Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1090 - Immunology | 70% |
| 212 - Pathogens and Nematodes Affecting Plants | 4010 - Bacteria | 1160 - Pathology | 10% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1081 - Breeding | 10% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1160 - Pathology | 10% |
Huanglongbing (HLB) is currently the most destructive disease of citrus. There is an urgent need to develop therapies to treat HLB in existing groves and develop citrus with resistance to this disease. Plants can recognize diverse pathogens using immune receptors. Surface localized receptors can recognize conserved pathogen features and activate defense responses. Pathogen features are called pathogen associated molecular patterns (PAMPs); PAMP perception leads to pattern-triggered immunity. Pattern-triggered immunity induces immediate defense responses and protects against subsequent infection.The overall goals of the project are to harness pattern-triggered immune responses to control HLB and develop resistant citrus. PAMPs perceived in cultivated citrus will be used to develop therapies to boost citrus defense and induce HLB resistance in existing groves. We will identify and test pathogen PAMPs for their perception in citrus relatives with HLB resistance for use as markers to accelerate citrus breeding. We will employ state-of-the art biotechnological approaches to transfer stacks of immune receptors into citrus varieties to generate HLB resistant material. Furthermore, we will leverage multiple approaches to engage stakeholders and educate end-users (citrus industry, growers, breeders, residential citrus owners, and the scientific community) of our project findings. We will use a variety of multi-media outlets and connections with our extension team in Florida, Texas, and California. Taken together, the completion of these experiments will facilitate near term solutions for HLB management while accelerating breeding efforts for delivery of new disease resistant cultivars.
The general scientific methods to be employed for each objective as well as how the outputs will be evaluated is described below:Objective 1: Systematic identification of CLas and psyllid PAMPs. We will test known PAMPs as well as identify novel CLas and psyllid PAMPs to identify those that can be perceived in cultivated citrus and citrus relatives. CLas PAMPs will be identified by homology and molecular signatures of natural selection using comparative genome analyses. Psyllid PAMPs will be identified by mass spectrometry based approached. PAMPs will be tested for perception on leaf tissue using a luminometer-based assay to measure reactive oxygen species (ROS) production, a plant defense hallmark. The output will be identification of 15 new CLas and psyllid PAMPs.Objective 2: Synthesize and test PAMP compounds to boost HLB resistance. We will use the citrus-hairy root system to rapidly screen for the ability of perceived PAMPs to inhibit CLas proliferation. Cocktails of 3-5 PAMPs that can be perceived in commonly grown sweet orange varieties will be screened. CLas titers will be assessed by qPCR. The output will be identification of three distinct PAMP formulations with promise based on the hairy root system. Next, we will investigate the ability of these PAMP formulations to suppress CLas pre- and post-infection under greenhouse and field conditions. Foliar application will be examined, followed by trunk injection and nanoparticles as delivery vehicles. If successful, these experiments will provide a much-needed therapy to combat HLB in existing groves.Objective 3: Utilize recognition of CLas and psyllid PAMPs to accelerate citrus breeding. We will screen early rootstocks, hybrid populations, and backcross progeny that are segregating for HLB susceptibility using PAMPs that can be differentially percieved in parental lines PAMPs will be tested for perception on leaf tissue using a luminometer-based assay to measure ROS production. The output will provide an understanding of the correlation between PAMP responses and disease resistance. The output will also identify specific PAMPs whose perception can be used as markers for HLB resistance to accelerate citrus breeding.Objective 4: Generate novel PAMP recognition in cultivated citrus. In order to generate durable resistance, multiple immune receptors can be combined into one genetic background. PAMP immune receptors have been identified in multiple plant species and can be transferred between diverse plant families. We will generate novel pathogen recognition by staking different PAMP receptors into Valencia and Navel orange and test for HLB resistance. We will use a GAANTRY stacking system that facilitates transfers of gene stacks into the Agrobacterium T-DNA plasmid for simultaneous transfer into plants. Resulting plant material will be tested for HLB resistance under greenhouse conditions. The output will be the generation of plant material expressing two different stacks of PAMP receptors. A similar strategy will be used to identify PAMP receptor orthologs from citrus relatives with HLB resistance to transfer into sweet orange.Objective 5: Extension and Outreach. We will make sure information concerning efforts to utilize pattern-triggered immunity for HLB control are understood by stakeholders and that they are able to use the information in strategic planning for the future of citrus production. We will use translational research methodology in which the outreach specialist provides the interconnection between the basic research and the end-users. The effectiveness of our strategy will be quantified by the use of pre- and post-evaluation surveys. We aim to provide key decision-makers with the information they need to evaluate the potential for PAMP therapies in state-specific HLB management strategies. Information will be presented in person and online. A workshop will be developed on advanced approaches for introduction of specific traits into citrus that will be held as a satellite meeting in conjunction with an industry research organization (Citrus Research Board or Citrus Research and Development Foundation).
Target Audience
The target audience for this project includes scientists, regulatory agencies, members of the citrus industry, and the general public. We have engaged the scientific community through presentations at scientific meetings and peer-reviewed publications. We have engaged members of the citrus industry through workshops and presentations. We have engaged the general public through presentations and general publications focused on biotechnology in combatting HLB.
Changes / Problems
Nothing Reported
Training & Professional Development
All team members and postdocs had opportunities to present at multiple scientific conferences and workshops such as the Annual Meeting of the American Phytopathological Society, ASM Microbe, the International Society for Plant-Microbe Interactions, and the International Society for Citrus Huanglongbing and Phloem-Colonizing Bacterial Pathosystems (IS-CHPP). Each group was present at IS-CHPP and we had a separate half-day meeting with all project members in 2023. Scientists/technicians working on this project have received training in sample collection, processing, data analyses, and standardization of assay conditions. Graduate students (Jessica Franco and Jessica Trinh), as well as one postdoc (Tania Toruno), have visited UC Riverside and trained a total of five technicians in utilizing PAMP protocols to assess ROS response in citrus relatives to facilitate citrus breeding. Two PhD students in Coaker's lab graduated and obtained positions as postdocs or in industry, one postdoc obtained a permanent position in industry. All scientists trained had one-on-one mentoring with their respective PI or co-PIs as well as mentoring from the group when presenting research results.
