Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 206 - Basic Plant Biology | 920 - Orange | 1030 - Cellular biology | 40% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 930 - Lemon | 1160 - Pathology | 30% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1040 - Molecular biology | 30% |
We have initiated a pipeline for developing products for HLB treatment, HLB prevention, and ACP control and propose to expand it under this CAP. Toward this goal, we have assembled an entrepreneurial team of scientists with expertise in disease biology and therapy, regulatory consultants with experience in obtaining product approval from federal agencies, and business experts with a track record in commercialization of agricultural products. Our team will register a product for HLB treatment in 3 years. We will be able to register a transgenic product for HLB prevention in 7 years first by collecting efficacy, toxicity, and safety data in 5 years (2 years after this pilot CAP) and then by collecting citrus health and fruit quality data in next 2 years. In addition, we will create a "Knowledge Base" for researchers, which will describe our concept and its application in therapy of HLB and other plant diseases. Finally, our CAP will include a component on "Education and Outreach" that will involve two important functions: training of students, technicians, and extension professionals on the "Knowledge Base" and holding onsite and online meetings/workshops to communicate to the growers and stakeholders the scope, deliverables, and impact of our CAP.Following the recommendation of the reviewers, the objectives have been revised as constrained by the reduced total budget (from 11.8M to 4.75M) and project duration (3 years instead of 5). However, the project theme remains the same, i.e., to demonstrate that enhancement of citrus innate immunity via delivery of the citrus derived helix-turn-helix (HTH) peptides and protein chimeras leads to efficacious HLB therapy. The focus is on completing and publishing the ongoing greenhouse and field studies to unequivocally show that the citrus derived helix-turn-helix (HTH) peptides and protein chimeras (i) clear Candidatus Liberibacter asiaticus (CLas), (ii) suppress HLB symptoms, (iii) block CLas transmission by Asian Citrus Psyllid (ACP), (iv) show no human and plant toxicity, (v) augment citrus innate immunity during CLas infection, and (vi) maintain homeostasis of microbiome beneficial to citrus. We will also concurrently
Finally, we will prepare an information package describing the foundation of our technology, its application in HLB therapy, and its relevance to sustaining the US citrus industry. Both the training modules and the information package will be developed and regularly updated using inputs from all the CAP team members.8. We will incorporate two important functions: (i) training of students, technicians, and extension professionals on the "Knowledge Base" and (ii) holding onsite and online meetings and workshops to communicate to the growers and stakeholders the scope, deliverables, and impact of our CAP. We will gather feedback from the growers and stakeholders to improve performance of the CAP.
(Objectives 1: HLB treatment). We will complete exogenous application studies on both greenhouse and field trees in 3 years on two HTH peptides, 28P-2 and 36P-1, already identified to be potent CLas killers by laboratory experiments. For the greenhouse studies, we will deliver the HTH peptides by trunk microabraison and foliar spray with the aid of laser etching. For field studies, we will use peptide delivery by trunk injection and foliar spray with the aid of laser etching. From our field studies conducted, so far, laser etching appears to facilitate phloem-specific delivery of the HTH peptides consistent with the published data on the delivery of small fluorescently labeled molecules.(Objective 2: HLB prevention). For transgenic HLB-resistant citrus rootstocks (Carrizo and Sour Orange) and scions (Hamlin), we have already constructed four different chimeras BT, TB, ST, and TS wherein B = citrus bacterial permeability increasing/liposaccharide bind protein (BPI/LBP), S = citrus subtilisin, T = citrus Thionin. These transgenics will be tested in the greenhouse in years 1 and 2 and planted for field studies in year 3. Two greenhouse experiments will be of critical importance for scree. In one experiment, transgenic and untransformed citrus will be fed with CLas+ caged psyllid for 1month and the level of CLas in the leaves will be monitored by qPCR over 3-6 months post-inoculation to determine whether the expressed chimeras lower or eliminate the bacterial load. In another experiment, CLas-infected rough lemon will be grafted on the HLB-resistant Carrizo rootstocks and CLas level in the leaves and roots and disease symptoms will be monitored over a year. Finally, we will initiate and complete the construction of HLB-resistant Sour Orange rootstocks expressing BT, TB, ST, and TS. The ultimate goal is to examine whether HLB resistance is transferred to the untransformed scion grafted on the rootstock. Note that commercially viable HLB-resistant Sour Orange rootstocks are important to the Texas citrus industry.(Objective 3: Blocking of ACP transmission). Initial studies indicate that the chimeras expressed by the transgenic citrus can lower the CLas level in Asian Citrus Psyllid (ACP). There are two possible ways the chimeras may kill the ACP either by lysing gram-negative ACP endosymbionts or by destroying protective matrices in the ACP gut. We will analyze the ACP microbiome (including endosymbionts) by metagenomics and the anatomy of the gut matrix by microscopy after feeding CLas +/- psyllids on untransformed infected/uninfected and transgenic infected/uninfected citrus. Analysis of metagenomics and microscopic data will reveal the mechanism of ACP killing by the chimeras and the possibility of their use in blocking CLas transmission by ACP.The quantitative studies on the role of (i) the HTH peptides in HLB treatment, (ii) the citrus derived chimeras in HLB resistance, and (iii) the chimeras in CLas transmission by ACP will be published in peer-reviewed journals to validate our concept that enhancing citrus innate immunity is a viable approach for HLB therapy.