Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 4010 - Bacteria | 1100 - Bacteriology | 40% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1080 - Genetics (excludes breeding) | 40% |
| 212 - Pathogens and Nematodes Affecting Plants | 4010 - Bacteria | 1040 - Molecular biology | 10% |
| 502 - New and Improved Food Products | 999 - Citrus, general/other | 3080 - Sociology | 10% |
Huanglongbing (HLB) has caused unprecedented crisis to the citrus industry worldwide. In the US, HLB is associated with the phloem-colonizing, insect-transmitted bacterium Candidatus Liberibacter asiaticus (Las). Since no cures for HLB are available and true resistance in citrus has not been found, methodologies that effectively detect Las in large-scale set-ups and the development of resistant citrus varieties are urgently needed for a long-term solution.One of the most important virulence mechanisms utilized by bacterial pathogens is manipulation of host immunity and physiology through the function of secreted effector proteins. Las possesses the Sec secretion system, through which a variety of Sec-delivered effectors (SDEs) could be secreted into the citrus phloem. Research findings from our research team and other laboratories strongly suggest that SDEs are promising detection markers for robust HLB diagnosis and excellent molecular probes to identify key components required for HLB development.In this project, we will systematically characterize SDEs produced by Las isolated from HLB-infected citrus in the three major producing states, FL, CA and TX. A "core" (likely essential) SDEs produced by all Las isolates will be identified and further used to develop antibody cocktails for HLB detection. Robust HLB detection is essential for the timely removal of HLB-infected trees in order to: 1) prevent the spread of HLB in California; 2) implement therapies and nutritional management programs to extend the productive life of trees before they decline in Texas; and 3) reinforce the replanting effort in Florida. Furthermore, the core set of SDEs will be investigated to understand the molecular basis of their virulence functions. In particular, we will identify the citrus targets of these SDEs and then generate genome-edited citrus to achieve enhanced resistance to HLB. The availability of HLB-resistant citrus would represent a major milestone on combating HLB. Therefore, the outcome of this project will benefit citrus production and profitability over the long term.
This Standard Research and Extension Project (SREP) directly addresses critical stakeholder needs represented by two of the four priority areas identified by the Citrus Disease Sub-committee, i.e. "Development of methodologies that allow for the early detection of Las" and "Development of rootstocks resistant to, or tolerant of, Las".
Objective 1. Systematic analysis of Sec-delivered effectors from various Las isolatesWe will obtain full genome sequences of Las isolated from Texas, Florida and California using next-generation sequencing including Illumina and PacBio. Potential Sec-delivered effectors (SDEs) will be bioinformatically predicted from the genomes. The expression of SDEs will be determined from HLB-infected trees in the field and using greenhouse experiments.Objective 2. Antibody development targeting the core SDEs for HLB detectionWe will screen a library of synthetic, monoclonal antibodies for those that specifically bind to individual "core" SDEs with high affinity. These antibodies will be used as a cocktail for HLB detection by direct tissue imprint assay and enzyme-linked immunosorbent assay (ELISA).Objective 3. Identification of SDEs that contribute to HLB pathogenesisWe will characterize the impact of SDEs on citrus development and immunity using transgenic citrus expressing individual "core" SDEs. The transgenic citrus plants will be examined for susceptibility to Las infection and monitored for developmental phenotypes reminiscent to HLB symptoms.Objective 4. Development of genome-edited citrus with HLB resistanceWe will: 1) identify targets of SDEs using yeast two-hybrid screening and co-immunoprecipitation followed by mass spectrometry; 2) develop genome-edited citrus plants that interrupt SDE targets using a CRISPR/Cas9 system, which enables simultaneous manipulation of multiple targets; 3) examine HLB resistance of the genome-edited citrus. Note: genome-editing does not involve introducing foreign genes into citrus; genome-edited crops are not considered "GMO" and therefore expected to be better accepted by customers.Objective 5. Sociological analysis of consumer responses to genome-edited citrusWe will investigate how the use of biotechnology and the source of genetic modification influence consumer reactions to citrus products from genome-edited plants when the motivation for the use of biotechnology is to preserve a crop and no external genes are used.Objective 6. Extension and outreachWe will collaborate with communication strategists to recommend antibody-based HLB detection methods and to introduce the concepts and uses of genome-edited citrus through workshops, field day events, grower meetings, and websites.
Target Audience
The PD and co-PDs are from four research institutions in California, Florida and Texas, the three largest citrus-producing states. Three PD/co-PDs are affiliated with minority-serving universities. Therefore, this project reaches a large, diverse body of students and trainees through the teaching activities of the PD and co-PDs. Science-based knowledge has been delivered to a broad audience including researchers and citrus growers through extension and outreach activites, as well as presentations in scientific conferences (local, national and international) and grower meetings.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided excellent training opportunities to five postdoc, three Ph.D students, one research assistant, and one undergraduate student in all participating institutions. The participants have been exposed to experiments using interdisciplinary approaches, and gained experience on grant writing, manuscript preparation, and research presentation. The trainees also got the unusual opportunities to work with a multi-institutional team and directly communicate with growers. We were unable to train as many undergraduate students as expected due to the pandemic.
Dissemination Streams
Knowledge obtained from this project has been widely disseminated to a broad audience. We published an impressive 23 research articles and three Ph.D/MSc thesis, and presented our project in numerous national and international conferences, as well as grower meetings. The results have also been disseminated through seminars given by the PD/co-PDs and the participating researchers in universities and research institutions in the US and abroad. The PD was frequently invited to talk to the public about the current status of HLB through his interviews by local newspapers and national broadcast venues.
