman teaches at least one course every year: MCBL 126, an undergraduate course in microbiomes. See Other Products or Outputs for more detailed information on project relevant teaching. Changes/Problems:The COVID-19 pandemic hindered progress during this last reported period. What opportunities for training and professional development has the project provided?Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate level formal teaching. For example, James Borneman teaches at least one course every year: MCBL 126, an undergraduate course in microbiomes. See Other Products or Outputs for more detailed information on project relevant teaching. How have the results been disseminated to communities of interest?Project updates and results were regularly disseminated among the team members. Project findings was also periodically shared with the scientific community, students, citrus growers and stakeholders in TX, CA and FL at various grower and extension meetings/conferences (see Other Products section). The broader scientific community and the citrus stakeholder community were also reached out through publications, newsletters (e.g., Citrograph), and the project's website (modelinghlb.weebly.com) which is under construction. Other outreach activities include among others creating an App that can be used on phones, tablets, and computers to teach the general public about HLB and our solutions for managing HLB (this has not been completed). This is work is being done in collaboration with colleagues from UC Berkeley's Lawrence Hall of Science museum. What do you plan to do during the next reporting period to accomplish the goals?OBJECTIVE 1: Design, Construct, And Test Peptides That Specifically Target And Kill CLas To Create Highly Effective Prophylactic And Curative HLB Treatments We will continue to move all of the research described above forward as follows. The aforementioned new targeted antimicrobial peptides will be put into CTV and tested in greenhouse and field experiments. We will continue to design optimized CLas targeted peptides. We will continue to test newly designed CLas-targeted antimicrobial peptides using in planta screening with the novel hairy root methodology. We will continue to test newly design CLas-targeted peptides and CLas targeted antimicrobial peptides using in vitro methods. We will continue to Translate these findings into Highly Effective HLB Treatments that can be used by citrus growers using the following activities: 1. Patent Disclosure. Intellectual property protection is critical for creating products that growers can use. As is routine for our university systems, we have submitted an invention disclosure on the aforementioned new results and we will work with our industrial partners on product development. 2. Stakeholder meetings. Team members have participated in meetings with stakeholders (e.g., Southern Gardens Citrus) and informed them of our promising results and plans. To reach a wider audience, we have also published a research snapshot related to our efficacy screening system in Science for Citrus Health, which is an online resource growers. 3. Advisory Committee. We are arranging a meeting with our advisory committee to discuss our new findings and to seek their input to ensure the effective translation of these findings into products for HLB treatments. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM We will validate the newly constructed first metabolic model of citrus using biological data. We may also construct tissue-specific models for roots, leaves, etc. We will continue to collect and analyze biologic and multi-omics datasets to train the integrated HLB pathosystem model. For example, see below. The grad student (Kwak) discovered a new Liberibacter species (Liberibacter capsica) in a new psyllid pest from South America (Brazil) unexpectantly while conducting a microbiome analyses of Liberibacter psyllid vectors to further understand the metabolomics of psyllids that vector Liberibacter (published, Kwak et al. 2021. Frontiers in Microbiology (see full citation above)). Kwak also finished conducting a comparative genomic analysis of psyllid Liberibacter vectors and identified insect-microbe genes that are under selection in ACP that are important for metabolic interactions and insect immunity (paper submitted, Kwak et al. 2022). Kwak also finished transcriptome trials on ACP symbiont cells versus body tissues and successfully sequenced these data and is currently conducting data analysis. Kwak is also currently setting up trials for Liberibacter infected and uninfected psyllids to further understand how Liberibacter infection modifies the psyllid's metabolic symbiosis with the psyllid host. By spring 2023 we should be done analyzing metabolomic data from psyllids and their microbiome with and without Liberibacter in addition to free amino acid concentrations within sap from leaves and psyllid bodies to further understand the metabolic flux within this Liberibacter vector and its crosstalk with its symbiont community when feeding on infected and uninfected plants. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE Once the model described in Section 5A is completed, the simulated costs and returns from adopting strategies derived from the model driven, systems biology approach that cure or prophylactically treat CLas infection in citrus groves and those based on current practices can then be compared to determine the potential gains from the derived strategies. The general structure of the model is expected to be completed in the coming months. When the strategies are identified and their cost and yield effects parameterized, the estimation of the net economic gains from adopting these strategies can be conducted. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN. We will continue our outreach efforts by disseminating the findings of this project to the scientific community, students, citrus growers and stakeholders in TX, CA and FL via various grower and extension meetings/conferences, publications, and newsletters (e.g., Citrograph). We will also make our website (modelinghlb.weebly.com) more complete and more useful. Here, we will aim to promote communication and networking between researchers and growers via an interactive web-based platform created for this project (managed by Georgios Vidalakis). Here, we will also endeavor to stimulate research initiatives and collaborations by crowd-sourcing our data and research results. Our website will inform the stakeholders and the general public on the progress and outcomes of our research. In addition, this website will serve as a hub to existing HLB online resources (Industry, californiacitrusthreat.org/; State, freshfromflorida.com; USDA, saveourcitrus.org; fruitmentorTM, www.fruitmentor.com/; etc.) to inform the public about the enormous threat HLB poses to the citrus industry, how they can identify HLB disease systems and ACP in their neighborhoods, and how they can communicate this information to the appropriate officials. We will incorporate a blog to disseminate updated information about our deliverable solutions so that end users such as growers can provide feedback to facilitate the development of products that will actually be useful for them. The blog will also allow us to communicate information about our research with the citrus community in an interactive manner. We will use Google Analytics to determine who our blog/website audience is and what blog posts/webpages they are most interested in. Our website will also provide free access to protocols for the HLB solutions that are developed by this project along with the metabolic models and multi-omics data. It will also provide information about citrus events and access to our presentations. Other outreach activities will include among others creating an App that can be used on phones, tablets, and computers to teach the general public about HLB and our solutions for managing HLB. This is work is being done in collaboration with colleagues from UC Berkeley's Lawrence Hall of Science museum. We will also create videos. The creator of the fruitmentorTM website will design and construct videos designed to inform the general public, citrus industry, and other scientists about the two science-based objectives of this USDA proposal.