(Objective 4: Effect of therapeutics on citrus innate immunity during infection). Field studies are underway in which leaf and root samples are collected (see above in Objective 1 revised) after HTH peptide treatment. These samples will be analyzed to determine the genome-wide expression of mRNA/miRNA by RNA-seq and proteins by nano LC-MS/MS at different post-treatment times comparing controls only to the foliar delivery by laser etching. We will compare the gene networks in treated and untreated samples to examine whether the HTH peptides do indeed augment, as hypothesized by us, the innate immune defense in citrus during CLas infection. Similar studies will be performed in the greenhouse to investigate the effect of the HTH peptides on the innate immune defense in citrus during CLas infection.(Objective 5: Effect of therapeutics on citrus and soil microbiome). We will conduct metagenome analysis on the field and greenhouse samples collected for the 'omics studies discussed above to determine the effect of the HTH peptides in maintaining the homeostasis of the microbiome beneficial to citrus vis-Ã -vis augmenting innate immunity during infection.(Objective 6: Collection of data for regulatory processes). In this 3-year CAP, we have significantly reduced the budget for collecting the EPA, FDA, and APHIS recommended data on the effect of the patented HTH peptides and protein chimeras on human health/safety, citrus health, and fruit quality. Instead we will focus on standardizing a method for producing citrus derived HTH peptides and chimeras from transgenic tobacco, which will be necessary for collecting the EPA, FDA, and APHIS recommended data by certified laboratories. During the three years of this project, we will, however, collect preliminary human and plant toxicity data on these peptides and chimeras. Innate Immunity LLC will provide additional funding and resources to manage and guide the regulatory strategy and processes for product registrations and approvals of the therapeutic products.(Objective 7: Creation of a knowledge base). We will create a "Knowledge Base", which will describe our concept and its application in therapy of HLB and other plant diseases. Specifically, we will store, integrate, and analyze 'omics and microbiome data as they relate to HLB therapy using the HTH peptides and chimeras. Additionally, we will create training modules focusing on the "Knowledge Base" and hold an annual workshop for students. Finally, we will prepare an information package describing the foundation of our technology, its application in HLB therapy, and its relevance to sustaining the US citrus industry. Both the training modules and the information package will be developed and regularly updated using inputs from all the CAP team members.(Objective 8: Education and Outreach). We will incorporate two important functions: (i) training of students, technicians, and extension professionals on the "Knowledge Base" and (ii) holding onsite and online meetings and workshops to communicate to the growers and stakeholders the scope, deliverables, and impact of our CAP. We will gather feedback from the growers and stakeholders to improve performance of the CAP.
Target Audience
Citrus growers and stakeholders, representatives from industries and from the federal regulatory agencies, consumers, citrus researchers and other plant disease researchers, and public.
Changes / Problems
Nothing Reported
Training & Professional Development
The creation of a knowledge base on HLB biology and therapy (Objective 3) provided the source materials (i.e. novel concepts and technical approaches) for training of the students, post-docs, and techs, associated with the project team. The execution of the training was performed under education and outreach (Objective 5). Education component involved training of students, post-doc, and techs and allowing their direct involvement in the project. At the NMC, Elena Sineva led the work on exosome delivery system. Mikhael Sinev was involved in the production of recombinant chimeras. Liza Ngyuen was involved determining the activity of citrus-derived peptides. Narattam Sikdar worked on extracting protein chimeras from transgenic plants and participated in field studies. Supratim Basu (as a NMC consultant) helped in conducting field and laboratory efficacy studies. Please see the reports from other Co-PIs for the role of other students, post-docs, and techs from their participating organizations.
Dissemination Streams
Education and outreach (Ed Stover, UFL and the project team) Education component involved training of students, post-doc, and techs and allowing their direct involvement in the project. At the NMC, Elena Sineva led the work on exosome delivery system. Mikhael Sinev was involved in the production of recombinant chimeras. Liza Ngyuen was involved determining the activity of citrus-derived peptides. Narattam Sikdar worked on extracting protein chimeras from transgenic plants and participated in field studies. Supratim Basu (as a NMC consultant) helped in conducting field and laboratory efficacy studies. Please see the reports from other Co-PIs for the role of other students, post-docs, and techs from their participating organizations. The outreach component involved engaging the citrus stakeholders. First, project news and progress were posted on various websites and in national and international symposia. Second, Ed Stover (Co-PI) met with the stakeholders on regular (monthly or quarterly) basis to discuss the current development in the field of HLB disease therapy vis-Ã -vis our contributions through this project. Finally, the stakeholders were part of the advisory board, which also included leading researchers in the field of plant disease biology and therapy. The advisory board played a key role in making sure that the project was on track in meeting the deliverables appropriately and on time.
Next Reporting Steps
Follow on field studies.
Target Audience
Citrus growers and stakeholders, representatives from industries and from the federal regulatory agencies, consumers, citrus researchers and other plant disease researchers, and public.