Dissemination Streams
Throughout this project, we have engaged the citrus industry and the public. We have shared research progress in real time. Multiple extension seminars were presented to citrus growers in California, Florida, and Texas. Team members engaged with citrus industry stakeholders by participating in board meetings and individual meetings (Texas Citrus Pest and Disease Management Corporation, Texas Citrus Mutual, Florida Citrus Mutual, Southern Gardens Citrus, National Plant Diagnostic Network). Research Results were presented annually at UC Riverside's citrus day event to scientists, growers and extension personnel. Specific dissemination, as mentioned above, has been reported for progress reports. Included below are how results have been disseminated during the last year of the project: Two seminars were presented to citrus growers by Nian Wang in 2023. Ramadugu presented a talk about HLB mitigation efforts in Riverside at the UCR Citrus day event, March 2024. 100 people attended
Next Reporting Steps
Nothing Reported
Target Audience
The target audience for this project include scientists, regulatory agencies, members of the citrus industry, and the general public. We have engaged the scientific community through presentations at scientific meetings and peer-reviewed publications. We have engaged members of the citrus industry through workshops and presentations. We have engaged the general public through presentations and general publications focused on biotechnology in combatting HLB.
Changes / Problems
Nothing Reported
Training & Professional Development
Team members and postdocs had opportunities to present at multiple scientific conferences and workshops such as the Annual Meeting of the American Phytopathological Society Caribbean Division (APS-CD) and International Society for Citrus Huanglongbing and Phloem-Colonizing Bacterial Pathosystems (IS-CHPP). Each group was present at IS-CHPP and we had a separate half-day meeting to discuss progress on the project and outline future steps. Scientists/technicians working in this project have received training in sample collection, processing, data analyses, and standardization of assay conditions. One graduate student, Jessica Trinh from the Coaker group has trained technicians in Ramadugu laboratory and conducted collaborative research with the Ramadugu group at UC Riverside. A total of five technicians were trained in Riverside in utilizing PAMP protocols to assess ROS response in citrus relatives. Three recent graduates and two technicians were exposed to conducting molecular assays using leaf discs, synthetic MAMPs and recording the response using a plate reader.
Dissemination Streams
Nian Wang presented an extension seminar to citrus growers on 09/21/2022. Team members organized and presented a citrus greening workshop/symposium at the Annual Meeting of the American Phytopathological Society Caribbean Division (APS-CD). Feb 9-10, 2023, South Padre Island, TX. Kranthi Mandadi team members engaged with various citrus industry stakeholders by participating in board meetings or individual meetings (Texas Plant and Disease Management Corporation, TX and Southern Gardens Citrus, FL). Chandrika Ramadugu delivered a talk at the UCR citrus day event on March 3, 2023. Title: Citrus huanglongbing: finding solutions for disease management and long-term cultivation. The UCR citrus day event was organized by CAPCA. Concepts and results of Citrus Immunity project were presented (along with development of resistance). About 120 people (scientists, growers, extension personnel, citrus industry members) attended the event. Chandrika Ramadugu conducted a two-day workshop in Riverside, CA to discuss NIFA project progress on January 18th and 19th 2023. The meeting was attended by 47 people (33 in person and 14 by zoom; consisted of scientists, growers, advisory board members, Citrus industry leaders from CA, FL and TX). Sixteen scientists and six growers/citrus industry leaders talked about the research work or industry perspectives about the citrus HLB situation in USA. In her presentation, Ramadugu discussed about the Citrus immunity project to the select group of audience interested in developing solutions for citrus HLB.
Next Reporting Steps
Our primary focus during the next reporting period will be on testing stacked PAMP receptors in citrus for HLB disease control and continue to accelerate citrus breeding using PAMP perception. We have prioritized 22 new MAMP epitopes to be screened across six citrus genotypes in the next reporting period. The data generated will be useful in screening novel hybrids generated in Ramadugu's breeding program at Riverside. <br><br>
<br>What was accomplished under these goals? Plants use pattern triggered immunity to recognize components from pathogens and insects as non-self to trigger plant defenses and clear pathogen infection. This project aims to harness immune responses in citrus to control HLB. We have finished screening a diverse set of citrus genotypes to determine variation in immune perception that can be used to generate resistant plant material. In the past year, we have identified additional Liberibacter features present in outer membrane vesicles (Obj 1), analyzed additional PAMP for their ability to boost plant defense responses (Obj 2), completed proposed screens of CLas and psyllid PAMPs in diverse germplasm (Obj 3), and generated four new stacked immune receptor lines in citrus. Objective 1: Systematic identification of CLas and psyllid PAMPs. The goal is to identify components from pathogens and insects that can be perceived by plant immune receptors. These components are called pathogen associated molecular patterns (PAMPs). We have identified one Asian citrus psyllid secreted protein that triggers cell death in plants, thus activating plant immunity. We have identified three PAMPs from Liberibacter that are capable of being recognized by plant immune receptors, these receptors are being transferred into citrus (Objective 4). Because CLas resides in the phloem tissues, it is expected that phloem cell might recognize outer membrane proteins, outer membrane vesicle (OMV) proteins and extracellular proteins of Las to contribute to the immune responses. Because CLas has not been cultivated, we usedLiberibacter crescens(Lcr) as a surrogate to identify proteins in the OM fraction, OMV proteins and extracellular proteins by liquid chromatography with tandem mass spectrometry (LC-MS/MS). We observed OMVs of Lcr under scanning electron microscope, representing the first experimental evidence that Liberibacter can deliver proteins to the extracellular compartment. Our study provides valuable information regarding the biology ofCa.Liberibacter species and identifies many putative proteins that may