Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 999 - Citrus, general/other | 1020 - Physiology | 100% |
So far there is no cure for HLB nor is there any HLB-resistant citrus. Therefore, protection measures are urgently needed to save the infected trees and to protect the healthy ones. In this CAP, we will perform systems level studies to understand the battle between Liberibacter and citrus, i.e., Liberibacter attempting to propagate its lifecycle while citrus is trying to clear Liberibacter by mounting innate immune defense. We will use this knowledge to make the innate immune system of citrus stronger to win the battle against Liberibacter. This will be achieved by designing two classes of citrus or citrus-like proteins, i.e., one that directly targets and clears Liberibacter from the phloem and the other that prevents detrimental Liberibacter proteins from disrupting innate immune defense in citrus. We propose to develop delivery strategies for short-term (6 months), intermediate-term (1-2 years), and long-term protection (3-5 years) protection. With these robust and cost-effective tools, the growers will be able to save the infected trees and protect the healthy ones.
This CAP focuses on engineering novel innate immunity in citrus via the design and delivery of therapeutic proteins that facilitate rapid clearance of Liberibacter and inhibition of key steps in the development of Huanglongbing (HLB). Unlike small molecule antibiotics and RNAi, the proposed therapy is based on endogenous proteins derived from the citrus innate immune repertoire. The main goals are to develop: (i) a novel strategy for designing therapeutic proteins based on genome-wide studies on Liberibacter-citrus interactions and (ii) transgenic and non-transgenic delivery methods that offer short-, intermediate-, and long-term solutions for Huanglongbing (HLB) protection. This CAP specifically addresses the areas 1 and 4 of the call (page 6 in the call):Bacterial therapy systems that either kill or suppress Candidatus Liberibacter asiaticus (Liberibacter)Development of citrus scions and rootstocks resistant to, or tolerant of, Liberibacter that are suitable for a wide range of growing environments.
Objective 1 (Target Identification)(Team: Mikeal Roose, UC Riverside; Rakesh Kaundal, UC Riverside; Goutam Gupta, NMC/NMC; Geoffrey Waldo, NMC/NMC; Hau Ngyuen, NMC)For Liberibacter killer proteins, we will first identify the molecular entities on the Liberibacter membrane that can be targeted by specific citrus proteins. Two high priority targets are the outer-membrane proteins (OMPs) and the conserved lipopolysaccharide (LPS) core, which, as we have shown (see below), can be recognized respectively by citrus proteases and LPS-binding proteins (LBP). For the chimeras, lysis domains will be citrus linear or disulfide-bridgedantimicrobial peptides (AMP), which bind and rupture gram-negative bacterial membranes. We will perform structural and computational analyses to prioritize Liberibacter recognition and lysis domains. For the protein inhibitors of HLB, we will first identify the critical protein-protein interactions in Liberibacter-citrus interactions, for example the ones in the signaling pathways involving pathogen-associated molecular pattern triggered (PTI) and effector triggered signaling. PTI and ETI signaling constitute an important part of plant innate immune defense. Specific pathogen proteins (virulence factors and effectors) tend to disrupt one or more steps in the PTI/ETI signaling pathway, and inhibiting this disruption will be an effective strategy for HLB protection.Specific Tasks will include:Task 1a: (i) To analyze Liberibacter genomes to identify the conserved outer-membrane proteins (OMP) and lipopolysaccharides (LPS); (ii) To screen citrus genomes to identify citrus proteases that cleave Liberibacter OMP and LBP that bind LPS; (iii) To analyze citrus genomes to select citrus AMPs that lyse gram-negative bacteria such as Liberibacter.Task 1b: (i) To measure and analyze dual Liberibacter-citrus transcriptome at different stages of infection (early to late) to predict candidate interactions between Liberibacter effectors and citrus innate immune defense proteins; (ii) To validate the predicted protein-protein interaction pairs by a three-body split-GFP reporter assay.Objective 2 (Design and expression of therapeutic proteins for HLB Protection)(Team: Goutam Gupta, NMC/NMC; Hau Nguyen, NMC/NMC; Geoffrey Waldo, NMC/NMC)We will use structure-based algorithms to design Liberibacter killers and HLB-blockers and will express the therapeutic proteins in tobacco BY-2 cell lines.Specific Tasks will include:Task 2a: To design protein chimeras with recognition (e.g., protease and LBP) and lysis (AMP) domains to rapidly clear Liberibacter. We have already designed three chimeras: (i) tobacco Thionin-D4E1 chimera (tobacco Thionin is 70% identical in sequence to citrus Thionin; D4E1 is a citrus-friendly synthetic AMP); (ii) citrus Thionin-LBP1 chimera; and (iii) citrus Thionin-LBP2 (LBP1 and LBP2 are two citrus LBPs). Note that Thionins