Changes / Problems
Updated Project Goals (Participating Team Members) Design and Laboratory Testing of the Host-derived Peptides and Chimeras (Participating Team Members: Goutam Gupta, NMC; Pankaj Trivedi, CSU; Madhura Kunta, TAMUK) Greenhouse and field efficacy studies ((Participating Team Members: Ed Stover & Joseph Krystel, USDA-ARS, Ft. Pierce, FL; Madhura Kunta, TAMUK; Pankaj Trivedi, CSU, CO; Goutam Gupta, NMC) Creation of a knowledge base for HLB therapy (Participating Team Member: Lukas Mueller, BTI, Cornell, NY) The EPA regulatory processes (Participating Team Member: Michael Braverman, IR4, Rutgers, NJ) Education and outreach (Participating Team Member: Ed Stover, UFL and the project team) Summary: Our team introduced the concept and successful applications of "Precision Design" for understanding biology and developing therapy of plant and human diseases [1-5]. "Precision Design" is executed in the following steps. First, genome sequences of the pathogens and host-pathogen biology are studied to identify the root cause of infection/disease due to a given pathogen. The root cause is often a pathogen membrane component or a protein that is critical to pathogen survival and onset/progression of infection. Second, the host sequence and immune system are surveyed to identify a host protein of the immune system that initially evolved to eliminate the root cause but rendered inactive by pathogenic resistance. Third, the identified host protein is modified to overcome pathogenic resistance, rescue its activity to clear the pathogen while ensuring the modified host protein enhances host immune defense during infection and protects the beneficial host microbiome. Finally, replicated experiments are performed to validate the predicted efficacy of the modified host protein in curing the disease and restoring the health of the host including enhancing immune defense and protecting beneficial microbiome. "Precision Design" was applied to deliver practical solutions for HLB treatment and prevention. First, two pathogenic elements, namely lipid membrane and outer membrane protein (OMP), on the HLB causing bacterium, CLas, were identified as the root causes of HLB. Second, host peptides were designed to rupture the membrane and clear CLas while overcoming bacterial resistance. The activity of the designed host peptides was further increased by linking them to modified host proteins that specifically recognized CLas OMP. Third, laboratory, greenhouse, and field efficacy studies were performed on the host-derived peptides and (peptide-recognition protein) chimeras to validate their role in curing the disease and restoring the health of the host including enhancing immune defense and protecting beneficial microbiome.
Training & Professional Development
The creation of a knowledge base on HLB biology and therapy (Objective 3) provided the source materials (i.e. novel concepts and technical approaches) for training of the students, post-docs, and techs, associated with the project team. The execution of the training was performed under education and outreach (Objective 5). Education component involved training of students, post-doc, and techs and allowing their direct involvement in the project. At the NMC, Elena Sineva led the work on exosome delivery system. Mikhael Sinev was involved in the production of recombinant chimeras. Liza Ngyuen was involved determining the activity of citrus-derived peptides. Narattam Sikdar worked on extracting protein chimeras from transgenic plants and participated in field studies. Supratim Basu (as a NMC consultant) helped in conducting field and laboratory efficacy studies. Please see the reports from other Co-PIs for the role of other students, post-docs, and techs from their participating organizations.
Dissemination Streams
The concepts and the application tools developed for HLB therapy were communicated to the citrus researchers and stakeholders. Education component involved training of students, post-doc, and techs and allowing their direct involvement in the project. At the NMC, Elena Sineva led the work on exosome delivery system. Mikhael Sinev was involved in the production of recombinant chimeras. Liza Ngyuen was involved determining the activity of citrus-derived peptides. Narattam Sikdar worked on extracting protein chimeras from transgenic plants and participated in field studies. Supratim Basu (as a NMC consultant) helped in conducting field and laboratory efficacy studies. Please see the reports from other Co-PIs for the role of other students, post-docs, and techs from their participating organizations. The outreach component involved engaging the citrus stakeholders.First, project news and progress were posted on various websites and in national and international symposia.Second, Ed Stover (Co-PI) met with the stakeholders on regular (monthly or quarterly) basis to discuss the current development in the field of HLB disease therapy vis-Ã -vis our contributions through this project.Finally, the stakeholders were part of the advisory board, which also included leading researchers in the field of plant disease biology and therapy. The advisory board played a key role in making sure that the project was on track in meeting the deliverables appropriately and on time.