Impacts
What was accomplished under these goals?
OBJECTIVE 1: Design, Construct, And Test Peptides That Specifically Target And Kill CLas To Create Highly Effective Prophylactic And Curative HLB Treatments We have New Results demonstrating that we have created much more effective anti-CLas peptides - which is a Major Step toward creating Highly Effective HLB Treatments. Given that our newly designed CLas-targeted antimicrobial peptides (AMPs) are able to kill L. crescens at concentrations where non-targeted AMPs have little effect - this suggests that delivery of these new targeted AMPs into citrus phloem using the Citrus tristeza virus (CTV) will Transform an AMP-based treatment that may be moderately effective in plants into one that is Highly Effective in plants. To Translate these findings into Highly Effective HLB Treatments that can be used by citrus growers, we have implemented the following activities: 1. Patent Disclosure. Intellectual property protection is critical for creating products that growers can use. As is routine for our university systems, we have submitted an invention disclosure on the aforementioned new results and we will work with our industrial partners on product development. Mandadi, K., Borneman, J., Fernando, S., Irigoyen, S., Padilla, C., Ramasamy, M., Mallawarachchi, S. (2022). Development of specifically targeted binders and antimicrobial peptides (STAMPs) to control Candidatus Liberibacter spp. Invention Disclosure (6003AGLR22). 2. Stakeholder meetings. Team members have participated in meetings with stakeholders (e.g., Southern Gardens Citrus) and informed them of our promising results and plans. To reach a wider audience, we have also published a research snapshot related to our efficacy screening system in Science for Citrus Health, which is an online resource growers. 3. Advisory Committee. We are arranging a meeting with our advisory committee to discuss our new findings and to seek their input to ensure the effective translation of these findings into products for HLB treatments. OTHER RESULTS FROM THIS PAST YEAR ARE DESCRIBED BELOW DESIGNING CLAS TARGETED PEPTIDES A novel approach was used to identify a set of new short peptides which had a very high affinity towards BamA protein of CLas bacteria - which we call the targeted peptides. This approach was based on creating peptides using amino acids which had strongest affinity towards BamA. Initially, peptide-probes modified to emulate bound amino-acid activity were docked using Schrodinger Glide on the target domain of BamA. Docking results of probes showed three closely located clusters on BamA. Peptides were designed by selecting the strongest binding probes in each cluster based on docking scores, and linking them using linkers consisting of 1-3 amino acids depending on the distances between clusters. Two types of linkers were used for peptide design: conventional linkers GSG and GGS, and linkers consisting of strongest binding probes. The affinity of the designed peptides was evaluated based on docking scores and MMGBSA free energies. Based on docking results, several peptides which had a very high affinity towards BamA could be identified. The screened peptides had average docking scores around -9 kcal/mol, and average MMGBSA free energies around -80 kcal/mol. Out of 19 peptides, Peptide 12 had the most negative docking score (-10.132 kcal/mol), and Peptide 18 had the most negative binding free energy (-84.067 kcal/mol). Based on docking scores and MMGBSA energies, six peptides were screened for in-vitro testing using Bio-layer Interferometry (BLI). BLI results showed that four of the screened peptides: Peptide 10, Peptide 11, Peptide 12 and Peptide 18, had very strong affinity towards BamA. All four of these peptides had dissociation rates less than 10-7 s, and affinity constants in picomolar level, indicating extremely tight binding to BamA. Thus, those four peptides were identified as potential candidates for the CLas targeted peptides. Peptides are presented in codes since intellectual property (IP) protection is being pursued. EVALUATING CLAS TARGETED ANTIMICROBIAL PEPTIDES A) Testing newly designed targeted peptides for their ability to bind to CLas and surrogates (completed). In total, we completed testing of ~10 putative targeted peptides using in vitro binding assays. Of them, ~2 showed good binding to three CLas surrogates and CLas from enriched plant extracts. B) Targeted antimicrobial peptides comprising N- and C-terminus conjugates of the two targeted peptides and a broad-spectrum antimicrobial peptides were synthesized (completed). C) Evaluation of the efficacy of the targeted peptides and targeted antimicrobial peptides (ongoing). To date, we completed efficacy testing of ~6 different targeted peptides in various combinations and orientations of targeted antimicrobial peptides against three culturable surrogates of CLas and in vitro CLas-citrus hairy root (HR) assays. The results are promising and show that some binders and targeted antimicrobial peptides had enhanced efficacy against the targets. We will continue the optimization of the targeted antimicrobial peptides and complete efficacy evaluations in the next reporting period. OBJECTIVE 2: CREATE AN IN SILICO METABOLIC MODEL OF THE HLB PATHOSYSTEM BENEFITS TO THE COMMUNITY. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivate CLas in vitro. In this project period, we have completed the construction of the first metabolic model of citrus, which we are now validating using the biological data described below. Side projects derived from this work include speeding up the process of making engineered citrus by increasing the growth rates of citrus at the various steps in the process; this is a project that was recently funded by the California Department of Food and Agriculture, and which is utilizing the aforementioned model. We have also performed several sets of experiments to create multi-omics datasets to train the integrated HLB model. This includes, among others, detailed time series experiments starting with the initial infection event, and then coupling these results with model-based bioinformatic analyses. We have generated sequence data from these samples and an IsoSeq resource to improve mapping transcriptomic data for this project and future related experiments. Informatics analyses have identified differentially expressed genes across treatments at various time points in the infection process. These genes have been mapped to known enzymatic functions in silico as well as known citrus metabolic pathways to derive biologically-relevant information. OBJECTIVE 3: PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE Our work on this project began in the spring of 2021 with revising a dynamic bioeconomic model to be used to simulate citrus production when HLB is present or a threat. During this time we also surveyed growers, industry representatives, farm advisors, research collaborators, and others to guide adaptation of the model strategies to combat HLB and ACP. Additional data for the simulation model has been collected from the scientific literature, USDA NASS, CDFA, UCANR, UF/IFAS, DATOC, and other such sources. OBJECTIVE 4: IMPLEMENT OUR OUTREACH PLAN. See Section 5 C below.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Younghwan Kwak, Penglin Sun, Venkata RamaSravani Meduri, Diana M. Percy, Kerry E. Mauck, Allison K. Hansen. 2021. Uncovering symbionts across the psyllid tree of life and the discovery of a new Liberibacter species, Candidatus Liberibacter capsica. Frontiers in Microbiology. 29 September (12). 2927. https://doi.org/10.3389/fmicb.2021.739763
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Margaret W. Thairu, Venkata Rama Sravani Meduri, Patrick H. Degnan, Allison K. Hansen. 2021. Natural selection shapes maintenance of orthologous sRNAs in divergent host-restricted bacterial genomes. Molecular Biology and Evolution. Volume 38, Issue 11, November 2021, Pages 47784791, https://doi.org/10.1093/molbev/msab202.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Mandadi, K., Irigoyen, S., Ramasamy, M. (2021). Novel tools for antimicrobial testing and discovery of new HLB therapies. Science for Citrus Health (Research Snapshot).