interact with host proteins in the phloem tissues. Objective 2: Synthesize and test PAMP compounds to boost HLB resistance. The goal of this objective is to test the ability of PAMP formulations to boost HLB resistance when externally applied. The impact will be to produce a formulation that can be applied in the field to defend against HLB. Lipopolysaccharide (LPS), harpin, and chitin were selected and applied to sweet orange leaves by syringe infiltration and foliar spray in different concentrations. Defense-associated gene expression was transiently induced after treatment and reduced to basal levels shortly (24 hr after injection and 7 days after foliar spray). Defense-associated gene induction was also dosage-dependent. By comparing two different spray adjuvants affecting the ratio of PTI-associated gene induction, we found that Induce Nonionic Low Foam Wetter can promote defense induction. Furthermore, we treated HLB-infected sweet orange with PAMPs in the greenhouse by foliar spray and monitored CLas titer by qPCR. We were unable to reduce the titer of Liberibacter after treatment in sweet orange. We hypothesize this could be due to two reasons: (1) Liberibacter is already in the phloem in the citrus trees. Any activation of defenses may need to occur early on such as when the psyllid is feeding in order to stop transmission as the phloem is a protective environment for the pathogen. (2) Sweet orange cannot robustly perceive any Liberibacter PAMPs, so the application of this formation as a therapeutic may not induce a strong enough response for pathogen clearing. Given our promising results in other Objectives we have focused our efforts on other objectives. Objective 3: Utilize recognition of CLas and psyllid PAMPs to accelerate citrus breeding. The goal of this objective is to screen breeding material with CLas and psyllid PAMPs. The impact of this objective is the identification of phenotypic markers for a more rapid selection of promising citrus genotypes to accelerate citrus breeding. Evaluation of the breeding population for HLB disease response is a slow process. Having an initial and rapid phenotypic assay to determine promising genotypes will accelerate citrus breeding. Previously, we utilized six PAMPs to conduct replicated ROS assays in 86 citrus and related accessions belonging to different tribes of the subfamily Aurantioideae, identified receptors capable of recognizing flagellin and chitin, as well as citrus accessions that can recognize Liberibacter-specific PAMPs. We developed immune assays for diverse citrus genotypes and are using those in breeding efforts. We have identified 22 new MAMP epitopes to be screened across six citrus genotypes in the next reporting period. Objective 4: Generate novel PAMP recognition in citrus Objective 4 is the employment of state-of-the-art biotechnological approaches to control HLB. The impact will be to produce existing citrus cultivars that are resistant or tolerant to HLB and other diseases. We plan to stack PAMP receptors in citrus using biotechnologies. These receptors are from other plants that have the potential to control HLB but are not found in cultivated citrus. We noticed that when strong promoters drive the expression of multiple receptors this has a negative impact on citrus transformation/growth. Due to the effect of strong promoter driven receptor expression in citrus we have rebuilt four favorable stacks using promoters that produce lower rates of expression. We have generated transgenic citrus for those four stacks and will test them soon for resistance to psyllid feeding and Liberibacter proliferation. Objective 5: Extension and Outreach Nian Wang presented an extension seminar to citrus growers on 09/21/2022. Team members organized and presented a citrus greening workshop/symposium at the Annual Meeting of the American Phytopathological Society Caribbean Division (APS-CD). Feb 9-10, 2023, South Padre Island, TX. Kranthi Mandadi team members engaged with various citrus industry stakeholders by participating in board meetings or individual meetings (Texas Plant and Disease Management Corporation, TX and Southern Gardens Citrus, FL). Chandrika Ramadugu delivered a talk at the UCR citrus day event on March 3, 2023. Concepts and results of Citrus Immunity project were presented (along with development of resistance). About 120 people (scientists, growers, extension personnel, citrus industry members) attended the event. Chandrika Ramadugu conducted a two-day workshop in Riverside, CA to discuss NIFA project progress on January 18th and 19th 2023. The meeting was attended by scientists, growers, advisory board members, Citrus industry leaders from CA, FL and TX. <br><br><b>Publications</b><br>
Target Audience
The target audience for this project include scientists, regulatory agencies, members of the citrus industry, and the general public. We have engaged the scientific community through presentations at scientific meetings and peer-reviewed publications. We have engaged members of the citrus industry through workshops and presentations. We have engaged the general public through presentations and general publications focused on biotechnology in combatting HLB.
Changes / Problems
receptor stacks may have a limit (too many receptors stacked together can give yield issues). Therefore, we are working with existing stacks containing less than four receptors and we are finessing promoters to finely regulate receptor expression. PAMP treatment as a prophylactic does not work, but I think we can get around this with other positive results from the other objectives.
Training & Professional Development
Scientists in the project have received training in sample processing, data analyses, and standardization of assay conditions between laboratories. Two scientists from the Coaker group have cross-trained and conducted research with the Ramadugu group at UC Riverside. Three technicians in Riverside were trained in utilizing PAMP protocols to assess ROS response in citrus relatives. The three recent graduates were exposed to conducting molecular assays using leaf discs, synthetic MAMPs and recording the response using a plate reader. Graduate students and postdoctoral scientists also had the opportunity to present their research at laboratory meetings and virtual meetings. Several presentations at conferences were canceled in the summer and fall due to the pandemic. Multiple students and postdoctoral scientists are scheduled to present their research at scientific conferences this summer at the APS meeting in Pittsburg, PA (3 individuals). The PI and co-PIs are planning to attend (along with group members) the International Conference on Citrus Huanglongbing and Phloem-Colonizing Bacterial Pathosystems in Florida in September, 2022.