Next Reporting Steps
Nothing Reported
Target Audience
This report period is thefinal stage of this project with an one-yearnon-cost extension from Feb 1st, 2021 to Jan 31st,. 2022. As a whole, this project has reached a large, diverse body of students and trainees through the research, teaching and outreach activities of the PD and co-PDs. Despite the continuedimpact of the covid-19 pandemic in the current reporting period, science-based knowledge was delivered to a broad audience including researchers and citrus growers through research and extension publications, presentations in scientific conferences and grower meetings, as well as additional extension and outreach activities. The PD and co-PDs are from four research institutions in California, Florida and Texas, the three largest citrus-producing states in the US. Three PD/co-PDs are affiliated with minority-serving universities.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided excellent training opportunities to three postdoc researchers, onePh.D students, and one research assistants. All these students/trainees have been exposed to experiments using interdisciplinary approaches, and gained experience on grant/manuscript writing and giving scientific presentations. They have also got the unusual opportunities to work with a multi-institutional team and directly communicate with growers. We were unable to train undergraduate students this year due to the pandemic.
Dissemination Streams
Knowledge obtained from this project has been widely disseminated to a broad audience. In the past reporting period, our team has published 6 research articles in well-respected journals and several are in the pipeline to be published within the next few months.Although in person meetings were mostly canceled during 2020, results related to this project were reported in virtual meetings and invited seminars, as well as grower meetings. The team has frequent, direct communications with stakeholders and the general public about the current status and research efforts on HLB through interviews by local media and national broadcast venues.
Next Reporting Steps
Nothing Reported
<br><br>
<br>What was accomplished under these goals? The current reporting period (February2021 to January 2022) was an non-cost extension of the project. We are grateful that NIFAgranted this extension, which was due to the significant impact ofour research and outreach activities by the covid-19 pandemic since early 2019. Although our research and extension activites continued to be affected by the pandemic, we have accomplished all the proposed research and made our best effort to disseminate our results and support growers. Objective 1 - Systematic analysis of Sec-delivered effectors (SDEs) from various Las isolates. The goal of this objective wasto develop a comprehensive understanding of Las and its effector repertoire. During the past years, we have completed the first comprehensive Las transcriptome analysis using a optimized procedure to enrich the bacterial cells from infected citrus tissues. Using this protocol, we were able to detect transcripts from >90% of the Las genes (compared to ~30% from previous studies) and identify the differentially expressed genes in citrus vs in psyllids. We also define the most highly expressed Las genes in citrus, which represent essential biological processes when the bactrium colonizes the plant host. Finally, we analyzed the effector expression repertoires in citrus and in psyllids. These results have been published in the journal Phytopathology (De Francescoet al., 2022). We also analyzed the gene expression profiles of three selected SDEs and five papain-like cystein proteases (PLCPs) in field trees in Texas by quantitative RT-PCR. We found that variety, season, and Las titers impacted SDE and the protease gene expression. A manuscript reporting these results is currently under revision and will be published soon. Objective 2 - Antibody development targeting a core group of SDEs for HLB detection. The goal of this objective was to develop antibodies that detect individual core SDEs for HLB detection. This objective was completed in 2020. Objective 3 - Identification of SDEs that contribute to HLB pathogenesis. The goal of this objective was to examine the virulence activity of core Las SDEs and identify how they affect citrus physiology and promote HLB. Previously, we characterized the first virulence-related Las factor, SDE1 (CLIBASIA_05315), and showed that it suppresses the citrus papain-like cysteine proteases (PLCPs).We also characterized another SDE, SDE15 (CLIBASIA_04025), which suppresses programmed cell death (PCD) by directly targeting ACD2 (ACCELERATED CELL DEATH 2).Expression of SDE1 or SDE15 accelerated HLB disease progression, indicating that they are important virulence factors of Las. We continue to analyze additional SDEs even though this project is coming to an end. For example,40 SDEs have been expressed in citrus, tobacco or arabidopsis to examinehow they many affect disease susceptibility. Many of these SDEs have also been investigated using yeast two-hybrid screens to identify their host targets. This research will continue beyond the current grant period. Objective 4 - Development of genome-edited citrus with HLB resistance. The goal of this objective was to establish the CRISPR-based genome editing technology in citrus to enhance HLB resistance. We have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we generated homozygous mutants of the SDE1 and SDE15 targets including PLCPs and ACD2. These plants are currently being examined for HLB resistance. Furthermore,we have successfully adapted the adenine base editors (ABE) to edit the TATA box in the promoter region of the canker susceptibility geneLOB1from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogenXanthomonas citrisubsp.citri(Xcc). In addition, cytosine base editors (CBE) was successfully used to edit theacetolactate synthase(ALS) gene in citrus.ALS-edited plants were resistant to the herbicide chlorsulfuron. TwoALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. TheCas9gene was undetectable in the herbicide-resistant citrus plants. This indicates that theALSedited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression. Objective 5 - Sociological analyses of consumer responses to genome-edited citrus. The purpose of this objective was to understand public responses to genetically modified citrus, especially under the current threat from HLB. The budget for this objective was three years. Due to the pandemic, the activities were delayed. However, the first paper was published this year (Hu, Y., L. House, B. McFadden, and Z. Gao. "The influence of choice context on consumers' preference for GM orange juice). This study offers important insight into how different communication methods work to influence acceptance of CRISPR and GM products. A second paper (Hu, Y., L. House, and Z. Gao.How do consumers respond to labels for CRISPR (gene-editing)?)is currently under revision will be soonresubmitted to the journalFood Policy. Objective 6 - Extension and outreach. Although extension and outreach activities were still limited during this reporting period, we were able to meet multiple times with various grower groups,individual growers, and other stakeholders to assess HLB damage, provide guidance on disease management and discuss about project results about differences in technologies for HLB-resistance development and consumer acceptance of genome-edited citrus.Information on this project was presented online at the University of California Cooperative Extension Citrus Production Course for New Growers on August 26, 2021 and the Citrus Clonal Protection Program (CCPP) shared HLB related information with 1,002 users of the online CCPP citrus budwood ordering system. <br><br><b>Publications</b><br>
Target Audience
This project reaches a large, diverse body of students and trainees through the research, teaching and outreach activities of the PD and co-PDs. Despite the significant impact of the covid-19 pandemic in the current reporting period, science-based knowledge was delivered to a broad audience including researchers and citrus growers through research and extension publications,presentations in scientific conferences and grower meetings, as well as extension and outreach activities. The PD and co-PDs are from four research institutions in California, Florida and Texas, the three largest citrus-producing states in the US. Three PD/co-PDs are affiliated with minority-serving universities.