Next Reporting Steps
Follow on field studies. <br><br>
<br>What was accomplished under these goals? Please note that the changes/edit to Major Goals/Objectives are outlined in the Changes section of the report. For Objective 1 Three types of host-derived peptides are reported here: helix-turn-helix (HTH) peptides, alpha/beta peptides, and thionins. The HTH and alpha/beta peptides were designed to overcome the bacterial resistance faced by the individual helical and beta-stranded peptides that are naturally present in citrus immune system. In addition, the HTH and alpha/beta peptides were shown to be more active and less toxic than their original host counterparts. Similarly, citrus thionin was modified to increase activity and lower toxicity. A laboratory assay was set up to screen non-toxic HTH and alpha/beta peptides, which included the following steps. First, a molecular modeling method was developed to design libraries of HTH and alpha/beta peptides and predict their activity on gram-negative bacteria such as CLas [14-15]. Second, the peptides with high antibacterial activity were chemically synthesized. Third, the synthesized peptides were tested for their activity on gram-negative and culturable plant and human bacteria; note that CLas is not culturable in the laboratory [11]. Fourth, the peptides with high antibacterial activity were tested for toxicity against human cells and plant (citrus, grape, tomato, tobacco) leaves [11]. Finally, non-toxic and active peptides were tested for their ability to clear CLas from infected grapefruit and Hamlin leaves from the field [11]. This enabled us to select a small set of citrus derived HTH and alpha/beta peptides for greenhouse and field studies. Transgenic citrus expressing the modified thionin represented the 1st generation [13]. Two generations of transgenics were subsequently generated. The 2nd generation expressed a chimera in which citrus thionin was linked to a peptide that bound to the lipid membrane whereas each of the 3rd generation transgenics expressed one of four different chimeras: BT, TB, ST, and TS (wherein T = citrus thionin; B = bacterial permeability increasing/lipid binding protein; S= subtilisin protease designed to target the CLas OMP). Comparison of bacterial loads in transgenic and untransformed citrus leaves showed anti-CLas activity in the order of 3rd > 2nd > 1st transgenics. The RNA from the HTH and alpha/beta peptide treated and untreated (control) leaf samples were analyzed by RT-qPCR of selected 50 citrus genes to show that citrus innate immune defense was enhanced upon treatment during CLas infection. Metagenome sequencing of the DNA isolated from the leaf samples also demonstrated that the beneficial microbes were unharmed by the treatment. For Objective 2 For field trials in FL, an HTH peptide, 28P-2, was delivered by ordinary foliar spray, trunk injection, and foliar spray with laser etching. Laser etching appeared to be most effective in clearing phloem limited CLas with two sprays one month apart. An alpha/beta peptide, a/bP-1, was also delivered to infected grapefruit in TX by foliar spray with laser etching and CLas loads in leaves were monitored every two weeks for 4 months after a single application. After 4 months of the spray, the bacterial load in leaves dropped to 85% relative to the untreated control. We plan to improve the efficacy in clearance by increasing the number of applications to two (one in March and the other in August) and by using a newly discovered and alpha/beta peptide, a/bP-2 with higher anti-CLas activity than a/bP-1. 1st and 2nd generation transgenics have been in the field for 30 months in the environment of natural infection by psyllids. After 2 years, the 2nd generation transgenics showed complete CLas clearance whereas untransformed and 1st generation citrus showed little CLas clearance. Statistically significant data on plant health and fruit quality will be obtained from larger replicated field trials. The 3rd generations are now being planted in the field. The most noteworthy discovery was the grape exosome-based delivery of the anti-CLas chimeras. Typically, chimera genes are delivered by transgenic citrus. However, we demonstrated by greenhouse studies that exosomes encapsulated with the pEFF plasmid carrying a GFP reporter gene can be delivered to citrus to facilitate in planta protein expression as evidenced by the appearance of green fluorescence. The non-GMO exosome delivery also demonstrated CLas clearing activity of the TS chimera discussed above, in a greenhouse study in which the pEFF plasmid carried the TS chimera gene and the exosomes were delivered by a trunk infusion device. For field studies the surface of the pEFF-chimera encapsulated exosomes were decorated with proprietary peptide to facilitate penetration of the greasy leaves upon foliar spray. Field studies, so far, are showing positive trends towards efficacy of the TS chimera delivered by exosomes. Further field validation studies are underway. For Objective 3 Diverse datasets from 1 & 2 were stored, curated, and integrated. Analysis tools were developed to interpret the efficacy, immune response, and microbiome data. Diverse datasets from 1 & 2 were stored, curated, and integrated. Analysis tools were developed to interpret the efficacy, immune response, and microbiome data. For Objective 4 The active ingredients (i.e., therapeutic products) are the citrus-derived peptides and chimeras and the inactive ingredients are the delivery systems, i.e., foliar spray with laser etching and foliar spray exosomes-pEFF-chimera. A subset of toxicity data was collected under this project and so were the data for the product and delivery system formulation. Additional data will be collected with a different source of funding and completed by 2024. For Objective 5 Education component involved training of students, post-doc, and techs and allowing their direct involvement in the project. At the NMC, Elena Sineva led the work on exosome delivery system. Mikhael Sinev was involved in the production of recombinant chimeras. Liza Ngyuen was involved determining the activity of citrus-derived peptides. Narattam Sikdar worked on extracting protein chimeras from transgenic plants and participated in field studies. Supratim Basu (as a NMC consultant) helped in conducting field and laboratory efficacy studies. Please see the reports from other Co-PIs for the role of other students, post-docs, and techs from their participating organizations. The outreach component involved engaging the citrus stakeholders. First, project news and progress were posted on various websites and in national and international symposia. Second, Ed Stover (Co-PI) met with the stakeholders on regular (monthly or quarterly) basis to discuss the current development in the field of HLB disease therapy vis-Ã -vis our contributions through this project. Finally, the stakeholders were part of the advisory board, which also included leading researchers in the field of plant disease biology and therapy. The advisory board played a key role in making sure that the project was on track in meeting the deliverables appropriately and on time. <br><br><b>Publications</b><br>
Target Audience
Citrus growers and stakeholders, representatives from industries and from the federal regulatory agencies, consumers, citrus researchers and other plant disease researchers, and public.