https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/Tools/Novel_tools_for_antimicrobial_testing_and_discovery_of_new_HLB_therapies/
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Passi, A., Tibocha-Bonilla, J.D., Kumar, M., Tec-Campos, D., Zengler, K., Zuniga, C. (2022) Genome-scale metabolic modeling enables in-depth understanding of big data. Metabolites 22:14.
|
Progress 02/01/20 to 01/31/21
Outputs
Target Audience:2A. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE WORK YOU ARE DOING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we arein silicodesigning and experimentally selecting for peptides that will selectively bind to the HLB associated pathogen (CLas). Besides being used in our work - which will involve using them to construct peptides that specifically and effectively kill CLas - such peptides could also be used by other scientists in a variety of ways. For example, they could be used to (i) create CLas detection assays or (ii) to immuno-capture viable CLas for in vitro cultivation inoculum. 2B. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE TOOLS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we are using several different methods to design and select for peptides that will selectively bind to the HLB associated pathogen (CLas). Other scientists could learn from the methods that we are using to create such peptides. These methods include (i)in silicodesign, (ii) peptide aptamer selection and, (iii) Nanobody selection. In addition, other scientists will also be able to use the most effective and rapidin plantamethod for analyzing the effect of molecules on CLas. This is the hairy root method created by Kranthi Mandadi. OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is to construct an integrated metabolic model of the HLB pathosystem - citrus, CLas, APC - and their molecular interactions. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivateCLas in vitro. This will be accomplish by, for example, analyzing omics datasets in the context of the model. This type of analysis greatly extends such datasets. For example, if an RNAseq dataset from infected citrus is analyzed in this way, the output is increased from a list of RNA transcripts to a dataset that includes all of the other reactions and pathways that are turned on or off in CLas and citrus under the conditions when that sample was collected - along with all of the interactions among the pathosystem organisms. 2C. STAKEHOLDERS THAT COULD BENEFIT FROM THE SOLUTIONS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS This objective is designed to benefit the citrus industry in the near-, intermediate-, and long-term by creating both prophylactic and curative HLB management strategies. This is because this objective is to create highly effective peptides that will specifically kill the HLB associated pathogen. We will deliver these peptides to CLas' habitat (citrus phloem) in several ways: (i) using CTV which is a near-term solution, (ii) using other vehicles that are not completely developed such as the novel virus-like RNA that Anne Simon is developing - this is an intermediate-term solution and, (iii) engineering citrus which is a longer-term solution. OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is designed to benefit the citrus industry as we envision this model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivateCLas in vitro. OBJECTIVE 3:PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This objective is designed to benefit the citrus industry by ensuring that the solutions we create are commercially viable, and to steer us away from solutions that are not commercially viable. OBJECTIVE 4:IMPLEMENT OUR OUTREACH PLAN This objective is designed to benefit the citrus industry in several ways. First, since we have included members of the industry in the development of this project, and as members of this project, we are confident that we are taking the necessary steps to make sure the solutions created by this project are commercially viable in terms of things like licensing and economic feasibility. Second, our outreach team is and will maintain communications to members of the industry concerning the progress we are making and the solutions that get developed. Third, we have a public outreach component that is to broadly educate the general public about HLB. We believe that if the general public was more aware of the HLB situation, they would be more inclined to contact their local, state and federal representatives and ask them to do what is needed to solve this problem. We are doing this through a variety of means including creating an App that will explain the HLB pathosystem, why it is a problem, and what researchers and the citrus industry are doing to solve this problem. 2D. STUDENTS FROM YOUR FORMAL TEACHING AND OTHER EDUCATION ACTIVITIES Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate level formal teaching. For example, James Borneman teaches two courses every year: MCBL 126, an undergraduate course in microbiomes and MCBL 226, a graduate course in microbiomes. See Other Products or Outputs for more detailed information on project relevant teaching. OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM OBJECTIVE 3:PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE OBJECTIVE 4:IMPLEMENT OUR OUTREACH PLAN Changes/Problems:THE COVID-19 PANDEMIC CONSIDERABLY HINDERED PROGRESS DURING THIS LAST REPORTED PERIOD. What opportunities for training and professional development has the project provided?Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate level formal teaching. For example, James Borneman teaches two courses every year: MCBL 126, an undergraduate course in microbiomes and MCBL 226, a graduate course in microbiomes. See Other Products or Outputs for more detailed information on project relevant teaching. How have the results been disseminated to communities of interest?Project updates and results were regularly disseminated among the team members. Project findings was also periodically shared with the scientific community, students, citrus growers and stakeholders in TX, CA and FL at various grower and extension meetings/conferences (see Other Products section). The broader scientific community and the citrus stakeholder community were also reached out through publications, newsletters (e.g., Citrograph), and the project's website (modelinghlb.weebly.com). Other outreach activities include among others creating an App that can be used on phones, tablets, and computers to teach the general public about HLB and our solutions for managing HLB (this has not been completed). This is work is being done in collaboration