Dissemination Streams
Ramadugu: Presented a poster titled ""How do Citrus Plants recognize Pathogens?". UC Riverside Citrus Field day for Growers and Industry Members" organized by UC Riverside and CAPCA SoCal chapter.attended by about 60 growers, citrus industrymembers and scientists. March 10, 2022 in UCR, Riverside,CA. A Research Snapshot was developed and is available for download on the Science for Citrus Health website. Mandadi, K., Irigoyen, S., Ramasamy, M. (2021). Novel tools for antimicrobial testing and discovery of new HLB therapies. https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/Tools/Novel_tools_for_antimicrobial_testing_and_discovery_of_new_HLB_therapies/ McRoberts group presented their work on characterizing the collaboration network of the project to USDA-ARS and USDA-NIFA National Program Leaders, and members of the USDA-APHIS HLB MAC steering group at two webinars in early 2022. The Coaker group presented research goals and progress to K-12, undergraduate students and the general public: 12/7/22: Virtual career panel in collaboration with Young Women in Bio - about pursuing a career in life (plant/microbial) science 2/9/22: Los Altos High School STEAM Week talk - In the Middle: Between undergrad and doctorate, studying interactions between plants and microbes(career talk+ short research talk about HLB) 2/16/22: Virtual chat with local Cub Scouts - about citrus research & how we research plant diseases (virtual "lab tour"+ mini science talk) 4/23/22: Picnic Day 2022 atUC Davis - created demos for attendees to talk about the way we communicate science about agriculture/food and technology to your average audience (one of the demos included a section on HLB).
Next Reporting Steps
Our primary focus during the next reporting period will be on both stacking and testing PAMP receptors for HLB disease control as well as accelerating citrus breeding in the Ramadugu group using knowledge transfer. We now have several plant immune receptors in hand that are capable of recognizing Liberibacter and psyllids. We will focus on testing existing stacks and generating stacks of receptors in different combinations and under different expression levels (high/medium) and initiate tests for HLB resistance. extenstion and outreach future directions:The network analysis methods we are developing have been included in a new proposal for a Research Coordination Network proposal for the 2022 ECDRE funding round. If the proposal is successful the methodology will have broad application across the ECDRE program. There is interest from USDA-ARS National Program Leaders to adopt our approach in strategic assessment of their research commissioning process. When our analysis of the connectivity of the current project to the wider network of citrus research is complete we will assess opportunities for improving the flow of information from the project into translational research and technology development. <br><br>
<br>What was accomplished under these goals? There is a pressing need to protect citrus trees against HLB in existing groves and develop new commercial varieties that can resist HLB. Plants use pattern-triggered immunity to recognize components from pathogens and insects as non-self to trigger plant defenses and clear pathogen infection. This project will harness immune responses in citrus to control HLB and develop resistant germplasm. During the last year, we finished screening a diverse set of citrus genotypes to determine variation in immune perception that can be used to generate resistant plant material. We have generated five stacks of immune receptors, have generated two stacks in citrus and are currently optimizing additional stacks. We have also identified three immune receptors that can recognize the HLB pathogen and are using these for stacking as well. These results identify specific genes that can be used in citrus varieties to confer resistance to HLB. Objective 1: Systematic identification of CLas and psyllid PAMPs. We have completed this objective with promising results. The goal is to identify components from pathogens and insects that can be perceived by plant immune receptors. These components are called pathogen associated molecular patterns (PAMPs). We have identified one Asian citrus psyllid secreted protein that triggers cell death in plants, thus activating plant immunity. We have identified three PAMPs from Liberibacter that are capable of being recognized by plant immune receptors, these receptors are being transferred into citrus (Objective 4). Objective 2: Synthesize and test PAMP compounds to boost HLB resistance. The goal of this objective is to test the ability of PAMP formulations to boost HLB resistance when externally applied. The impact will be to produce a formulation that can be applied in the field to defend against HLB. Cultivated sweet orange can weakly perceive flg22 and chitin PAMPs. We have tested these PAMP treatments in both citrus hairy roots as well as in infected trees in Florida. In both cases, we were unable to reduce the titer of Liberibacter after treatment. We hypothesize this could be due to two reasons: (1) Liberibacter is already in the phloem in the infected hairy root or citrus trees. Any activation of defenses may need to occur early on such as when the psyllid is feeding in order to stop transmission as the phloem is a protective environment for the pathogen. (2) Sweet orange cannot robustly perceive any Liberibacter PAMPs, so the application of this formation as a therapeutic may not induce a strong enough response for pathogen clearing. Given our promising results in other Objectives we have focused our efforts in these areas. Objective 3: Utilize recognition of CLas and psyllid PAMPs to accelerate citrus breeding. The goal of this objective is to screen breeding material with CLas and psyllid PAMPs. The impact of this objective is the identification of phenotypic markers for a more rapid selection of promising citrus genotypes to accelerate citrus breeding. In parallel projects, we are crossing citrus cultivars with HLB-resistant accessions to introgress disease resistance traits into the breeding progeny. Production of a large number of progeny is desirable since the aim of the breeding projects is to generate novel accessions with disease tolerance/resistance and acceptable horticultural traits. Evaluation of the breeding population for HLB disease response is a slow process. Having an initial and rapid phenotypic assay to determine promising genotypes will accelerate citrus breeding. We have utilized six PAMPs to conduct replicated ROS assays in 86 citrus and related accessions belonging to different tribes of the subfamily Aurantioideae. We have now identified citrus relative accessions with the ability to perceive CLas csp22 (Liberibacter PAMP) and chitin (insect PAMP). Perception of these two PAMPs is currently being used in screening breeding materials. Our study has generated information on plant responses to pathogen-derived receptors in Citrus and related plants. This knowledge can be utilized for identification of putative disease resistant progeny. Objective 4: Generate novel PAMP recognition in citrus Objective 4 is the employment of state-of-the-art biotechnological approaches to generate HLB-resistant citrus. The impact will be to produce existing citrus cultivars that are resistant or tolerant to HLB. To accomplish this goal several components are required. First