Changes / Problems
We applied for a one-year non-cost extension because our research and extension activities have been severely affected by the pandemic. This application has been approved. The previous PD Wenbo Ma has moved to the Sainsbury Laboratory in the UK. Professor Georgios Vidalakis is now the PD of the project.
Training & Professional Development
This project has provided excellent training opportunities to five postdoc researchers, four Ph.D students, and two research assistants. All these students/trainees have been exposed to experiments using interdisciplinary approaches, and gained experience on grant/manuscript writing and giving scientific presentations. They have also got the unusual opportunities to work with a multi-institutional team and directly communicate with growers. We were unable to train undergraduate students this year due to the pandemic.
Dissemination Streams
Knowledge obtained from this project has been widely disseminated to a broad audience. In the past reporting period, our team has published 10 research articles in well-respected journals including Molecular Plant, Molecular Plant-Microbe Interactions, Plant Physiology, and Molecular Plant Pathology. We have also published one extension publication on CRISPR technology in citrus improvement (N Wang, F Gmitter, M Dutt. Update on CRISPR research for citrus improvement in the HLB era. EDIS 2020). Although in person meetings have all been canceled, results related to this project were reported in virtual meetings and invited seminars, as well as grower meetings. The team has frequent, direct communications withstakeholders and the general public about the current status and research efforts on HLBthrough interviews by local media and national broadcast venues.
Next Reporting Steps
We are entering the last year of this project (with non-cost extension). Objectives 2 and 3 have been completed. We will focus on data analyses and manuscript preparation/publicationforObjective 1and 5. We will continue research activities in Objective 4, which aims to develop genome-edited citrus and test them for HLB response. Research activities in this objective representlong-term efforts that will continue even after the completion of this project. We will also continueoutreach activities and disseminating our findings to a broad audience. Objective 1 We will finish the RNA-seq analysis of Las genes that was initiated in the current reporting period. We haveobtained all the data and started the data analysis. We will soon start writing themanuscript for publication. In addition, we will finish the SDE gene expression analysis in the different instars of psyllid's life cycleand prepare a manuscript reporting these findings. Objective 4 Research in this objective is a rather long-term effort. We will continue optimizing CRISPR mediated genome editing in citrus and generating genome modified citrus plants for putative HLB susceptibility genes.For the transgenic citrus plants that have already been generated, we will continue the research of testing them for HLB resistance. Objective 5 We will complete the analysis of the survey focusing on customer willingness to pay for orange juice produced with CRISPR, GM, or no genetictechnology.We will also conduct outreach activities that have been affected in the past year. Our sociology studies offer support to customer acceptance of engineered citrus, which has important implications to growers' choice in growing GM citrus. Objective 6 We will continue our extension and outreach activities to advocate citrus HLB to a broad audience, communicate with growers on our new findings and technologies. <br><br>
<br>What was accomplished under these goals? In the current reporting period (January 2020 to January 2021), our research and outreach activities have been significantly affected by the covid-19 pandemic. Therefore, we applied for a one-year non-cost extension and we are grateful that NIFA approved our application. Despite the impact from the pandemic, we continued to make progress on all objectives. Objective 1 -Systematic analysis of Sec-delivered effectors (SDEs) from various Las isolates. The goal of this objective is to develop a comprehensive understanding of Las and its effector repertoire. During the past years, we have completed the genome analysis of 24 Las strains including 11 newly sequenced strains isolated from infected trees in CA, TX, FL and Mexico. We also determined SDE repertoires from each Las strain and determined the expression of 27 core SDEs in citrus vs psyllids using qRT-PCR. These results have been published in the journal Molecular Plant Pathology (Thapa et al., 2020). We conducted RNA-seq analysis to quantitative evaluate Las gene expression with a focus on SDEs. This approach remediates the issue that some SDEs couldn't be effectively amplified using qRT-PCR. RNA-seq will also provide accurate evaluation of relative expression levels between SDEs. This work has been slowed down by the pandemic and will be continuednext year. We analyzed the gene expression profiles of three selected SDEs and five cystein proteases in field trees in Texas by quantitative RT-PCR.We found that variety, season, and Las titers impacted SDE and the protease gene expression. A manuscript reporting these results is in preparation. We also conductedgreenhouse assays in infected grapefruit and orange trees infested with the Asian citrus psyllid to analyze the gene expression of SDE's in the different instars of psyllid's life cycle.This experiment will increase our understanding of the role of SDEs in pathogen transmission and survival in alternate insect-host. Objective 2 - Antibody development targeting a core group of SDEs for HLB detection. The goal of this objective is to develop antibodies that detect individual core SDEs for HLB detection. During the past four years of this project, we have generated polyclonal antibodiestargeting an effector SDE1 and developed enzyme-linked immunosorbent assay (ELISA) protocols. The ELISA has been extensively evaluated using thousands of field samples from TX and CA. Objective 3 - Identification of SDEs that contribute to HLB pathogenesis. The goal of this objective is to examine the virulence activity of core Las SDEs and identify how they affect citrus physiology and promoteHLB. Previously, we