with colleagues from UC Berkeley's Lawrence Hall of Science museum. What do you plan to do during the next reporting period to accomplish the goals?We will continue to move all of the research described above forward. OBJECTIVE 1: DESIGN AND SELECT FOR CLAS TARGETING PEPTIDES 1. IN SILICO DESIGN. Outer membrane porin protein also was identified as a potential target, and a group of short peptides which show high affinity towards the porin protein were identified via STRING database searching for protein screening, identification of potential peptide sequences using Schrodinger protein-protein docking tool and AutoDock vina docking. The five screened peptides are SVDLPPVIARVS, KFFSGEEPILSDTVE, IVTSGQFHMG, FQMQLQDTGIVVSRIGDMITCYIP, and YKYPIDTNDTKVGRQNNQRI from the following proteins, ACT57173.1, ACT57173.1, ACT57173.1, ACT57426.1, ACT57426.1, respectively. The use of these peptides to improve the affinity of anti-microbial peptides towards porin protein will be studied by comparing the binding affinities of original AMPs and the STAMPs created by fusing AMPs with screened peptides towards the outer membrane porin protein.Autodock vina docking scores and Prime MMGBSA binding free energies will be used as quantitative measures on binding affinity. In addition, searching for other potential drug targets will be conducted through literature searches. Regarding the ongoing work on BamA, we plan to validate the findings experimentally and through molecular dynamics. Two STAMP sequences which have much higher affinity towards BamA compared to the unmodified AMPs have been identified. Molecular dynamics related to STAMP binding will be evaluated using Schrodinger Desmond simulations.Once a viable STAMP is synthesized, the authors also plan to experimentally determine the association and dissociation constants of the AMPs and STAMPs using biolayer interferometry and compare whether the experimental results are consistent with the simulations. We will continue to conduct MD simulations coupled BLI studies for screening and verifying the activity of AMPs and STAMPs against CLas BAMa. 2. NANOBODY SELECTION. To create Nanobodies that selectively bind CLas, we will determine whether we have successfully immuno-captured and purified CLas. When we have successfully optimized this work, we will scale-up the experiments and then ship fixed CLas cells to Geoffrey Chang (UC San Diego) to select for CLas-binding Nanobodies. DESIGN STAMPS We will continue with the design the CLas-selective STAMPS for testing in the hairy root system. EVALUATE STAMPS USING NOVEL HAIRY ROOT SYSTEM We will evaluate efficacy of the STAMP conjugates against Liberibacter crescens or Rhizobium spp. that are culturable surrogates of CLas. The in vitro assays will allow us to perform dose-response assays and resolve better the efficacy of the AMP alone vs. the STAMP. Next, we will also use the ex vivo CLas-citrus hairy root (HR) assay to confirm the efficacy of the STAMP in citrus plant tissues. OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM Now that we have constructed a draft of the citrus metabolic model, we will next construct the tissue specific models for roots, leaves, etc. We will continue to collect and analyze multi-omics datasets to train the integrated HLB pathosystem model. We will also investigate how they metabolically interact in Liberibacter positive and negative psyllids. The graduate student that is training on this project is currently taking her Ph.D. candidate exams spring 2021 but she will start trials end of this spring using RNAseq sequencing. Within fall - winter we will then analyze metabolomic data from psyllids and their microbiome with and without Liberibacter in addition to free amino acid concentrations within sap from leaves and psyllid bodies to further understand the metabolic flux within this Liberibacter vector and its crosstalk with its symbiont community when feeding on infected and uninfected plants. OBJECTIVE 3:PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This work will be initiated in Year 3 of this project OBJECTIVE 4:IMPLEMENT OUR OUTREACH PLAN. We will continue our outreach efforts by disseminating the findings of this project to the scientific community, students, citrus growers and stakeholders in TX, CA and FL via various grower and extension meetings/conferences, publications, and newsletters (e.g., Citrograph). We will also make our website (modelinghlb.weebly.com) more complete and more useful. Here,we will aim to promote communication and networking between researchers and growers via an interactive web-based platform created for this project (managed by Georgios Vidalakis). Here, we will also endeavor to stimulate research initiatives and collaborations by crowd-sourcing our data and research results. Our website will inform the stakeholders and the general public on the progress and outcomes of our research. In addition, this website will serve as a hub to existing HLB online resources (Industry, californiacitrusthreat.org/; State, freshfromflorida.com; USDA, saveourcitrus.org; fruitmentorTM, www.fruitmentor.com/; etc.) to inform the public about the enormous threat HLB poses to the citrus industry, how they can identify HLB disease systems and ACP in their neighborhoods, and how they can communicate this information to the appropriate officials. We will incorporate a blog to disseminate updated information about our deliverable solutions so that end users such as growers can provide feedback to facilitate the development of products that will actually be useful for them. The blog will also allow us to communicate information about our research with the citrus community in an interactive manner. We will use Google Analytics to determine who our blog/website audience is and what blog posts/webpages they are most interested in. Our website will also provide free access to protocols for the HLB solutions that are developed by this project along with the metabolic models and multi-omics data. It will also provide information about citrus events and access to our presentations. Other outreach activities will include among others creating an App that can be used on phones, tablets, and computers to teach the general public about HLB and our solutions for managing HLB. This is work is being done in collaboration with colleagues from UC Berkeley's Lawrence Hall of Science museum. We will also create videos. The creator of the fruitmentorTMwebsite will design and construct videos designed to inform the general public, citrus industry, and other scientists about the two science-based objectives of this USDA proposal.
Impacts
What was accomplished under these goals?