is knowledge of resistance genes from other plants that have the potential to control HLB but are not found in cultivated citrus. In the past year, we have prioritized three new receptors that can recognize PAMPs from Liberibacter and one that can recognize damage induced by psyllid feeding that are not found in citrus or sexually compatible citrus relatives. We have isolated, cloned, and sequence confirmed six different receptors. Four different receptors were modified to provide strong expression in citrus and stacked together using the GAANTRY system for transformation. We have successfully transformed two independent stacks into citrus. We are also transforming potatoes with a subset of stacks in order to obtain faster results as Candidatus Liberibacter solanacearum (CLso) can be used as a surrogate system and inform our approach in citrus. Our initial results have identified the transfer of one receptor that results in enhanced resistance to CLso in potatoes (reduced bacterial titer, enhanced tolerance to the pathogen), indicating this approach is likely to work. Wenoticed that when strong promoters drive the expression of a large numbers of receptors (>4) this has an impact on potato growth with similar results reported (above) for citrus transformation Due to the effect of strong promoter driven PAMPs expression in both potato and citrus we have rebuilt the favorable stacks using promoters that produce lower rates of expression. We are testing these new stacks for resistance to psyllid feeding and Liberibacter proliferation. Objective 5: Extension and Outreach Team members organized and presented a citrus greening workshop/symposium at the Joint Annual Meeting of the American Phytopathological Society Caribbean Division (APS-CD) and Entomological Society of America South-Eastern Branch (SEB), San Juan, Puerto Rico. Its titled "Citrus greening or Huanglongbing (HLB): Strategies for mitigation and control." The goal was to provide an update on the HLB status and ongoing R&D efforts on new strategies and therapies to control the vector and the pathogen. We covered HLB epidemiology, insect vector management, RNAi, and new therapeutic screening and delivery systems to control HLB. Attendees: ~30.https://esa.confex.com/esa/2022seb/meetingapp.cgi/Session/38474 Published a Research Snapshot on new tools used in this project and were made available for download on the Science for Citrus Health website. Attendees: World wide webMandadi, K., Irigoyen, S., Ramasamy, M. (2021). Novel tools for antimicrobial testing and discovery of new HLB therapies. Science for Citrus Healthhttps://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/Tools/Novel_tools_for_antimicrobial_testing_and_discovery_of_new_HLB_therapies/ Team members engaged with citrus industry stakeholders by participating in board meetings or individual meetings (Texas Plant and Disease Management Corporation, TX and Southern Gardens Citrus, FL) McRoberts group produced a project impact evaluation questionnaire which was circulated to other project co-PIs to establish the knowledge baseline from which the project started in each key area of science. McRoberts group produced a social network analysis of research collaboration with the project PIs as its focus, using a literature snowball search process in which the PIs were used as the starting nodes to scrape literature sources for publications. <br><br><b>Publications</b><br>
Target Audience
The target audience for this project include scientists, regulatory agencies, members of the citrus industry, and the general public. We have engaged the scientific community through presentations at scientific meetings and peer-reviewed publications. We have engaged members of the citrus industry through workshops and presentations. We have engaged the general public through presentations and general publications focused on biotechnology in combatting HLB.
Changes / Problems
The past year has had multiple changes due to the COVID-19 pandemic. During the first six months of the project, scientists were either unable or severely impacted in their ability to perform research in person. At UC Davis and UC Riverside, we are only in the last several weeks at 50% research capacity on campus. In addition, we were restricted in hiring new personnel, including undergraduate researchers, at the start of the project as they would not be able to work in person. Due to these restrictions we focused some of our efforts on genome comparisons between different citrus varieties and relatives, which enabled us to identify and subsequently validate citrus immune receptors under objective 4. The PD (Gitta Coaker) and co-PDs prioritized individuals working on this project for early return to research and as a result, we are not behind in our research progress. Due to Covid19-imposed restrictions, planned outreach activities like dissemination of information to CA citrus industry personnel during UCR citrus day (generally held in January of every year in Riverside, CA) or fruit display events in Riverside and Lindcove were not possible. Therefore, we focused on dissemination through print, online, and virtual events. The co-PD Wenbo Ma left UC Riverside last year and has obtained a position in the United Kingdom. She was unable to take this funding with her and her portion of the research project (as well as one graduate student) transferred back to UC Davis under the direction of the PD Gitta Coaker. The PD and Wenbo Ma are still in frequent contact and Professor Ma is providing scientific input. In Feb 2021, Texas went through a severe winter storm, Uri, that devastated the citrus industry and orchards in South Texas. https://citrusindustry.net/2021/03/05/freeze-damage-update-on-texas-citrus/ Younger trees and new plantings were decimated. Older trees and groves survived the freeze but are severely damaged and are currently recovering. The regional ACP population is significantly down, and CLas status of the trees is perturbed/variable. Many of these citrus/ACP materials are needed for our research. This effects our experiments related to CLas bioassays, until the trees can fully recover and ACP/CLas infection status re-established. Therefore, bioassays are currently being performed in co-PD Wang's laboratory.
Training & Professional Development
This project has provided research training and mentoring opportunities to one undergraduate student, six graduate students, five postdoctoral scientists, and two technicians. Five of these trainees are Hispanic minorities. Scientists in the project have received training in sample processing, data analyses, and standardization of assay conditions between laboratories. Two postdoctoral scientists in the Thomson and Coaker labs have participated in 10 joint meetings over zoom and have cross-trained in each research group. Graduate students and postdoctoral scientists also had the opportunity to present their research at laboratory meetings. Multiple students and postdoctoral scientists are scheduled to present their research at scientific conferences this summer. Several presentations at conferences were cancelled in the summer and fall due to the pandemic. One graduate student has been learning programming skills needed for the social network analysis and text scraping and background theory on information analysis in preparation for modeling the flow of information from the empirical research to potential deployment in practice.