characterized the first virulence-related Las factor, SDE1 (CLIBASIA_05315), and showed that it suppresses the citrus papain-like cysteine proteases (PLCPs). These results were published in 2018(Clark and Franco et al., Nat Comm). In the past reporting period, we continued the characterization of SDE1. By using transgenic citrus expressing SDE1, we show that SDE1 can promote Las infection and accelerate HLB symptom development. We also did transcriptome analysis and found enhanced expression of senescence-related genes in SDE1-expressing citrus, linking accelerated senescence by SDE1 to HLB progression. These findings have been published in the journal Molecular Plant-Microbe Interactions. We thenexpanded our work on SDE1-PLCP interaction to investigate the role of citrus proteases during HLB disease progression. Proteomics was performed in phloem enriched tissue, revealing specific proteases and protease inhibitors induced in the vasculature. These specific proteases and protease inhibitors represent potential targets for disease control. These data were published in 2020 in the journal Molecular Cellular Proteomics. Wealso characterized another SDE, SDE15 (CLIBASIA_04025), which suppresses programmed cell death (PCD) by directly targetingACD2 (ACCELERATED CELL DEATH 2), a known regulator of PCD. Transgenic expression of SDE15 in citrus promotes Las multiplication. We show that SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB.This work has been published in the journal Plant Physiology. Objective 4 -Development of genome-edited citrus with HLB resistance. The goal of this objective is to modify SDE targets in citrus using CRISPR-based genome editing to enhanceHLB resistance. We have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus. Using this technology, we generated homozygous mutants of the SDE1 and SDE15 targetsincluding PLCPs and ACD2. These plants are currently being examined for HLB resistance. Objective 5 -Sociological analyses of consumer responses to genome-edited citrus. The purpose of this objective is to understand public responses to genetically modified citrus, especially under the current threat from HLB. Our activities this year focused on analyzing data collecting in the prior year. The focus is on developing an understanding of how different communication methods work to influence acceptance of CRISPR and GM products. We also considered how information is delivered to respondents about what CRISPR and GM are to see if information formatimpacts willingness to pay with complex scientific concepts. A manuscript (?Hu, Y., L. House, B. McFadden, and Z. Gao. "The influence of choice context on consumers' preference for GM orange juice.") has been accepted for publication by the Journal of Agricultural Economics. Objective 6 -Extension and outreach. Although extension and outreach activities were limited during 2020, we were able to meet multiple times with individual growers to assess HLB damage, provide guidance on disease management and discuss about project results about differences in technologies for HLB-resistance development and consumer acceptance of edited citrus. Some of the stakeholder/grower presentations made by the teaminclude Citrus Expo (August, 2020), Texas Citrus Pest and Disease Management Corporation (June, October 2020), Citrus Center Winter Festival (February 2020), UC-ANR Master Gardeners (March 2020) and Citrus Production Course (July 2020), California Citrus Mutual Citrus Showcase (March 2020), and WERA 20 (May 2020). Co-PDLisa House also made a presentation at an online Workshoporganized by the USDA, Economic Research Serviceentitled: Consumers and Citrus Greening: Are Geneticsthe Answer? Like in previous years the Citrus Clonal Protection Program (CCPP) distributed HLB educational materials via 1,544 orders for pathogen-tested citrus budwood to 1,152 users of its online system. <br><br><b>Publications</b><br>
Target Audience
This project reaches a large, diverse body of students and trainees through the research, teaching and outreach activities of the PD and co-PDs. Science-based knowledge has been delivered and will continue to be delivered to a broad audience including researchers and citrus growers through presentations in scientific conferences (local, national and international) and grower meetings, as well as extension and outreach activities with individual growers and citrus commodities in California, Florida and Texas. The PD and co-PDs are from four research institutions in California, Florida and Texas, the three largest citrus-producing states in the US. Three PD/co-PDs are affiliated with minority-serving universities.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided excellent training opportunities to five postdoc researchers, four Ph.D students, and four undergraduate students. In particular, the co-PD Vidalakis co-superviseda team of ~30 high school students including members from different states in the US and also from South Korea. The team worked on a project aiming to use RNA silencing to controlHLB. Theycompeted in the iGEM comptition and won a bronze award.All these students/trainees have been exposed to experiments using interdisciplinary approaches, and gained experience on grant/manuscript writing and giving scientific presentations. They have also got the unusual opportunities to work with a multi-institutional team and directly communicate with growers. A group meeting with the grad student/postdoc participants from all groups was held at the 6thInternational ResearchConferenceon Huanglongbing(IRCHLB) in March, 2019 at Riverside, CA.
Dissemination Streams
Knowledge obtained from this project has been widely disseminated to a broad audience. In the past reporting period, one manuscript has been accepted by the jounral Molecular Plant Pathology and another one was submitted. Results related to this project were presented in several national and international conferences, as well as grower meetings. The results have also been disseminated through seminars given by the PD/co-PDs and the participating researchers in universities and research institutions in the US and abroad. The team has frequent, direct communications withstakeholders and the general public about the current status and research efforts on HLBthrough interviews by local media and national broadcast venues.