Changes / Problems
Considering the urgency of finding a solution, our focus is shifted from transgenic (GMO) to non-GMO products. Specifically, we conducted field studies to demonstrate that the citrus derived protein chimeras expressed in transgenic citrus promise to show sustainable and robust HLB resistance. However, transgenic citrus requires 5-10 years to complete efficacy testing and even then, it is hard to receive consumer acceptance. Therefore, we switched to exosome (non-GMO) mediated delivery system for the chimeras that showed HLB resistance in transgenic (GMO) citrus. Preliminary studies showed that exosome (non-GMO) mediated delivery can achieve an efficacy similar to that obtained by transgenic route but it may take only 4-5 months to show the efficacy.?
Training & Professional Development
From the NMC, Elena Sineva, Narattam Sikdar, Liza Nguyen, and Mikhail Sinev were supported by the NIFA-CAP and were assigned to carry out specific tasks under this project. Supratim Basu (now at the Wash U) was involved in the beginning stages of this project while still at the NMC. Now Basu is continuing in this project as a NMC consultant. The NMC research group made significant contributions in the following areas: mechanism of action of host plant antimicrobial peptides, rational design of antimicrobial peptides and their applications in HLB treatment, roles of CLas effectors in HLB pathogenesis and disruption of citrus innate immune response, and exosome delivery systems for treatment of plant diseases. Jeongyun Choi (a graduate student of the CSU supervised by Pankaj Trivedi) and Jong Won Park (Research Assistant Professor at TAMUK supervised by Madhura Kunta) joined the NMC team to study the effect of the host derived a/b peptides on bacterial clearance and host innate immunity. Jeongyun Choi was recruited as a Ph. D student to work on the effect of the citrus derived peptides and chimeras on the homeostasis of citrus microbiome. Joseph Krystel, a USDA-ARS researcher, is being groomed to assume an independent role to carry out the work on the transgenic (GMO) and exosome mediated (non-GMO) delivery of the citrus derived chimeras for HLB treatment and prevention.
Dissemination Streams
Interested communities include: citrus growers and stakeholders, citrus industries and related organizations in the main citrus producing states (FL, CA, TX, AZ), researchers and consumers. Our team communicated mainly though Ed Stover (Lead, Education and Outreach of the CAP). Stover participated in meetings and proposed that the SCH website (Science for Citrus Health (ucanr.edu) ) have an additional Research Snapshot category providing one-page summaries of ongoing HLB-focused NIFA projects. He developed such Snapshots on the first for the Gupta CAP, project 2020-70029-33199) and on the current one and posted on Science for Citrus Health (ucanr.edu). Our team has submitted abstracts for the 2022 California Citrus Congress and the International Citrus Congress summarizing the Gupta NIFA CAP (project 2020-70029-33199) and an updated poster will be presented at the Conference and Congress. Stover has also served as a liaison with the Florida citrus industry, maintaining visibility and contacts as a credible citrus expert, discussing the Gupta NIFA CAP (project 2020-70029-33199) at 4 major Florida citrus grower conferences and attending zoom meetings of the Citrus Research and Development Foundation.
Next Reporting Steps
Objectives 1-3: Foliar spray with laser etching is planned in coming months for the field efficacy studies on the a/b peptides. Note that our previous studies (reported for the 1st year progress) showed effectiveness of foliar spray with laser etching for delivery of peptides in citrus. For field efficacy studies on the chimera, we have encapsulated a plasmid with the chimera gene in exosomes decorated with basic peptides for citrus leaf penetration. We have demonstrated that the spray of surface decorated exosomes carrying a GFP reporter on lemon leaves shows green fluorescence after 3-6 days following sprays. Therefore, for field efficacy studies, we will spray the surface decorated exosomes carrying the chimera gene. For this, we have constructed novel chimeras by linking the a/b peptide (CLas killer) with a recognition domain that specifically binds the CLas-OMP (OMP = outer membrane porin). Objectives 4-5: Genome wide transcription studies by RNA-seq will be performed to measure the changes in citrus mRNA and microRNA upon the treatment of a/b peptides in the detached leaf assay of infected citrus. mRNA and microRNA with statistically significant changes in the level of expression upon treatment will be validated by real-time qPCR. Immune response network upon treatment of infected citrus will be predicted emphasizing on the coupled PTI, ETI, SA, JA, ET networks involving pattern recognition receptors, signaling/transcription factors, pathogenesis-related (PR) defense genes. The predicted networks changes will be validated by measuring the levels of key pattern recognition receptors, signaling/transcription factors, and pathogenesis-related (PR) defense proteins. Similar genome wide 'omics will be performed to analyze the effect of greenhouse/field treatment of the a/b peptides or protein chimeras on the innate immune system of infected citrus. Microbiome studies will be performed to identify the effect of greenhouse/field treatment of the a/b peptides or protein chimeras on the bacterial and fungal communities in healthy and infected citrus. Finally, the effect of treatment of the a/b peptides or protein chimeras on the CLas clearance, citrus immune response, and homeostasis of the citrus microbiome will be correlated. Objective 6: Study design and results/data from the experiments on the effect of treatment of the a/b peptides or protein chimeras on the CLas clearance, citrus immune response, and homeostasis of the citrus microbiome will be stored, analyzed, and integrated. Objective 7: We will proceed with EPA approval of the most promising peptide and chimera for HLB treatment. Objective 8: We will keep updating stakeholders on our progress and seek feedback from the citrus growers, stakeholders/industries, researchers, and consumers. <br><br>