Dissemination Streams
Due to the pandemic, dissemination of results in the past year was conducted virtually. Results were disseminated to the scientific community through peer reviewed publications and scientific presentations. Results were disseminated to the citrus industry through publication in trade journals (e.g., Citrograph). Knowledge transfer to citrus growers was facilitated by the Citrus Research Board, public presentations on the project and strategies to combat HLB, and engagement with citrus industry stakeholders in California, Texas and Florida. News articles also featured progress related to breeding for disease resistance in citrus. See the products section of this report for specific examples.
Next Reporting Steps
We will complete Objective 1 by screening through any remaining PAMPs identified from Liberibacter and screen the psyllid protein eliciting cell death on different citrus relatives. We will initiate and monitor the promise of external application of PAMPs to restrict HLB development and Liberibacter titers pre- and post-infection in citrus under greenhouse and field conditions. We will generate transgenic plants expressing stacks of immune receptors for HLB resistance. We will also generate a subset of transgenic potato plants expressing the same stacks of immune receptors in order to have more rapid results for disease phenotyping and functional analyses (these experiments can be performed in parallel with citrus experiments. As part our work in a parallel project (co-PD C. Ramadugu), we have developed complete genome sequences for three citrus relative genera that have either complete resistance or high level of tolerance to CLas. During the next reporting period, a postdoctoral scientist appointed recently will conduct bioinformatics analysis of genome sequences of Microcitrus australasica, Microcitrus inodora and Eremocitrus glauca and identify novel genes that may have a role in pattern-triggered immunity. The condition change questionnaire will be circulated to project Co-PIs. The resulting data on changes will be extracted, coded and used for statistical analysis to quantify impact from the project. This work will be combined with strand of research on the social network around the project by projecting the pathways through which the knowledge from the project will propagate on the network. <br><br>
<br>What was accomplished under these goals? Huanglongbing (HLB) or citrus greening disease has severely impacted the US citrus industry in Florida and is spreading in Texas and California. There is a pressing need to protect citrus trees against HLB in existing groves and develop new commercial varieties that can resist HLB. Our approach to address this challenge is to increase the potential of citrus' own innate immunity. Plants use pattern-triggered immunity to recognize components from pathogens and insects as non-self to trigger plant defenses and clear pathogen infection. This project will harness immune responses in citrus to control HLB and develop resistant germplasm. During the last year, we have screened a diverse set of citrus genotypes to determine variation in immune perception that can be used to generate resistant plant material. Citrus relatives can perceive pathogen components and trigger defense responses. We have generated five stacks of immune receptors and are transferring them into two sweet orange varieties. We have also identified a novel receptor that can recognize the HLB-associated pathogen. These results identify specific genes that can be used in citrus varieties to confer resistance to HLB. Objective 1: Systematic identification of CLas and psyllid PAMPs.The goal is to identify components from pathogens and insects that can be perceived by plant immune receptors. These components are called pathogen associated molecular patterns (PAMPs). The impact will be to have a set of PAMPs in hand to induce disease resistance. We have identified one Asian citrus psyllid secreted protein that triggers cell death in plants, thus activating plant immunity. We have identified plants that can recognize Liberibacter specific cold-shock protein (CLas csp22) and flagellin (CLas flg22) PAMPs. We have also identified a novel receptor that can recognize Liberibacter flagellin and elicit a defense response. This receptor is being transferred into citrus (Objective 4). We have screened 38 Liberibacter genomes for candidate PAMPs and tested 36 candidates for recognition in a panel of citrus and tomato genotypes. We have not detected recognition of new Liberibacter PAMPs from this screen. We plan to alter the pipeline and add more genomes related to Liberibacters to finalize this Objective. Objective 2: Synthesize and test PAMP compounds to boost HLB resistance.The goal of this objective is to test the ability of PAMP formulations to boost HLB resistance when externally applied. The impact will be to produce a formulation that can be applied in the field to defend against HLB. Cultivated sweet orange can weakly perceive flg22 and chitin PAMPs. Perception induces the production of reactive oxygen species and defense gene induction. We are testing these PAMPs in different formulations to suppress HLB infection both in hairy root cultures and intact trees. Citrus hairy roots (HR) have intact vasculature and provide an approach for faster efficacy screening and functional studies of various PAMP and antimicrobial therapies. Ex vivo CLas-citrus HR cultures from sour orange and grapefruit were generated, validated and used to test the effects of two PAMPs in inducing defense in citrus. Both PAMPs induced the production of reactive oxygen species, but were not able to reduce the titer of CLas in hairy roots. This suggests that the PAMP treatments (or the triggered ROS) alone may be insufficient as a therapeutic to lower CLas in an infected citrus tissue. Because citrus hairy roots are a proxy for plant experiments, we have also initiated field experiments in Florida to test the effect of pre and post-treatment of recognized PAMPs in restricting CLas titer or HLB development. We anticipate that activation of defense responses should result in decreased HLB development, since PAMP recognition leads to activation of defense and systemic acquired resistance. Recently, transgenic citrus expressing NPR1, which activates defense were shown to exhibit HLB resistance (PLOS ONE 10(9):e0137134). Objective 3: Utilize recognition of CLas and psyllid PAMPs to accelerate citrus breeding.The goal of this objective is to screen breeding material with CLas and psyllid PAMPs. The impact of this objective is the identification of phenotypic markers for more rapid selection of promising citrus genotypes to accelerate citrus breeding. It is possible to produce 1,000's of citrus progeny each season, but it is not possible to evaluate them all. Having an initial and rapid phenotypic assay to determine promising genotypes will accelerate citrus breeding. We have utilized six PAMPs identified earlier by Coaker group to conduct ROS assays in a total of 106 citrus accessions. The PAMPS tried so far are: csp22, CLas csp22, chitin, flg22, CLas flg22 and peptidoglycan. Twenty four citrus accessions representing the various Aurantioideae sub groups were chosen for the study. Sixteen hybrids generated by crossing citrus with Australian citrus relative genera were included. 