Next Reporting Steps
We are entering the last year of this five-year project. We are excited to note that all of our proposed experiments are going very well and all objectives are in the near completion stage. Scientific discoveries from this project have provided key fundamental insight in HLB biology and offered important opportunities to engineer disease resistant citrus. Our sociology studies also offer support to customer acceptance of engineered citrus, which has important implications to growers' choice in growing GM citrus. Objective 1 The proposed experiments have been mostly completed. We will finish the SDE expression analysis using root tissues and focus on compiling the data and preparing manuscripts for publication. Objective 2 We will continue to improvethe current ELISA protocol using monoclonal antibodies and including a cocktailof antibodies targeting different antigens. We realize there is still a significant gap before this method can be used for HLB diagnosis. However, since robust diagnosis is such an important step in HLB management, we have a strong commitment to continue our effort in this key area of HLB control. Objective 3 We will finish the functional characterization of SDE1 and SDE15. We will focus on compiling the data, finish data analysis and distribute the results. Objective 4 We will finish the generation and characterization of transgenic citrus with genetically modified targets of SDE1 and SDE15, i.e. PLCP and ACD2respectively. These plants will be monitored in developmental and HLB resistancephenotypes.Research in this objective is a rather long-term effort. We will continue generate edited and transgenic citrus lines as new SDE targets are identified. All these genome-edited plants will be tested for HLB resistance. Objective 5 We will complete the analysis of the second survey focusing on customer willingness to pay for orange juice produced with CRISPR, GM, or no genetictechnology. In this survey, we also consider how information is delivered to respondents about what CRISPR and GM are to see if information formatimpacts willingness to pay with complex scientific concepts. We will submit the manuscript focusing on trade-offs between country of origin and production method to a journal. Objective 6 We will continue our extension and outreach activities to advocate citrus HLB to a broad audience, communicate with growers on our new findings and technologies, and pursue applications of the newly developed ELISA-based HLB detection methods. <br><br>
<br>What was accomplished under these goals? In the current reporting period (fourth year of the five-year project), we have made significant progress on all six objectives. Objective 1 - Systematic analysis of Sec-delivered effectors (SDEs) from various Las isolates. In the past reporting period, we have analyzed the genome sequences of 24 Las strains. Eleven of these strains were isolated from infected trees in CA, TX, FL and Mexico. The complete genomes of these strains were sequenced and then analyzed together with 13 previously sequenced strains. The phylogenetic relationship of these HLB-associated strains was determined using a set of 765 housekeeping genes. Our results support three subgroups of Las isolates in CA (indicating three separate introduction events from different geographic areas) and the spread of the pathogen from FL to TX. We have also performed genomic analyses of diverse Liberibacter species and their relatives. We have determined SDE repertoires from each Las strain. SDEs were manually curated for stringency and also validated using PCR and sequencing across different strains. We identified 31 SDEs, of which 27 are core SDEs. The expression profiles of the 27 core SDEs was determined using qRT-PCR. Our results show extensive differential expression of SDEs in citrus vs psyllid hosts. Some SDEs are exclusively expressed in one host. Many SDEs exhibit significantly higher expression levels in citrus. We also analyzed the expression of three SDE's during four seasons in symptomatic and asymptomatic tissues with different bacterial titers.The datashowhigher expression of SDEs in asymptomatic tissuewith lower bacterial titers as compared to symptomatic tissues. These results suggest a link between SDEs and Las colonization/HLB progression. Objective 2 - Antibody development targeting a core group of SDEs for HLB detection. The goal of this objective is to develop antibodies that detect individual core SDEs for HLB detection. During the first three years of this project, we have generated polyclonal antibodiestargeting an effector SDE1 and developed enzyme-linked immunosorbent assay (ELISA) protocols. In the past reporting period, we extensively evaluated the Sandwich ELISA protocol using an antibody cocktail, which includes antibodies targeting both SDE1 and Las cell surface structures (including outer membrane proteins and lipopolysaccharides). Using thousands of field samples from TX and CA, wefound that the sensitivity of the procedure needs improvement. Our current effort focuses on generating and testing monoclonal antibodies to increase the sensitivity. Objective 3 - Identification of SDEs that contribute to HLB pathogenesis. The goal of this objective is to examine the virulence activity of core Las SDEs and identify how they affect citrus physiology and promoteHLB. Previously, we characterized the first virulence-related Las factor, SDE1, and showed that it suppresses the citrus papain-like cysteine proteases (PLCPs). These results were published in 2018(Clark and Franco et al., Nat Comm) and a patent application was filed. In the past reporting period, we continued the characterization of SDE1. By using transgenic citrus expressing SDE1, we show that SDE1 can promote Las infection and accelerate HLB symptom development. We also did transcriptome analysis and found enhanced expression of senescence-related genes in SDE1-expressing citrus. These results directly link SDE1 to HLB progression. A manuscript is in preparation to report these findings. Our work on SDE1 suggeststhat SDEs can interfere with citrus enzymes. Furtherproteomic profiling analysis revealed significant changes in serine proteases and peroxidasesupon infection. Activity based profiling of serine proteases revealed diverse patterns of activation and inactivation. While peroxidase transcript and protein accumulation is increased during infection, the peroxidase enzymatic activity does not increase. Thus, peroxidases are likely targets of Las SDEs. A manuscript is in preparation to report these findings. We have also characterized another SDE, SDE15, which suppresses programmed cell death (PCD) by directly targetingACD2 (ACCELERATED CELL DEATH 2), a known regulator of PCD. Transgenic expression of SDE15 in citrus promotes Las multiplication. It also promotes the chlorophyll break-down and contributes to the development of yellowing symptom associated with HLB. Characterization of SDE15 unravels a new virulence mechanism of Las. A manuscript has recently been submitted to report these findings. Objective 4 - Development of genome-edited citrus with HLB resistance. The goal of this objective is to modify SDE targets in citrus using CRISPR-based genome editing to enhanceHLB resistance. We have successfully developed CRISPR-based genome editing technology in citrus. In the past reporting period, weexpanded the tool box by includingCRISPR-LbCas12a (LbCpf1), an enzymefrom Lachnospiraceae bacterium ND2006. Using this method, we successfully edited a susceptibility gene of citrus canker disease with no potential off-targets observed.We have also generated homozygous mutants of the SDE1 and SDE15 targetsincluding PLCPs and ACD2. These plants are currently being examined for HLB resistance. Objective 5 - Sociological analyses of consumer responses to genome-edited citrus. The purpose of this objective is to understand public