<br>What was accomplished under these goals? Objectives 1-3: (A) Constructed a library of citrus-derived a/b peptides; performed laboratory tests to screen peptides that are active on gram-negative bacteria and non-toxic to human and plant cells; chose a subset of the most promising non-toxic bactericidal peptides and tested their CLas-clearing activity on infected citrus (grapefruit and sweet orange) by a detached leaf assay; performed a greenhouse test on a/bP-1 (the most active) peptide by trunk infusion in infected Valencia, monitored bacterial load on the infected leaves for 2 months post-treatment, and demonstrated in planta CLas clearance. A US provisional patent was submitted on the citrus-derived a/b peptides in April 2022 and a manuscript has been submitted and is currently in review. (B) Three generations of transgenic citrus were constructed expressing: (1st generation) a modified tobacco Thionin (MThionin), modified to increase lytic activity and lower toxicity; (2nd generation) a chimera that links citrus MThionin with a bacterial recognition peptide from citrus which causes permeability in bacteria; (3rd generation) BT, TB, ST, and TS chimeras wherein T =citrus MThionin, lysis domain; B or T = BPI/LBP (B) or Subtilisin (S), CLas recognition domain. The 1st and 2nd generation Hamlin transgenic scions have been planted in the field, CLas clearance and plant health parameters (canopy volume, tree height, and trunk diameter) have been monitored for 2 years. Both the 1st and 2nd generation transgenics show better tree health than the untransformed Hamlin. However, the 2nd generation transgenics show better CLas clearance than the 1st generation transgenics and the untransformed Hamlin. Detached leaf assay was performed in the greenhouse on the 3rd generation ST transgenics infested by CLas+ psyllids. We observed a significantly higher CLas clearance in the 3rd generation ST transgenics (Ct value for a CLas marker = 38) than that in the untransformed Hamlin (Ct value for the same CLas marker = 32). Note that our design strategy predicts on an activity scale, 1st <br><b>Publications</b><br>
Target Audience
Citrus researchers specially those working on HLB, citrus growers, citrus industry personnel, regulators (federal and state and local), consumers of fresh fruit and juice, citrus workers in the field, packing and processing companies.
Changes / Problems
We made significant progress with the original team that included Innate Immunity especially in developing the HTH peptides as viable candidates for safe and efficacious HLB treatment. However, after April 21, Innate immunity ceased to be a partner in the project. This led us to stop working on the HTH peptides, which are owned by Innate Immunity as their proprietary property. In July 21 after obtaining USDA-NIFA approval, we started working on the novel citrus derived a/b peptides as candidates for HLB treatment, which avoided any infringement of Innate Immunity IP. As a matter of fact, we already identified one a/b peptide, which promises to be as good if not better than the HTH peptide 28P-2 we studied. The work is planned to discover additional a/b peptides that are superior to any HTH peptides studied so far. A new research component is added that involves exosome mediated (non-GMO) delivery of the citrus derived chimeras with CLas recognition and lysis domains. We noted ten- to fifteen-fold higher in vitro antibacterial activity of ST/TS chimeras over T. This prompted us to explore shorter delivery mode of the chimeras than achieved by the transgenic route. We have provided a proof-of-concept for exosome mediated delivery of the anti-CLas chimeras. For this, we extracted exosomes (100+45 nm in diameter) by ultracentrifugation of Grapefruit juice, then encapsulated a plasmid carrying the eGFP gene, delivered the plasmid to HEK cells, and showed expression of eGFP with a transfection efficiency comparable to that reported in the literature. We will conduct the following steps to improve the in planta efficiency of the anti-CLas chimera gene. First, we will combine ultracentrifugation and density gradient sedimentation to obtain more homogenous exosomes and more uniform grapefruit exosomes. Second, we will increase the encapsulation of the chimera-plasmid DNA (35PS) and the in planta transfection efficiency of the chimera possibly by coating the plasmid DNA with Arginine-rich peptides. Finally, we will test the efficacy of exosome mediated deliver by greenhouse studies in which will deliver the chimera-plasmid DNA (35PS) encapsulated exosome to infected citrus by leaf infiltration and monitor the CLas load over time. If the greenhouse studies are successful, we will perform field studies on the delivery of the chimeras encapsulated in exosomes and their transient expression using foliar spray with or without laser etching. This will accomplish shorter timeframe for HLB therapy than achieved by transgenic citrus.
Training & Professional Development
Note that Gupta (the PI) was jointly employed by NMC and Innate Immunity at the start of this project. As the CSO of Innate Immunity, he recruited and trained 3 researchers (Supratim Basu, Brian Jenkins, and Lucas Ribeiro) in protein design, microbiology, nanotechnology, immunology, and plant pathology as a part of this project. During this time, Innate immunity used the facility at the NMC Biolab, Los Alamos NM. In August 2020, Innate Immunity moved to Fort Collins CO to set up a new facility, which never materialized. Thus, the three employees decided to leave Innate Immunity in February 2021 because the company failed to provide them with any facility/resource to conduct their research. Interestingly, they quickly got new employment at reputed organizations because of the skill set they acquired while working on NIFA projects at the NMC. After severing ties with Innate Immunity and moving back to the NMC facility, Los Alamos in April 2021, Gupta recruited and trained five researchers (Wataru Nishima, Elena Sineva, Liza Nguyen, Narattam Sikdar, Mikhail Sineva), who have already made significant contributions toward accomplishing various project objectives. Madhura Kunta, Texas A&M (a Co-PI) recruited a post-doc, Jing-Won Park, to work in collaboration with the NMC group to study the effect of the peptides/chimeras on citrus immunity during CLas infection. Pankaj Trivedi, CSU (a Co-PI) recruited Jeongyun Choi (GRA) and Jing Yuan (post-doc) to work jointly with the NMC and Texas A&M groups on the relationship among HLB therapy, citrus immunity, and microbiome using genomics, biochemical, and machine learning tools. During the 1st year of this project, Joseph Krystel, USDA-ARS, FL has been trained to take over from Ed Stover to lead the transgenic work. Also, Neftali Panitz, BTI made the transition to replace Surya Saha to assist Lukas Mueller in developing the Knowledge Base.