66 accessions belonged to other genera of the subfamily. We identified citrus relatives with the ability to perceive CLas csp22 and chitin. Perception of these two PAMPs has promise in screening breeding material. Objective 4: Generate novel PAMP recognition in citrus. Objective 4.1: Stacking immune receptors for transfer into citrus.The objective 4 goal is the employment of state-of-the art biotechnological approaches to generate HLB resistant citrus. The impact will be to produce existing citrus cultivars that are resistant or tolerant to HLB. To accomplish this goal a number of components are required. First is knowledge of esistance genes from other plants that have potential to control HLB but are not found in cultivated citrus. We identified a novel receptor that can recognize CLas flg22, an epitope of flagellin not recognized in most plants. We gained a greater understanding of how CLas regulates flagellin production in the psyllid and the plant. Seven genes, including the receptor for CLas flg22, were isolated, cloned and sequence confirmed. These genes were modified to provide strong expression in citrus and stacked together using the GAANTRY system for transformation. We have generated five different stacks of PAMP receptors from other plants and are transferring them into two sweet orange varieties. Four immune receptor stacks are being transformed into potato. This system will allow for more rapid evaluation and should provide important information about the ability of immune receptor transfer in conferring resistance to Liberibacter. Objective 4.2: identification of citrus immune receptors for transfer into sweet orange. We have performed comparative genomics of Valencia sweet orange, two mandarin varieties, and Lisbon lemon. We have identified two citrus PAMP immune receptors and validated their function. Objective 5: Extension and Outreach.Due to the pandemic, most extension and outreach in the last year was conducted virtually. Outreach occurred through knowledge transfer to citrus growers facilitated by the Citrus Research Board, public presentations on the project and strategies to combat HLB, and engagement with citrus industry stakeholders in California, Texas and Florida. News articles also featured progress related to breeding for disease resistance in citrus. Knowledge or products generated under Objectives 1-4 will progress towards use by passing along the network of scientific research and technology development. To understand this network better we are constructing a social network of collaboration among citrus researchers based on text scraping from the authorship (1139 authors) and addresses of published research articles (569 articles). We have also devised a simple questionnaire to assess the impact of the work carried out in Objectives 1-4 by using a self-assessment of condition-change for the project Co-PIs to complete. Condition changes will be recorded as changes in: technical capacity, knowledge, and technology. <br><br><b>Publications</b><br>
Target Audience
The first year of the project has mainly focused on generating scientific results and sharing scientific discoveries with the team as well as other scientists. Project updates and results were also periodically disseminated to stakeholders in Texas, Florida, and California as well as the broader scientific/citrus stakeholder community through publications and newsletters (e.g., Citrograph). Co-PD Neil McRoberts has been working to train a new graduate student for outreach efforts, which will involve interviewing all researchers involved in the project and distilling project findings into layperson terms for industry and the general public.
Changes / Problems
Due to the coronavirus pandemic, there have been some unforeseen changes. The entire research team was scheduled to meet in person on May 18, 2020 on the UC Davis campus for a two-day period to share progress. This meeting was cancelled and held virtually at the end of April. Unfortunately, this did not allow for as much interaction with junior scientists working on the project as an in-person meeting. We plan to reschedule the in-person meeting in 2021. The coronavirus pandemic has also hampered outreach efforts to target audiences as meetings and workshops through the summer of 2020 were cancelled. Depending on the duration of the pandemic, we may need to shift to an online platform to reach target audiences. Research has continued during this time, with some researchers shifting to more computationally intensive investigations. Although there are some delays for generating new transgenic lines, existing lines are being advanced.
Training & Professional Development
This project has provided research training and mentoring opportunities to two Hispanic minority undergraduate researchers and three postdocs that are Hispanic and African American minorities. Scientists in the project have received training in sample processing, data analyses, and standardization of assay conditions between laboratories. Graduate students and postdoctoral scientists at the University of California, Davis visited collaborator laboratories at the University of California, Riverside twice to exchange information and cross-train. Graduate students and postdoctoral scientists also had the opportunity to present their research in oral or poster format as documented in the publications section. One graduate student employed on the project has started to receive training in methodologies for social science research and computational methods.
Dissemination Streams
The first year of the project has mainly focused on generating scientific results and sharing scientific discoveries with the team as well as other scientists. Project updates and results were also periodically disseminated to stakeholders in Texas, Florida, and California as well as the broader scientific/citrus stakeholder community through publications and newsletters (e.g., Citrograph). Co-PD Neil McRoberts has been working to train a new graduate student for outreach efforts, which will involve interviewing all researchers involved in the project and distilling project findings into layperson terms for industry and the general public. Initial discussions have been held between the PD, Co-PDs, and the graduate student researcher to outline the approach to be taken to gather relevant data from the research team about their progress. The technology to be deployed in the project for HLB control was presented at the American Society for Horticultural Science conference.