responses to genetically modified citrus, especially under the current threat from HLB. In the past reporting period, we have continued our analysis of data collected in the prior year. This data is from asurveydesigned to understand how consumers make trade-offs when making decisions regarding genetically modified foods. Participants competed a choice experiment where they selected orange juice at different prices given different production methods (GM versus not GM) and countries (forced trade-off between preferred country of origin and preferred technology). Our results show that demographics do influence willingness to pay for GM juice, but the main finding is that consumers are more likely to select country of origin over production technology when forced to make a trade-off. We think these results will provide important implications to growers' choice in growing GM citrus. A manuscript is currently in preparation to report these findings. In addition, we conducted a second survey to specifically look at consumer willingness to pay for orange juice produced with CRISPR-based genome editing. Further data analysis is being conducted on this data. Objective 6 - Extension and outreach. Together, the PD and co-PDs participated innumerous extension and outreach events. Through presentations at these venues and one-on-one interactions, the scientific findings and technologies developed through this project have been introduced to a diverse body of stakeholders. Educational materials have been distributed via the weekly Citrus Clonal Protection Program (CCPP) budwood shipments to over 3,500 customs. Some of the stakeholder/grower presentations made by the teaminclude Texas Citrus Pest and Disease Management Corporation (February, June and August,2019),Wonderful citrus(October, 2019), and California Citrus Nursery Society Conference (November, 2019). The co-PDVidalakis also organized the 6thInternational ResearchConferenceon Huanglongbing(IRCHLB) in March, 2019. The co-PDVidalakis also co-sponsored and co-supervised a high school students team, Biotech Without Boarders (https://2019.igem.org/Team:Bio_Without_Borders), that competed at the 2019 International Genetically Engineered Machine Giant Jamboree (iGEM) on aproject related to controlling HLB. The teamreceived a bronze award in this competition. <br><br><b>Publications</b><br>
Target Audience
This project reaches a large, diverse body of students and trainees through the research, teaching and outreach activities of the PD and co-PDs. Science-based knowledge has been delivered and will continue to be delivered to a broad audience including researchers and citrus growers through presentations in scientific conferences (local, national and international) and grower meetings, as well as extension and outreach activities with individual growers and citrus commodities in California, Florida and Texas. The PD and co-PDs are from four research institutions in California, Florida and Texas, the three largest citrus-producing states in the US. Three PD/co-PDs are affiliated with minority-serving universities.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided excellent training opportunities to five postdoc researchers, four Ph.D students, and seven undergraduate students in all participating institutions. In particular, a Ph.D student Kelley Clark in the PI Wenbo Ma's group worked with 48 UC Riverside undergraduate students to study PLCPs in different citrus varieties. These students/trainees have been exposed to experiments using interdisciplinary approaches, and gained experience on grant writing, manuscript preparation, and research presentation. They have also got the unusual opportunities to work with a multi-institutional team and directly communicate with growers. A group meetings with the grad student/postdoc participants from all groups was held at the ICPP congress in Boston, MA in August 2018.
Dissemination Streams
Knowledge obtained from this project has been widely disseminated to a broad audience. In the current reporting period, we published two research articles in highly regarded journals and one project report in a grower's magazine. Results related to this project were presented in numerous national and international conferences (including all the major research conferences in the general discipline of Plant Pathology), as well as grower meetings. The results have also been disseminated through seminars given by the PI/co-PIs and the participating researchers in universities and research institutions in the US and worldwide. The team has also talked to the public about the HLB in general as well as the current research efforts through interviews by local media.
Next Reporting Steps
Objective 1 We will complete the genome analyses of Las isolates. The focus of our research will be to determine the expression profiles of core SDEs in different citrus varieties with different HLB susceptibility levels, at different infection stages (early vs late), with or without HLB symptoms, in different tissues (roots vs leaves), and during different seasons. These results will provide insight into basic biology in HLB pathogenesis and help understand the differences that have been observed in citrus response and HLB progression. Two manuscripts will be submitted to report the Las genome sequence analysis and SDE expression profiling respectively. Objective 2 We will continue evaluating the current ELISA protocol using field samples. We will focus on optimizing a Sandwich ELISA protocol using a cocktail of antibodies targeting different antigens. One manuscript is currently in preparation to report the newly developed sandwich ELISA. Objective 3 We will focus onfocused on detailed characterization of the PLCP-SDE1 interaction and ACD2-SDE15 interaction. Additional core SDEs will be investigated by using transgenic plants, expression profiles and target identification. Other proteases that might regulate citrus defense response and targeted by SDE(s) will also be studied. Objective 4 We will monitor the phenotypes (development and HLB resistance) of the transgenic and genome-edited citrus lines with altered expression of PLCPs and ACD2. Additional transgenic lines will be generated when new detailed information about SDE targets is revealed. Research in this objective is a rather long-term effort. We will continue generate edited and transgenic citrus lines as more SDE targets are identified. All these genome-edited plants will be tested for HLB resistance. Objective 5 We will design the second survey, which will focus on different methods of explaining gene-editing technologies. In this survey, participants will be divided into four treatments: no information/control; a text description of the difference between transgenic genetic modification and gene-editing; a pictorial description of the difference; and a video describing the differences. Prior to, and after receiving, the information, participants will answer questions about willingness to pay for orange juice with different labels referencing technology. Objective 6 We will continue our extension and outreach activities to advocate citrus HLB to a broad audience, communicate with growers on our new findings and technologies, and pursue applications of the newly developed ELISA-based HLB detection methods. <br><br>