have both recognition and lysis functions.Task 2b. To design citrus protein mimics that bind and sequester Liberibacter effectors and inhibit their detrimental interaction with citrus proteins involved in plant innate immune defense.Task 2c. To use two-body split-GFP system to monitor expression therapeutic proteins in tobacco BY-2 cell expression system.Objective 3 (Delivery and efficacy testing of the therapeutic proteins)(Team: Ed Stover, USDA-ARS, Ft. Pierce, FL; William Belknap, Beltsville, MD; James Thomson, USDA-ARS, Albany, CA; Siddarame Gowda; University of Florida, Lake Alfred)We will develop short-, intermediate-, and long-term strategies for in planta delivery of the therapeutic proteins.Specific Tasks will include:Task 3a: To design lipid-based nanocapsules coated with citrus tristeza virus (CTV) capsid protein to deliver the therapeutic proteins; To test efficacy by determining Liberibacter level in greenhouse infected trees by qPCR at 0.5, 1, 2, 4, 8, and 12 months after initial delivery of the therapeutic proteins. Tree growth, health, and HLB symptoms will be monitored at 1, 2, 4, 8, and 12 months after the delivery of the therapeutic proteins.Task 3b: (i) To use CTV as a vector to express the therapeutic proteins for protection against HLB; (ii) To graft transmit this vector to express HLB therapeutic proteins in different infected citrus cultivars and monitor the efficacy as described in Task 3a.Task 3c: (i) To develop tools for more efficient and robust transgene expression (i.e., improved transcriptional control elements for optimal phloem-specific expression, CRISPR-like systems to repress, edit, or overexpress endogenous genes); (ii) To construct transgenic citrus lines expressing already designed Tobacco Thionin-D4E1, Citrus Thionin-LBP1, and Citrus Thionin-LBP2 chimera and test their efficacy in HLB protection. Note that we have already demonstrated that transgenic citrus (Carrizo) expressing Tobacco Thionin-D4E1 is protective against HLB. The other two (i.e., Citrus Thionin-LBP1, and Citrus Thionin-LBP2) chimeras have been designed to show higher efficacy than the Tobacco Thionin-D4E1 chimera.
Target Audience
This CAP focuses on engineering novel innate immunity in citrusviathe design and delivery of therapeutic proteins that facilitate rapid clearance of Liberibacter and inhibition of key steps in the development of Huanglongbing (HLB). Unlike small molecule antibiotics and RNAi, the proposed therapy is based on endogenous proteins derived from the citrus innate immune repertoire. The main goals are to develop: (i) a novel strategy for designing therapeutic proteins based on genome-wide studies on Liberibacter-citrus interactions and (ii) transgenic and non-transgenic delivery methods that offer short-, intermediate-, and long-term solutions for Huanglongbing (HLB) protection. This CAP specifically addresses the areas 1 and 4 of the call: Bacterial therapy systems that either kill or suppress CandidatusLiberibacter asiaticus (CLas) Development of citrus scions and rootstocks resistant to, or tolerant of, Liberibacter that are suitable for a wide range of growing environments. Objectives: Objective 1.Identify (a) Liberibacter genes/proteins and (b) citrus genes/proteins as potential targets for therapies to protect against HLB. Objective 2.Design and express HLB-protective proteins that either clear Liberibacter or block disease development. Objective 3. Deliver HLB-protective proteinsin plantafor short-, intermediate-, and long-term protection. What was accomplished under these objectives? Deliverables from Objective 1: Identification of the citrus defense genes/proteins that are suppressed due to CLas infection CLas-citrus protein-protein interactions that are detrimental to the host and beneficial to the bacterium 1(a): Determined expression of citrus genes during pre-symptomatic stages (2-24 weeks post-infection) and identified the innate immune genes that are suppressed. The important discovery was the suppression of citrus Thionins, which are known to be active against gram-negative bacteria such as CLas. 1(b): Performed dual transcriptomics (i.e., expression of CLas and citrus genes) during symptomatic and pre-symptomatic stages of infection and identified CLas-citrus gene/protein interactions that support the onset and progression of HLB. Also performed proteomic studies to determine the inhibitory interactions between important CLas effectors and citrus defense proteins that favor HLB infection. Deliverables from Objective 2: Candidates for CLas-killer proteins derived from the citrus proteome Candidates for HLB-blocker proteins also derived from the citrus proteome 2(a) The 1st generation CLas-killer consisted of a tobacco Thionin (with 70% identity with the citrus homolog), modified to increase activity and lower toxicity. The 2nd generation CLas-killer protein is a chimera consisting of citrus thionin and a citrus lipid-binding peptide (LBP); the Thionin again modified to to increase activity and lower toxicity and the LBP is added to increase the binding to CLas Liposaccharide (LPS) and subsequently the CLas killer activity. Indeed, preliminary studies reveal that the 2nd generation citrus transgenics is more efficacious in CLas killing than the 1st generation transgenics. We considered four types of the 3rd generation of anti-Liberibacter proteins, which are further improved in terms of CLas killer activity and are all derived from citrus. These are chimeras: Thionin-BPI/LBP, BPI/LBP-Thionin, Thionin-Subtilisin, and Subtilisin-Thionin. LBP/BPI and Subtilisin are CLas recognition proteins whereas Thionins are the CLas lysis proteins. We have shown that the synergy of lysis and recognition make the chimeras. 