Next Reporting Steps
Experimental: In the first year of the project, we have made strong progress on all objectives. In the second year, we will build upon this progress. We will focus on identification of immunogenic PAMPs from both CLas and D. citri for screening citrus genotypes, citrus relatives, and breeding populations. PAMP formulations will continue to be screened in both hairy root cultures and intact plants. All proposed immune receptor stacks will be generated and transformation will be initiated. Outreach: Co-PD Neil McRoberts and his team will interview the other project PD's to establish the baseline knowledge and technical accomplishments the project is expected to add. This baseline will allow us to measure benefit for the projects as milestones in the other objectives are accomplished. Furthermore, multiple researchers have extensive interaction with stakeholders at different events. Materials describing the project and results will be generated for dissemination at future public events. <br><br>
<br>What was accomplished under these goals? Huanglongbing (HLB) or citrus greening disease has severely impacted the US citrus industry in Florida and is spreading in Texas and California. There is a pressing need to identify therapies and treatment to protect citrus trees against HLB in existing groves as well as develop new commercial varieties that are tolerant or resistant to HLB in all production areas. Our approach to address this challenge is to increase the potential of citrus' own innate immunity. Pattern-triggered immunity is a robust immune system in plants. Activated by pathogen-associated molecular patterns (PAMPs), pattern-triggered immunity prevents infection from potential pathogens. This project will harness pattern-triggered immune responses in citrus to control HLB and develop resistant germplasm. The overall goals of the project are to utilize pattern-triggered immune responses for therapies to suppress HLB, as assays to accelerate citrus breeding, and for identification and transfer of immune receptor gene stacks to generate resistant citrus germplasm. We propose a systems-based, trans-disciplinary approach that will integrate both research and extension activities and leverages the strengths of our multidisciplinary team. Accomplishments on specific objectives of the project are included, below: Objective 1: Systematic identification of CLas and psyllid PAMPs. In order to identify novel PAMPs, 38 Liberibacter genomes were collected and annotated. A bioinformatics pipeline was implemented to predict immunogenic candidates using the following pipeline: identification of orthologous genes, determining codon-based substitution rates for each conserved (orthologous) gene, likelihood-ratio scores identified 88 genes with sites under positive selection, and further statistical analyses identified 24 proteins with residues having >95% probability of being positively selected. Using this pipeline, we have identified 24 candidate PAMPs. Seven peptides have been synthesized for functional characterization. The reactive oxygen species (ROS) assay was used to test synthesized candidate PAMP peptides in rough lemon, sweet orange, and Duncan grapefruit. No recognition was detected in these three genotypes. Additional citrus genotypes will be tested in the future. In order to identify novel psyllid PAMPs, 35 genes encoding putative Diaphorina citri (psyllid) secreted proteins were cloned and expressed in Nicotiana benthamiana. Protein expression was verified by western blotting and defense gene expression was detected using qPCR after expression of four D. citri proteins. One D. citri protein induced cell death after expression in N. benthamiana, indicating that it may be recognized by the plant immune system. Collectively, the experiments performed in this objective result in the identification of candidate PAMPs in both Liberibacter and D. citri that can be used to screen diverse citrus genotypes for perception and ultimately applied as formulations to boost HLB resistance. Objective 2: Synthesize and test PAMP compounds to boost HLB resistance. We are currently testing multiple PAMPs, including epitopes of flagellin (flg22, flg28), harpin, Ef-Tu, and cold shock protein (csp22) in different formulations to suppress HLB infection both in intact trees and in hairy root cultures. Because citrus hairy roots (HR) have intact vasculature and mimic in planta responses/environment for CLas, the CLas-citrus HR culture system provide a reliable approach for faster efficacy screening and functional studies of various PAMP and antimicrobial therapies. We generated ex vivo CLas-citrus HR cultures from sour orange and grapefruit. The CLas-citrus HR cultures were confirmed by diagnostic qPCR analysis for the presence and growth of CLas. The validated CLas-HR cultures are being used to test the effects of different PAMPs in inducing defense in citrus. We have identified defense elicitation for some features that are genotype dependent. Objective 3: Utilize recognition of CLas and psyllid PAMPs to accelerate citrus breeding. We have phenotyped 85 different genotypes, including cultivated citrus and related species, for the ability to perceive the following PAMPs: immunogenic epitopes of bacterial flagellin (including CLas specific flagellin epitopes), immunogenic epitopes of bacterial cold shock protein (including CLas csp22 epitopes), and chitin (present in D. citri). The majority of genotypes were able to perceive chitin, none elicited a robust ROS burst in response to CLas flagellin epitopes, 42 genotypes were able to reproducibly recognize general cold shock protein epitopes, and four genotypes were able to recognize CLas cold shock protein epitopes. While no cultivated citrus genotypes were able to perceive CLas csp22 epitopes, perception does exist in sexually compatible wild citrus species with existing breeding populations. Collectively, the experiments performed in this objective highlight diversity in immune perception in citrus relatives and identify particular genotypes that are sexually compatible with cultivated citrus that can perceive a Liberibacter specific PAMP, which has future promise for incorporation as a breeding marker. Objective 4: Generate novel PAMP recognition in cultivated citrus. This objective has two goals: (1) stacking verified immune receptors from other plants for transfer into citrus; (2) identification of citrus immune receptors for transfer into sweet orange based. We have begun generating stacks of PAMP receptors from other plants for transfer into cultivated citrus. We have isolated validated seven PAMP receptors and co-receptors from different species and cloned into GAANTRY vectors. A series of strong constitutive promoters were identified to drive immune receptor expression. Stacked gene cassettes were designed to minimize duplication of genetic elements to promote stable long-term genomic expression. In the last year, we have generated two stacks for bacterial and insect perception that are ready for transformation. In California, citrus transformation protocols have been established in the rootstock US802 and breeding cultivar pineapple orange. California scientists are working to establish transformation protocols for the elite cultivar Valencia and seedless cultivar Washington Navel. Florida scientists have established transformation protocols for elite cultivars and can initiate transformation of elite types. We have also made progress on identifying citrus immune receptors based on homology. Candidate receptors for one bacterial PAMP and one insect PAMP were identified and initial experiments indicate that these receptors are capable of PAMP recognition. Objective 5: Extension and Outreach. The first year of the project has mainly focused on generating scientific results and sharing scientific discoveries with the team as well as other scientists. Project updates and results were also periodically disseminated to stakeholders in Texas, Florida, and California as well as the broader scientific/citrus stakeholder community through publications and newsletters (e.g., Citrograph). We anticipate ramping up our extension and outreach efforts during years 2-4 to share results with all target audiences. Co-PD Neil McRoberts has been working to train a new graduate student for outreach efforts, which will involve interviewing all researchers involved in the project and distilling project findings into layperson terms for industry and the general public. Initial discussions have been held between the PD, Co-PDs, and the graduate student researcher to outline the approach to be taken to gather relevant data from the research team about their progress. The technology to be deployed in the project for HLB control was presented at the American Society for Horticultural Science conference. <br><br><b>Publications</b><br>