<br>What was accomplished under these goals? In the current reporting period (third year of the five-year project), we have made significant progress on all six objectives. Specific results for each objective are listed below. Objective 1 - Systematic analysis of Sec-delivered effectors from various Las isolates. The goal of this objective is to identify core Las SDEs using genome sequencing and analyze their expression pattern in different citrus varieties. In the current reporting period, we have analyzed the genome sequences of 23 Las strains. Eleven of these strains were isolated from infected trees in CA, TX and FL, and then sequenced in the co-PI Gitta Coaker's lab. These genomes were analyzed together with twelve previously sequenced strains and their phylogenetic relationship was determined using a set of 765 housekeeping genes. The results defined subgroups of Las isolates and suggested variable Las populations in CA. We have also determined SDE effector repertoires from each strain and 36 SDEs are found to be highly conserved across all Las isolates. This set of 36 SDEs is therefore defined as the "core SDEs". We have determined the expression profiles of the core SDEs. This experiment was conducted in the groups of PI Wenbo Ma and co-PI Nian Wang. Our results show extensive differential expression of individual SDEs in citrus vs psyllid hosts. Preliminary studies in co-PI Veronica Ancona's group also found that SDEs exhibited differential expression in different citrus varieties, seasons, and infection stages. These results suggest a link between SDEs and Las colonization/HLB progression. Objective 2 - Antibody development targeting a core group of SDEs for HLB detection. The goal of this objective is to develop antibodies that detect individual core SDEs for HLB detection. This research is mainly performed in the PI Wenbo Ma's lab. Previously, we have generated one polyclonal antibody targeting the core effector SDE1 (Pagliaccia et al., 2017) and developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) protocol using this antibody. In the current reporting period, we have developed a Sandwich ELISA procedure using an antibody cocktail, which includes antibodies targeting both SDEs and Las cell surface structures. This protocol has been extensively evaluated using field samples from FL, TX and CA. In particular, we participated in an "Early Detection Task" organized by Citrus Research Board and examined >1,500 field samples from CA. This method has also been used to monitoring the citrus germplasm culture collection trees on UC Riverside campus together with the K9 canine test and qPCR confirmation. Objective 3 - Identification of SDEs that contribute to HLB pathogenesis. The goal of this objective is to examine the virulence activity of core Las SDEs and identify those that affect citrus physiology and potentially promotes HLB. In the current reporting period, we have been focusing on two SDEs (SDE1 and SDE15), which are both core effectors produced by all Las isolates. We have completed the target characterization of both effectors, which provides important molecular insight into HLB pathogenesis. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants. These results have been published in Nature Communications (Clark and Franco et al., 2018) as a collaborative effort between Ma, Coaker, Wang and Ancona groups. A patent has been filed with Wenbo Ma as a primary inventor. The Co-PI Nian Wang's group has investigated the host target of the effector SDE15, which can suppress programmed cell death in citrus. SDE15 interacts with the host protein ACD2, which regulates programmed cell death. Importantly, transgenic citrus expressing SDE15 can enhance HLB progression. A manuscript describing this work has been submitted. Objective 4 - Development of genome-edited citrus with HLB resistance. The goal of this objective is to modify SDE targets in citrus using CRISPR-based genome editing for HLB resistance. Results from Objective 3 revealed that PLCPs, as the virulence targets of SDE1, are promising targets for genome editing. The co-PI Nian Wang's group has successfully developed CRISPR-based genome editing in citrus (Jia et al., 2017; Jia et la., 2019), and have initiated experiments to generate edited citrus plants targeting PLCPs. Other transgenic citrus plants over-expressing PLCPs or ACD2 have also been developed in Nian Wang's lab. Objective 5 - Sociological analyses of consumer responses to genome-edited citrus. The purpose of this objective is to understand public responses to genetically modified citrus, especially under the current threat from HLB. It is noteworthy that this objective started from 2017, so the current reporting period is the second year of this research, which is mainly conducted by the co-PI Lisa House. In the past reporting period, we conducted a thorough literature review and determined the best course of action for data collection. The first survey, designed to understand how consumers make trade-offs when making decisions regarding genetically modified foods, was developed and conducted. We examined the idea of having to trade off to get non-genetically modified orange juice by randomly assigning participants to one of three survey treatments. The treatments tested the trade-off between juice produced using biotechnology and juice produced in Brazil. Participants competed a choice experiment where they selected orange juice at different prices given different production methods (we only included GM versus not GM, not CRISPR for this stage of the project). One group faced a choice experiment with non-GM orange juice from both Brazil and the U.S. (the status quo/control group), the second group faced an experiment with GM US orange juice and non-GM Brazilian orange juice (forced trade-off between preferred country of origin and preferred technology), and the final group faced an experiment with all three options: non-GM US and Brazil orange juice and GM US orange juice. Our results show that demographics do influence willingness to pay for GM juice, but the main finding is that consumers are more likely to select country of origin over production technology when forced to make a trade-off, with willingness to pay for US GM orange juice higher than Brazilian non-GM orange juice. We think these results will provide important implications to growers' choice in growing GM citrus. A manuscript is currently in preparation to report these findings, and a presentation was developed for the Southern Agricultural Economics Association annual meetings in early 2019. Objective 6 - Extension and outreach. Together, the PD and co-PDs attended and made presentations at numerous extension and outreach events. Through these venues and one-on-one interactions with individual citrus growers, the project, as well as technologies used and are being developed through this project have been introduced to the community. Information of the project is also presented on the website http://citrushlb.org. One article (Franco et al., 2019) was published in the citrus industry magazine "Citrograph". <br><br><b>Publications</b><br>
Target Audience
This project reaches a large, diverse body of students and trainees through the research, teaching and outreach activities of the PD and co-PDs. Science-based knowledge has been delivered and will continue to be delivered to a broad audience including researchers and citrus growers through presentations in scientific conferences (local, national and international) and grower meetings, as well as extension and outreach activities with individual growers and citrus commodities in California, Florida and Texas. The PD and co-PDs are from four research institutions in California, Florida and Texas, the three largest citrus-producing states in the US. Three PD/co-PDs are affiliated with minority-serving universities.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided excellent training opportunities to five postdoc, four Ph.D students, and three undergraduate students in all participating institutions. The students have been exposed to experiments using interdisciplinary approaches, and gained experience on grant writing, manuscript preparation, and research presentation. The trainees also got the unusual opportunities to work with a multi-institutional team and directly communicate with growers. A group meeting with all the grad student/postdoc participants was held at the IRCHLB V in Orlando, FL in March 2017.