THE COVID-19 PANDEMIC CONSIDERABLY HINDERED PROGRESS DURING THIS LAST REPORTED PERIOD. OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILLCLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS RATIONALE. This objective will address four key obstacles that have been, or still are, preventing the creation of highly effective prophylactic and curative treatments for HLB including: TO ADDRESS DELIVERY, our project will focus on using the citrus tristeza virus (CTV), which provides a cost-effective strategy to deliver anti-CLas peptides to the phloem. We will also test other delivery methods. Finally, we will engineer and test citrus that express our newly created anti-CLas peptides, but this work will not be part of this proposed project. TO ADDRESS POTENCY AND SPECIFICITY, our project will create and use peptides that specifically target and killCLas, which we expect will have several important characteristics including greatly increased efficacy. TO ADDRESS TESTING, we developed an innovative plant-based assay that allows us to test anti-CLas peptides againstCLas at rates up to 4X faster that mature plant assays. DESIGN AND SELECT FOR CLAS TARGETING PEPTIDES 1. APTAGEN SELECTION. We contracted with Aptagen to have them select for peptide aptamers that bind to CLas, and this work has been completed. 2. IN SILICO DESIGN. Studies during the reporting period focused on the development of Specifically Targeted AntiMicrobial Peptides (STAMPs) for the CLas bacterium by fusing known antimicrobial peptides (AMPs) with high-affinity short peptides (HASPs) targeting the CLas BamA protein. Initially, three peptides, ACT56869.1_241-255, ACT56973.1_80-96 and ACT57020.1_94-118, which showed high affinity towards BamA were screened through database searching and molecular docking. The affinities of these HASPs towards BamA were evaluated using molecular docking and BioLayer Interferometry (BLI). Both in-silico and in-vitro results suggested that these peptides had a significant affinity towards BamA, with the docking scores ranging from -3.4 to -5.4 kcal/mol, MMGBSA free energies ranging from -21 to -40 kcal/mol, and affinity constants in the range of 200-300 nM. Molecular dynamic (MD) simulations also were conducted on these HASPs, which showed that the HASPsstayed steady at the binding site during the simulation. 3. NANOBODY SELECTION. To create Nanobodies that selectively bind CLas, we teamed up with Geoffrey Chang from UC San Diego, who is the world leader in this field. We have obtained APHIS and CDFA permits to receive fixed infected citrus phloem sap from Kranthi Mandadi (Texas A&M) at both UC Riverside (Borneman) and UC San Diego (Chang). The achieve our objective, we will immuno-capture and purify fixed CLas cells from phloem sap (Borneman). EVALUATE PUTATIVE CLAS TARGETING PEPTIDES FOR THEIR CLAS BINDING ABILITIES Testing CLas-Targeting Peptides for Their Ability to Bind CLas (completed). To determine if the newly designed CLas targeting peptides can bind to CLas, we optimized an in vitro fluorescently labelled binding assay. The assay was optimized using a FITC-labeled KH peptide that binds toPseudomonas mendocinaprovided by Borneman lab. Next, we optimized methods to enrich CLas from CLas-infected plant tissues. This CLas extract was subsequently used a source of CLas inoculum to perform binding assays with candidate CLas binding peptides. We completed testing of 9 binders and of them two appear to show some binding to CLas. STAMPs comprising N and C-terminus conjugates of the two binders and a broad-spectrum antimicrobial peptide were designed and synthesized for further efficacy testing. DESIGN STAMPS The effect of fusing the AMPs with HASPs was analyzed in-silico via Vina docking scores and MM-GBSA free energies for AMPs and STAMPs.Attachment of HASPs significantly reduced the docking scores and binding free energies for AMPs, suggesting that it would make it easier for the STAMPs to bind to BAMa. Attachment of HASP ACT56973.1_80-96 to the AMP gave the most favorable results, reducing the docking score from 12 kCal/mol to -1.5 kCal/mol. Visualizing of the binding poses showed thatthe HASPs portions of both STAMPs have plugged into the top part of the β-barrel, effectively giving specific access for the AMPs to interact with the targets at close proximity to BamA in the cell membrane. STAMPs may lead to a potentially dual-action method to inhibit CLas - by blocking BamA access and enhancing AMP action. EVALUATE STAMPS USING NOVEL HAIRY ROOT SYSTEM This work is pending the design of the STAMPS. OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM BENEFITS TO THE COMMUNITY. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivateCLas in vitro. Thus far, we have constructed genome-scale metabolic models for six CLas strains (A4, FL17, gxpsy, Ishi-1, psy62, and YCPsy), in addition to a model of the most closely related culturable microorganism, the culturableL. crescensBT-1 - See Zuniga et al. (2020) publication. CLas models were constrained using expression data obtained from CLas-infected citrus trees, as well as from CLas residing in the psyllid hostDiaphorina citriKuwayama, revealing host-dependent metabolic phenotypes. We also determined the unique metabolic capabilities for all strain-specific variants of the seven Liberibacter spp. Results identified conserved and unique metabolic traits among the bacterial strains and revealed strain-specific interactions between CLas and its hosts, laying the foundation for the development of model-driven HLB-management strategies. This work is described in a paper that is currently in the review process. We have also constructed a draft of the citrus metabolic model, which we expect to complete in the next few months and make available to the HLB community. Side projects derived from this work include speeding up the process of making engineered citrus by increasing the growth rates of citrus at the various steps in the process. We have also performed several sets of experiments to create multi-omics datasets to train the integrated HLB model. This includes, among others, detailed time series experiments starting with the initial infection event, and then coupling these results with model-based bioinformatic analyses. We have generated sequence data from these samples and an IsoSeq resource to improve mapping transcriptomic data for this project and future related experiments. Informatics analyses have identified differentially expressed genes across treatments at various time points in the infection process. These genes have been mapped to known enzymatic functionsin silicoas well as known citrus metabolic pathways to derive biologically-relevant information. We are also investigating how they metabolically interact in Liberibacter positive and negative psyllids. We have also developed an infected and uninfected psyllid lines from the same genetic background of psyllids to conduct RNAseq trials with to examine the metabolic impact of Liberibacter on the psyllid's nutritional microbiome. We have also optimized our detection of Liberibacter infection in psyllid bodies and plant leaves by designing new primers when using RT-qPCR since we observed high non-specific amplification when using previously published primers on psyllids due to mis-priming of their nutritional symbionts. OBJECTIVE 3:PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This work will be initiated in Year 3 of this project OBJECTIVE 4:IMPLEMENT OUR OUTREACH PLAN. See Section 5 C below.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Thairu M., Meduri V, Degnan, P, and A. Hansen. In revision. Small RNA regulation of nutritional symbionts from psyllid vectors of Liberibacter. Molecular Evolution and Biology.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zuniga C., Peacock B, Liang, B., McCollum, G., Irigoyen, S., Tec-Campos, D., Marotz, C., Weng, N-C., Zepeda, A., Vidalakis, G., Mandadi, K., Borneman, J., Zengler, K. (2020). Linking metabolic phenotypes to pathogenic traits among Candidatus Liberibacter asiaticus and its hosts. npj Systems Biology and Applications 6:1-12