Dissemination Streams
As an integral part of this project (Objective 8), the concept and tools behind HLB therapy are explained to the citrus industry personnel, growers, and stakeholders with the emphasis on how the therapy helps sustain a productive and profitable citrus industry in the US.
Next Reporting Steps
What do you plan to do during the next reporting period to accomplish the goals? Annual progress for Year 1 and plan for Year 2 (Objective 1: Year 1 progress). HTH peptides, 28P-2 and 36P-1, identified for field studies; demonstration of efficacy of 28P-2 in field trials in Florida;abpeptides with anti-CLas activity higher than 28P-2 identified for field trials. Isolation of exosomes from grapefruit, encapsulation of a plasmid carrying the GFP gene, and delivery of the GFP to a recipient cell. (Objective 1: Year 2 plan). Completion of field trials with theabpeptides with anti-CLas activity higher than that of the HTH peptides. Production of chimeras from HEK cells and transgenic tobacco and characterization of anti-CLas activity by detached leaf assay. (Objective 2: Year 1 progress). Construction and preliminary testing of citrus transgenics expressing anti-CLas ST and TS chimeras; computer design of citrus subtilisin (S) that specifically cleaves an extracellular loop of CLas-OMP; computer design of a chimera of an engineered S and anabpeptide with activity higher than thionin (T). (Objective 2: Year 2 plan). Greenhouse studies on ST and TS chimeras showing CLas clearance and suppression of HLB symptoms. Efficacy of genes encoding ST/TS and (S-abpeptide) chimeras delivered by exosomes and by conventional transgenic routes. (Objective 3: Year 1 progress). Preliminary data to show ACP killing by the ST/TS chimeras. (Objective 3: Year 2 plan). Quantitative studies to determine the mechanism of ACP killing by the ST/TS chimeras. Objective 3: Year 1 progress). Preliminary demonstration of augmentation of citrus innate immunity by the HTH peptides by gene expression analysis using qPCR. (Objective 4: Year 2 plan). Genome-wide transcriptome/proteome analysis of the leaf samples of the field citrus treated with theabpeptide(s). (Objective 5: Year 1 progress). Demonstration that the HTH peptide, 28P-2, does not affect citrus soil bacteria except those that are phylogenetically close to CLas. Similar studies on theabpeptide(s) are currently underway. (Objective 5: Year 2 plan). Collect and analyze citrus microbiome data from the field studies on HLB treatment and prevention. (Objective 6: Year 1 progress). A field book app for recording efficacy and safety data related to various HLB therapy, addition of transcriptome, proteome, and microbiome data. (Objective 6: Year 2 plan). Development of analysis and visualization tools the efficacy and safety data and identification of the best peptide/protein chimera for HLB treatment. (Objective 7: Year 1 progress). Obtained guidelineson how to consider the selection and development of the active ingredients that have the potential to impact regulatory requirements. (Objective 7: Year 2 plan). To decide on active ingredients, their manufacturing process, formulation and arrange meetings with regulatory agencies so that toxicology studies can begin. (Objective 8: Year 1 progress). Communicate the success in HLB treatment by citrus derived peptides. (Objective 8: Year 2 plan). Communicate the role and benefits of HLB therapy by peptides and protein chimeras. <br><br>
<br>What was accomplished under these goals? Major goals of the project have been modified since the time entered - seeChallenges/Problems. Objectives 1 and 4. Non-toxic anti-CLas HTH and ab peptide scaffolds, designed for HLB treatment, also augment citrus immunity. Field efficacy studies were performed on an HTH peptide, 28P-2, (at 20 mM dose) using three delivery methods: conventional foliar spray, trunk injection, and foliar spray with laser etching. Field studies were conducted in Florida in collaboration with Premier Citrus on their Grapefruit and Hamlin trees. CLas clearance and disease symptoms were monitored for 7 months during which the plants were treated 3 times. Leaf and root samples were collected and analyzed 30 days after the 1st and 2nd applications and 30 and 90 days after the 3rd application. Delivery of 20 mM 28P-2 by laser etching led to complete CLas clearance and suppression of HLB symptoms. Since July'21, we started our work on the ab peptide scaffolds in which antimicrobial a and b segments were covalently linked in the same molecule. We identified one such ab peptide (30P-3) that showed high anti-CLas activity and no toxicity. Next versions of ab peptides, predicted to show higher anti-CLas activity than 30P-3, have been chemically synthesized and soon be tested for their anti-CLas activity and then they will be used for field efficacy studies. We selected about 25 genes belonging to the CLas-affected PTI, ETI and plant hormone (SA, JA, ET) pathways and monitored their changes in expression in infected citrus leaves upon treatment with an HTH peptide (28P-2 or 28P-8). For this, we isolated RNA from the treated and untreated grapefruit leaves, collected from infected young trees in early and middle stages of infection. The change in the expression pattern of the selected genes indicated that citrus innate immunity is augmented in the treated infected leaves. Objectives 2 and 3. Transgenic chimeras prevent HLB and facilitates psyllid killing. We constructed BT, TB, ST, and TS chimeras by joining citrus Thionin (T) to citrus subtilisin (S) or bacterial permeability increasing/liposaccharide binding protein (BPI/LBP=B). Note that, T is selected as the CLas lysis domain whereas B/S is chosen as the CLas recognition domain. 