2(b). Designed HLB blocker proteins that inhibit the detrimental interactions between critical CLas effectors and citrus defense proteins. The HLB blockers are mimics of citrus defense proteins. Deliverables from Objective 3: Transgenic HLB-resistant citrus that are effective, consumer/environment friendly, and marketable Conferring HLB resistance in citrus by precision gene editing 3(a). Greenhouse studies are successfully completed for the 1st generation citrus transgenics. The field studies on the 1st generation citrus transgenics show efficacy in HLB resistance. The 2nd and 3rd generation citrus transgenics are ready for greenhouse efficacy studies. 3(b). A protocol for transient expression of Cas9/sgRNA in citrus has been developed to introduce specific mutations in a citrus gene. This method is expected to be applicable in introducing modified Thionin in citrus in a non-transgenic manner, which would be appealing to the industry/consumers.
Changes / Problems
Nothing Reported
Training & Professional Development
Two technicians Jefferson Shaw and Spencer Marshall (Stover Lab: USDA-ARS) were trained in the research associated with Objective 3(a). Not only did they make significant technical contributions but also did they participate in the conceptual development and refinement of the objective. Loan Huynh (Gupta lab: NMC) was trained in computational aspects of Objective 2(a, b) and made significant contributions and she has been promoted from a post-doc to a scientist. Supratim Basu (Gupta lab: NMC) was trained in experimental aspects of Objective 2(a, b) and made significant contributions and he has been promoted from a post-doc to a project coordinator. Shuijan Zhang, trained in NMC and USDA-ARS on constructing and evaluating transgenic citrus, is now a senior scientist with a biotechnology company.
Dissemination Streams
Our primary stakeholders were the citrus growers and citrus industries that are facing the HLB threat. We communicated to them the novelty and effectiveness of our technology. We also communicated with them how our technology will provide cost-effective and long-term protection against HLB and yet will pose no threat to human, plant, and environment. One of the prominent citrus industries is in the process of adopting our transgenic technology. It was also equally important to report that we communicated with the citrus and plant research community the foundation of our science that led to the technology of HLB protection. Similar science can be performed for other plant diseases of economic importance.
Next Reporting Steps
Nothing Reported
Target Audience
In this CAP, we aim to perform systemic studies to understand the battle between Liberibacter and citrus, i.e., Liberibacter attempting to propagate its lifecycle while citrus is trying to clear Liberibacter by mounting innate immune defense. During this reporting period, we have gained the knowledge of citrus immune system and will design strategies to make the innate immune system of citrus stronger to win the battle against Liberibacter. As our previous designed citrus thionin showing anti-Liberibacter activity in green house, we have planned the field trials with transgenic Carrizo and Hamlin. Also, one designed protein showed anti- Liberibacter activity in green house with hot psyllid innoculation. We are in the middle of production of large scale of proteins using tobacco then for spray treatment. Meanwhile, citrus thionin gene has been put into CTV (disarmed) and aim to express the gene in citrus phloem. The third generation of designed protein have been put into citrus (Carrizo and Hamlin, Valencia and Ray Ruby) and will test the efficacy against Liberibacter in greenhouse. With these robust and cost-effective tools/materials, the growers will be able to save the infected trees and protect the healthy ones.
Changes / Problems
Nothing Reported
Training & Professional Development
Research Associates, post-docs, lab assistants have been trained in Plant Physiology, Plant Pathology, Plant Biotechnology, Protein structure and design. They were send to attend professional conferences to present research progress and build up their network for future collaborations. The meetings opportunities are: Materials Innovation for Sustainable Agriculture (MISA) annual symposium, 2018 Plant and Animal Genome Conference, The 5th International Research Conference on Huanglongbing, Synthetic Immunity Symposium.