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Nichole A. Ginnan, Tyler Dang, Sohrab Bodaghi, Paul M. Ruegger, Greg McCollum, Gary England, Georgios Vidalakis, James Borneman, Philippe E. Rolshausen and M. Caroline Roper. 2020. Disease-induced microbial shifts in citrus indicate microbiome-derived responses to Huanglongbing across the disease severity spectrum. Phytobiomes Journal 4:375-387
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Mandadi, K., Irigoyen, S., Ancona, V., S�tamou, M., Coaker, G., Borneman, J., and Irey, M. (2020). Hairy roots to the rescue: Speeding up discovery for HLB Management. Citrograph 11:20-22
|
Progress 02/01/19 to 01/31/20
Outputs
Target Audience:2A. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE WORK YOU ARE DOING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we arein silicodesigning and experimentally selecting for peptides that will selectively bind to the HLB associated pathogen (CLas). Besides being used in our work - which will involve using them to construct peptides that specifically and effectively kill CLas - such peptides could also be used by other scientists in a variety of ways. For example, they could be used to (i) create CLas detection assays or (ii) to immuno-capture viable CLas for in vitro cultivation inoculum. 2B. OTHER SCIENTISTS THAT WOULD BENEFIT FROM THE TOOLS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS To accomplish this objective, we are using several different methods to design and select for peptides that will selectively bind to the HLB associated pathogen (CLas). Other scientists could learn from the methods that we are using to create such peptides. These methods include (i)in silicodesign, (ii) peptide aptamer selection and, (iii) Nanobody selection. In addition, other scientists will also be able to use the most effective and rapidin plantamethod for analyzing the effect of molecules on CLas. This is the hairy root method created by Kranthi Mandadi. OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is to construct an integrated metabolic model of the HLB pathosystem - citrus, CLas, APC - and their molecular interactions. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivateCLas in vitro. This will be accomplish by, for example, analyzing omics datasets in the context of the model. This type of analysis greatly extends such datasets. For example, if an RNAseq dataset from infected citrus is analyzed in this way, the output is increased from a list of RNA transcripts to a dataset that includes all of the other reactions and pathways that are turned on or off in CLas and citrus under the conditions when that sample was collected - along with all of the interactions among the pathosystem organisms. 2C. STAKEHOLDERS THAT COULD BENEFIT FROM THE SOLUTIONS YOU ARE DEVELOPING OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS This objective is designed to benefit the citrus industry in the near-, intermediate-, and long-term by creating both prophylactic and curative HLB management strategies. This is because this objective is to create highly effective peptides that will specifically kill the HLB associated pathogen. We will deliver these peptides to CLas' habitat (citrus phloem) in several ways: (i) using CTV which is a near-term solution, (ii) using other vehicles that are not completely developed such as the novel virus-like RNA that Anne Simon is developing - this is an intermediate-term solution and, (iii) engineering citrus which is a longer-term solution. OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM This objective is designed to benefit the citrus industry as we envision this model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivateCLas in vitro. OBJECTIVE 3:PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This objective is designed to benefit the citrus industry by ensuring that the solutions we create are commercially viable, and to steer us away from solutions that are not commercially viable. OBJECTIVE 4:IMPLEMENT OUR OUTREACH PLAN This objective is designed to benefit the citrus industry in several ways. First, since we have included members of the industry in the development of this project, and as members of this project, we are confident that we are taking the necessary steps to make sure the solutions created by this project are commercially viable in terms of things like licensing and economic feasibility. Second, our outreach team is and will maintain communications to members of the industry concerning the progress we are making and the solutions that get developed. Third, we have a public outreach component that is to broadly educate the general public about HLB. We believe that if the general public was more aware of the HLB situation, they would be more inclined to contact their local, state and federal representatives and ask them to do what is needed to solve this problem. We are doing this through a variety of means including creating an App that will explain the HLB pathosystem, why it is a problem, and what researchers and the citrus industry are doing to solve this problem. 2D. STUDENTS FROM YOUR FORMAL TEACHING AND OTHER EDUCATION ACTIVITIES Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate level formal teaching. For example, James Borneman teaches two courses every year: MCBL 126, an undergraduate course in microbiomes and MCBL 226, a graduate course in microbiomes. OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILL CLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM OBJECTIVE 3:PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE OBJECTIVE 4:IMPLEMENT OUR OUTREACH PLAN Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Within the project as defined by its four objectives listed below, this project is providing training and mentorship for students and other personnel in all of the various topics, methodologies, and experimental designs that are being used including (i) the host-microbe interactions associated with HLB, (ii) molecular biology experimentation including various omics analyses, (iii) microbiology experimentation, (iv) biochemistry experimentation, (v) in silico molecular interaction studies, (vi) plant rearing and various associated experiments, (vii) greenhouse experiments, (viii) field experiments, (ix) constructing in