17 different binary vectors were constructed and transformed into five commercially important citrus cultivars, generating approximately 1,000 transgenic lines. These generation transgenics (called the 3rd generation) are showing significantly strong results in detached leaf assay screening compared to the earlier designs, i.e., as demonstrated by the reduction of CLas transmitted to the leaf and by over 90% mortality in ACP after 7 days of feeding. Greenhouse trials for these plants are now underway and field trials will begin soon. The field trials will include assessments of ACP colonization and infectivity of ACP after feeding on transgenics, as the observed phenotypes may significantly reduce the ability/availability of ACP to continue spreading the disease. Further refinements are being made to the transgene components by replacing thionins with citrus derived non-toxic ab peptides with anti-CLas activity. Also, a modified citrus serine protease is engineered to specifically recognize the CLas outer membrane porin. This will provide the basis of future transgenics with higher anti-CLas activity and specificity. Objective 5. HLB therapy by citrus derived peptides maintains citrus microbiome homeostasis. We performed in vitro experiments to determine the impact of HLB treatment on the survival of bacterial and fungal isolates from the leaves and roots of citrus. We used the qPCR-based method to quantify the effect of HLB treatment on the 28 bacterial and 8 fungal families associated with the citrus "core" microbiota. Our results demonstrate that a HTH peptide, 28P-2, and an a/b peptide, 30P-1, at two concentrations (2.5mM and 5mM) had minor or no impact on the survival of over 90% of bacterial and fungal isolates. To evaluate the impact of 30P-1 on the phyllosphere microbiome, we sprayed 5.0 mM peptide on the leaves of Arabidopsis thaliana, Sugar beet and Nicotiana benthamiana. The a/b peptide 30P-1 has no impact on the microbial co-occurrence networks. Overall preliminary results strongly suggest that the therapeutic peptides have a non-significant impact on the plant-associated microbiome. Objective 6. Creation of a knowledge base for HLB therapy. The knowledge base involves collection and annotation of data from Objectives 1-5 as well as analysis and visual tools. This will include data from the laboratory, greenhouse, and field testing on the citrus derived peptides and protein chimeras and their effect on citrus innate immunity and citrus beneficial microbiome. An HLB therapy website (https://hlbtherapy.citrusgreening.org/) was set up to store testing data from all research groups. We developed a trait ontology for efficacy and safety data collected using the Fieldbook app on a portable notebook device. The traits will include: (i) citrus cultivar chosen for therapy; (ii) dose/frequency/length of treatment; (iii) disease severity/CLas load in treated and untreated plants; (iv) tissue (root/leaf) sample collected for analysis; (v) fruit/juice quality in treated and untreated plants. The HLB therapy website also includes data for the citrus derived peptides and protein chimeras: (i) sequence/3D structure/stability; (ii) anti-CLas activity; (iii) transcriptome/proteome expression in treated vs. untreated citrus; and (iv) effect on the homeostasis of citrus microbiome. Training modules are being prepared by the PI and Co-PIs on: "bacterial pathogenesis and plant innate immune response"; "design and topical/transgenic delivery of plant-derived peptides and proteins for disease therapy"; "measurement of transcriptome and microbiome and analysis of their role in immune defense and plant health" and "commercialization of products: from discovery to marketplace". This HLB therapy website will also be the primary outreach platform and all training documents and videos from Objective 8 will be hosted on this site in the future. Objective 7. Formulation and collection of data on the therapeutic peptides/proteins to meet the EPA guidelines. The broad classes of anticipated data requirements were obtained that relate to Product Chemistry, Human Health Effects, and Environmental/Non-Target effects. In addition, a product label will be developed to explain application, rate, frequency, application methods and pre-harvest interval. The registration package will also be accompanied by a tolerance exemption petition. Collection of field data for the peptides will be completed by the end of 2022. We expect to complete collection of the toxicity/safety data and submit the package for registration for the a/b peptides by 2023. For transgenic citrus this will involve the 3 agencies, APHIS-BRS, EPA and FDA. A rough estimate of the timeline for a decision for first generation data development is 2 years with a subsequent regulatory review of 2-3 years. This translates into completing registration through all three agencies in late 2027. Objective 8. Education and outreach. Ed Stover of USDA-ARS will take over from Monique Rivera of UC Riverside as the co-PI. Throughout his career Stover has worked closely with growers. The goal is to summarize plans for the CAP, relevance to commercial citrus production, and progress in project components. These materials will be shared through grower publications, on-line resources and at grower meetings. This will be done in collaboration with university extension personnel, as well as the CAP team. <br><br><b>Publications</b><br>