Dissemination Streams
Presented our results in national and international symposia, such as the 5th International Research Conference on Huanglongbing grower's meeting, Citrus Disease Subcommittee (CDS) of the National Agricultural Research, Education, Extension and Economics (NAREEE) Advisory Board. Our annual progress will be presented to the Advisory Board.
Next Reporting Steps
We will design the therapy strategies based on our finding on the targets of Liberibacter effectors in objective 1; We will product large scale of protein chimeras for leaf disc assay and topical treatment in objective 2; We will test the efficacy of Liberibacer clearance using detached leaf assay with hot psyllids before we propagate the transgenic plants to speed up the sections of events for greenhouse and filed testing and also set up the field trial on some constructs in objective 3. We will show the results of field trial on modified thionin transgenic plants (Carrizo and Hamlin). <br><br>
<br>What was accomplished under these goals? Objective 1: For objective 1a: Understanding citrus immune responses upon Liberibacter infection. We have gained deeper understanding of how citrus immune responses react to Liberibacter infection and found 5 signaling pathways were involved in immune networks. The specific genes and miRNAs were identified to be targets for designing therapy. For objective 1b: Identifying Liberibacter-Citrus interactions. We have characterized two Liberibacter intracellular effectors (P235 and Effector 3) and their targets, including several citrus defense proteins such as thinion, superoxide dismutase, aldehyde dehydrogenase, lipid transfer protein, Aspartyl protease, gycosyl hydrolase. Liberbacteria utilizes multiple mechanism (regulating specific citrus miRNAs and secreting effectors) to subvert the citrus innate immune system, thereby contributing to disease development; Design therapeutic proteins to target specific detrimental miRNAs and specific effectors for blocking HLB pathogenesis are underway. Objective 2: Design and production of therapeutic protein chimeras for rapid clearance of Liberibacter? A total of 8 therapeutic protein chimeras have been designed and one has been overexpressed in tobacco BY2 cells. This chimera showed anti-Liberibacter activity. Production of the others chimeras are underway and will be used for spray treatment. Objective 3. Deliver HLB-protective proteinsin plantafor short-, intermediate-, and long-term protection. Plasmids with a total of 8 therapeutic protein chimeras driving under two types of promoters were constructed and complete citrus (Carrizo, Hamlin, Ray Ruby and tobacco) transformation. Seven constructs have transgenic Carrizo plants confirmed and other varieties are under testing. ACP feeding on detached assay showed one transgenic construct (expressing thionin and lipid-binding protein domain) exhibited Liberibacter clearance. The modified thionin gene was put into CTV vector to express it in citrus phloem. <br><br><b>Publications</b><br>
Target Audience
The target audiences reached by our efforts are citrus grower, citrus industry, researchers, and public.
Changes / Problems
Nothing Reported
Training & Professional Development
Research associates and post-docs have been trained in plant physiology, plant transformation, nanotechnology, protein design 'omics studies
Dissemination Streams
Presented the results in national and international symposia. Manuscripts are being prepared. The annual progress will also be presented to the Advisory Board on March 13.
Next Reporting Steps
We will test thein plantaefficacy of protein chimeras containing thionin and lipid-binding protein domains. These chimeras have already been introduced into citrus and they are expected to show higher anti-Liberibacter activity than the modified thionin alone. Under objective 1, we will continue to identify the break-down of critical innate immune steps during Liberibacter infection and design/deliver proteins (called HLB blockers) that prevent such break-down. <br><br>
<br>What was accomplished under these goals? For Objective 1a: Understanding citrus immune response upon Liberibacter infection Determination of how early innate immune pathways in citrus are abrogated by Liberibacter infection thereby providing clues for developing therapy For Objective 1b: Identifying Liberibacter-Citrus interactions Experiments are underway. Objective 1C: Identification of critical Liberibacter-citrus protein-protein interactions critical to infection Initial citrus interactors to one effector p235 has been identified. Objective 2: Design and production of therapeutic protein chimeras for rapid clearance of Liberibacter A recombinant protein chimera has be overexpressed in tobacco BY2 cells. This chimera shows in planta anti-Liberibacter efficacy. Objective 3. In planta delivery of therapeutic proteins Transgenic sweet orange citrus is constructed. These transgenic express modified thionin and shows Liberibacter clearance and HLB protection. <br><br><b>Publications</b><br>