silico metabolic models, (x) App development, (xi) outreach to the citrus industry, (xii) writing papers, (xiii) creating and giving slide and poster presentations, (xiv) video development, among other things. In addition, several of the investigators integrate their research findings and related concepts into their undergraduate and graduate level formal teaching. For example, James Borneman teaches two courses every year: MCBL 126, an undergraduate course in microbiomes and MCBL 226, a graduate course in microbiomes. How have the results been disseminated to communities of interest?Project updates and results were regularly disseminated among the team members. Project findings was also periodically shared with the scientific community, students, citrus growers and stakeholders in TX, CA and FL at various grower and extension meetings/conferences (see Other Products section). The broader scientific community and the citrus stakeholder community were also reached out through publications, newsletters (e.g., Citrograph), and the project's website (modelinghlb.weebly.com). Other outreach activities include among others creating an App that can be used on phones, tablets, and computers to teach the general public about HLB and our solutions for managing HLB (this has not been completed). This is work is being done in collaboration with colleagues from UC Berkeley's Lawrence Hall of Science museum. What do you plan to do during the next reporting period to accomplish the goals?We will continue to move all of the research described above forward. OBJECTIVE 1: DESIGN AND SELECT FOR CLAS TARGETING PEPTIDES 1. IN SILICO DESIGN. Outer membrane porin protein also was identified as a potential target, and a group of short peptides which show high affinity towards the porin protein were identified via STRING database searching for protein screening, identification of potential peptide sequences using Schrodinger protein-protein docking tool and AutoDock vina docking. The five screened peptides are SVDLPPVIARVS, KFFSGEEPILSDTVE, IVTSGQFHMG, FQMQLQDTGIVVSRIGDMITCYIP, and YKYPIDTNDTKVGRQNNQRI from the following proteins, ACT57173.1, ACT57173.1, ACT57173.1, ACT57426.1, ACT57426.1, respectively. The use of these peptides to improve the affinity of anti-microbial peptides towards porin protein will be studied by comparing the binding affinities of original AMPs and the STAMPs created by fusing AMPs with screened peptides towards the outer membrane porin protein.Autodock vina docking scores and Prime MMGBSA binding free energies will be used as quantitative measures on binding affinity. In addition, searching for other potential drug targets will be conducted through literature searches. Regarding the ongoing work on BamA, we plan to validate the findings experimentally and through molecular dynamics. Two STAMP sequences which have much higher affinity towards BamA compared to the unmodified AMPs have been identified. Molecular dynamics related to STAMP binding will be evaluated using Schrodinger Desmond simulations.Once a viable STAMP is synthesized, the authors also plan to experimentally determine the association and dissociation constants of the AMPs and STAMPs using biolayer interferometry and compare whether the experimental results are consistent with the simulations. 2. NANOBODY SELECTION. To create Nanobodies that selectively bind CLas, we will determine whether we have successfully immuno-captured and purified CLas. When we have successfully optimized this work, we will scale-up the experiments and then ship fixed CLas cells to Geoffrey Chang (UC San Diego) to select for CLas-binding Nanobodies. EVALUATE PUTATIVE CLAS TARGETING PEPTIDES FOR THEIR CLAS BINDING ABILITIES We will scale up and/or modify our CLas enrichment approaches to increase CLas yield and recovery, which is necessary for the downstream CLas peptide binding assays. DESIGN STAMPS Once CLas-binding peptides have been identified, we will design the CLas-selective STAMPS for testing in the hairy root system. EVALUATE STAMPS USING NOVEL HAIRY ROOT SYSTEM We will evaluate the efficacy of new CLas-selective STAMPs, as they become available, in the CLas-citrus hairy root system. OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM Now that we have constructed a draft of the citrus metabolic model, we will next construct the tissue specific models for roots, leaves, etc. We will continue to collect and analyze multi-omics datasets to train the integrated HLB pathosystem model. OBJECTIVE 3:PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This work will be initiated in Year 3 of this project OBJECTIVE 4:IMPLEMENT OUR OUTREACH PLAN. We will continue our outreach efforts by disseminating the findings of this project to the scientific community, students, citrus growers and stakeholders in TX, CA and FL via various grower and extension meetings/conferences, publications, and newsletters (e.g., Citrograph). We will also make our website (modelinghlb.weebly.com) more complete and more useful. Here,we will aim to promote communication and networking between researchers and growers via an interactive web-based platform created for this project (managed by Georgios Vidalakis). Here, we will also endeavor to stimulate research initiatives and collaborations by crowd-sourcing our data and research results. Our website will inform the stakeholders and the general public on the progress and outcomes of our research. In addition, this website will serve as a hub to existing HLB online resources (Industry, californiacitrusthreat.org/; State, freshfromflorida.com; USDA, saveourcitrus.org; fruitmentorTM, www.fruitmentor.com/; etc.) to inform the public about the enormous threat HLB poses to the citrus industry, how they can identify HLB disease systems and ACP in their neighborhoods, and how they can communicate this information to the appropriate officials. We will incorporate a blog to disseminate updated information about our deliverable solutions so that end users such as growers can provide feedback to facilitate the development of products that will actually be useful for them. The blog will also allow us to communicate information about our research with the citrus community in an interactive manner. We will use Google Analytics to determine who our blog/website audience is and what blog posts/webpages they are most interested in. Our website will also provide free access to protocols for the HLB solutions that are developed by this project along with the metabolic models and multi-omics data. It will also provide information about citrus events and access to our presentations. Other outreach activities will include among others creating an App that can be used on phones, tablets, and computers to teach the general public about HLB and our solutions for managing HLB. This is work is being done in collaboration with colleagues from UC Berkeley's Lawrence Hall of Science museum. We will also create videos. The creator of the fruitmentorTMwebsite will design and construct videos designed to inform the general public, citrus industry, and other scientists about the two science-based objectives of this USDA proposal.
Impacts
What was accomplished under these goals?
UNFORESEEN ISSUES CAUSING DELAYS This project was started approximately 6-months after the start date because of federal legislation that was passed after we received the initial award notification. This legislation required us to obtain matching funds (cost-share) for the entire grant. We were eventually given an exemption, but this caused a 6-month delay in getting the funds to all of the project participants. Currently, we have another problem that is causing a lack of progress, and that is the project participants are unable to perform any or most of their research because of COVID-19 restrictions. Having said that, we have still made pretty good progress as described below. OBJECTIVE 1: DESIGN, CONSTRUCT, AND TEST PEPTIDES THAT SPECIFICALLY TARGET AND KILLCLAS TO CREATE HIGHLY EFFECTIVE PROPHYLACTIC AND CURATIVE HLB TREATMENTS RATIONALE. This objective will address four key obstacles that have been, or still are, preventing the creation of highly effective prophylactic and curative treatments for HLB including: TO ADDRESS DELIVERY, our project will focus on using the citrus tristeza virus (CTV), which provides a cost-effective strategy to deliver anti-CLas peptides to the phloem. We will also test other delivery methods. Finally, we will engineer and test citrus that express our newly created anti-CLas peptides, but this work will not be part of this proposed project. TO ADDRESS POTENCY AND SPECIFICITY, our project will create and use peptides that specifically target and killCLas, which we expect will have several important characteristics including greatly increased efficacy. TO ADDRESS TESTING, we developed an innovative plant-based assay that allows us to test anti-CLas peptides againstCLas at rates up to 4X faster that mature plant assays. DESIGN AND SELECT FOR CLAS TARGETING PEPTIDES 1. APTAGEN SELECTION. We contracted with Aptagen to have them select for peptide aptamers that bind to CLas. This was accomplished by having GenScript express CLas' BamA protein - which is located on the surface of cells. They have completed this work, and they determined that 12 peptides bind well to the BamA protein. When the COVID-19 restrictions to research are removed, we will start testing their abilities to bind CLas collected from phloem sap. 2. IN SILICO DESIGN. We used in silico analyses to design CLas-binding peptides. An in-silico evaluation was on the use of Smart Targeting Anti-Microbial Peptides (STAMPs) to inhibit citrus greening by attacking the BamA protein ofcandidatus liberibacter asiaticus(CLas).Initially 2 peptide segments, ACT56973_80-96 and ACT56869_241-255, which showed high affinity towards BAM-A were screened via molecular docking. In the second step,two antimicrobial peptides were modeled using SwissModel based on the gene sequences provided by Dr. Kranthi Mandadi. Both AMPs showed highly positive docking scores and MMGBSA binding free energies, indicating of very low affinity. Therefore, the potential of developing SMARTTargetting Anti Microbial Peptides (STAMPs) by fusing the AMPs with short peptide chains which have high binding affinity towardsBamAwas evaluated. Peptide fusion was done by joining the C terminus of the targeting peptide with the N terminus of the AMP using Chimera and affinity was evaluated via docking and MMGBSA calculations. 3. NANOBODY SELECTION. To create Nanobodies that selectively bind CLas, we teamed up with Geoffrey Chang from UC San Diego, who is the world leader in this field. We have obtained APHIS and CDFA permits to receive fixed infected citrus phloem sap from Kranthi Mandadi (Texas A&M) at both UC Riverside (Borneman) and UC San Diego (Chang). The achieve our objective, we will immuno-capture and purify fixed CLas cells from phloem sap (Borneman). These cells will then be shipped to the Chang lab for Nanobody selection. We optimized our protocols by immuno-capturing and purifying E. coli. We have also immuno-captured what appears to be purified CLas cells from phloem sap, but we haven't confirmed this result yet. When the COVID-19 restrictions to research are removed, we will perform these verification experiments and proceed with the rest of the steps. EVALUATE PUTATIVE CLAS TARGETING PEPTIDES FOR THEIR CLAS BINDING ABILITIES To determine if the newly designed or selected CLas targeting peptides described above can bind to CLas, we are optimizing anin vitrofluorescently labelled binding assay. Initial tests were performed using a FITC-labeled KH peptide that binds toPseudomonas mendocina. Binding assays were performed with KH peptide, followed by mounting and visualization using a fluorescence microscope. After a few optimizations, we were successful in visualizing specific binding of KH toP. mendocina, but not to an unrelated bacteriumE. coli. Because CLas bacterium is unculturable and there is no CLas axenic culture yet, we are also developing methods to enrich CLas from plant phloem sap using CLas-infected citrus. We were successful in isolating CLas containing phloem extract; however, the titers of CLas were lower. We will use the CLas-enriched phloem extract and the optimized binding assays to further test the various putative CLas binding peptide when the COVID-19 restrictions to research are removed. DESIGN STAMPS This work is pending the evaluation of the CLas-binding peptides. EVALUATE STAMPS USING NOVEL HAIRY ROOT SYSTEM This work is pending the design of the STAMPS. OBJECTIVE 2: CREATE ANIN SILICOMETABOLIC MODEL OF THE HLB PATHOSYSTEM BENEFITS TO THE COMMUNITY. This model, which will be made freely available once completed, will provide researchers, engineers and agribusinesses with a powerful suite of tools and an unprecedented base of knowledge - which we expect will enable them to create (i) anti-CLas molecules, (ii) HLB-tolerant/resistant trees (iii) early detection methods as well as (iv) media and conditions to cultivateCLas in vitro. Thus far, we have constructed genome-scale metabolic models for six CLas strains (A4, FL17, gxpsy, Ishi-1, psy62, and YCPsy), in addition to a model of the most closely related culturable microorganism, the culturableL. crescensBT-1. CLas models were constrained using expression data obtained from CLas-infected citrus trees, as well as from CLas residing in the psyllid hostDiaphorina citriKuwayama, revealing host-dependent metabolic phenotypes. We also determined the unique metabolic capabilities for all strain-specific variants of the seven Liberibacter spp. Results identified conserved and unique metabolic traits among the bacterial strains and revealed strain-specific interactions between CLas and its hosts, laying the foundation for the development of model-driven HLB-management strategies. This work is described in a paper that is currently in the review process. We have also constructed a draft of the citrus metabolic model, which we expect to complete in the next few months and make available to the HLB community. Side projects derived from this work include speeding up the process of making engineered citrus by increasing the growth rates of citrus at the various steps in the process. We have also performed several sets of experiments to create multi-omics datasets to train the integrated HLB model. This includes the novel use of TagSeq to perform detailed time series experiments starting with the initial infection event, and then coupling these results with pathway bioinformatic analyses. However, given the space limitations, we did not described any of these multi-omic experiments in this first progress report. OBJECTIVE 3:PERFORM ECONOMIC ANALYSES TO ENSURE OUR SOLUTION ARE COMMERCIALLY VIABLE This work will be initiated in Year 3 of this project OBJECTIVE 4:IMPLEMENT OUR OUTREACH PLAN. See Section 5 C below.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Zuniga, C., Peacock, B., Liang, B., McCollum, G., Irigoyen, S.C., Tec, D., Marotz, C., Weng, N-C., Zepeda, A., Vidalakis, G., Mandadi, K.K., Borneman, J., Zengler, K. (2020) Linking metabolic phenotypes to pathogenic traits among Candidatus Liberibacter asiaticus and its hosts. Nature's npj Systems Biology and Applications journal.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Mandadi, K., Irigoyen, S., Ancona, V., S�tamou, M., Coaker, G., Borneman, J., and Irey, M. (2020) Hairy roots to the rescue: Speeding up Discovery for HLB Management. (2020) Citrograph 11 (2) 20-22.
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