Dissemination Streams
Knowledge obtained from this project has been widely disseminated to a broad audience. In the current reporting period, we published two research articles and one comprehensive review article. Results related to this project were presented in numerous national and international conferences, as well as grower meetings. The results have also been disseminated through seminars given by the PD/co-PDs and the participating researchers in universities and research institutions in the US and abroad. The PD and co-PDs also talked to the public about the HLB in general as well as the current research efforts through interviews by local media and national broadcast venues.
Next Reporting Steps
Objective 1 We will complete the genome sequencing and analyses of the 10 field isolates from FL and TX groves. The isolates have been maintained in greenhouse after graft inoculation. The genome sequences of the isolates will greatly facilitate our research effort on determining the expression profiles of core SDEs in different citrus varieties at different infection stages (early vs late). This systematic analysis of SDE expression will provide essential information on Las pathogenesis. Objective 2 We will continue evaluating the current ELISA protocol using field samples. We will develop a Sandwich ELISA protocol using new monoclonal antibodies. We will develop monoclonal antibodies using SDE2 as the biomarker. For all the protocols, a cut-off value with reasonable specificity and sensitivity using statistical analysis will be determined. We will start to explore the utility of antibody cocktails to increase the detection sensitivity. Objective 3 We have generated multiple transgenic citrus plants expressing SDE1 and will characterize these plants for altered susceptibility to Las. Transgenic plants expressing other core SDEs will also be generated and characterized for development and disease resistance phenotypes. Objective 4 We will continue the functional characterization of core SDEs and their interactions with host targets. We have initiated the identification of host targets for five SDEs using in vitro and in vivo assays. We will generate genome-edited citrus with altered expression of PLCPs that are targeted by SDE1. These plants will be monitored for development and HLB resistance. Genome-edited citrus with 13 putative SDE targets modified have also been generated and will be extensively characterized. We will also optimize the CRISPR technology using Cas9 variants in citrus. Objective 5 We will conduct sociological analysis on consumer and grower responses to genome-edited citrus products. The information obtained from these analyses will be used to develop educational materials with the goal of filling gaps in knowledge between researchers, growers and consumers. Objective 6 We will continue our extension and outreach activities to advocate citrus HLB to a broad audience, communicate with growers on our new findings and technologies, and pursue applications of the newly developed ELISA-based HLB detection methods. <br><br>
<br>What was accomplished under these goals? IMPACT: Huanglongbing (HLB, aka citrus greening disease) is the most devastating disease that is currently threating the entire citrus industry in the US and worldwide. In the US, HLB is associated with an insect-transmitted bacterium called Candidatus Liberibacter asiaticus (Las), which specifically colonizes the citrus phloem (a vascular tissue). Las infection has caused enormous loss (billions of dollars and thousands of jobs) in Florida and the wholesale orange price has increased almost three times due to this disease. Importantly, there is no effective treatment to cure the trees after they are infected. Therefore, it is extremely important to: 1) develop robust detection methods in order to remove infected trees promptly, thereby restricting the further spread of the disease; 2) enhance HLB resistance through genetic approaches such as genome editing. This project addresses these two urgent needs by studying the Sec-delivered effectors (SDEs) of Las. Antibody-based detection methods using SDEs as the biomarkers will support field surveys and tree removal. Understanding the molecular mechanisms by which SDEs contribute to HLB not only provides key information of Las pathogenesis but also allows the identification of targets for genetic manipulation in order to control HLB in a sustainable manner. Sociological analysis and outreach activities will bridge effective communications between researchers, growers and consumers and help growers make informed decisions during the development of HLB resistant varieties. Six objectives were proposed in this project. In the current reporting period (second year of the five-year project), we have made significant progress on all objectives. Specific results for each objective are listed below. Objective 1 - Systematic analysis of Sec-delivered effectors from various Las isolates. The goal of this objective is to identify core Las SDEs using genome sequencing and analyze their expression pattern in different citrus varieties. In the current reporting period, we have sequenced ten Las strains isolated from infected trees in CA, FL and TX. These ten genomes were analyzed together with nine other previously sequenced strains. We analyzed the phylogenetic relationship between the 19 isolates and determined their SDE effector repertoire. Using stringent criteria, 23-35 SDEs were identified per genome. Current data using 13 isolates indicate that there are 15 core SDEs conserved across these strains. Our analyses reveal that the SDEs are small in size (less than 25kDa); thus, they should be capable of moving away from the site of primary infection to distal regions in citrus, which allows robust disease detection. 531 orthologous genes were used to resolve Las phylogeny from sequenced strains. This approach enabled the identification of strain variation from state-to-state, revealing that recently isolated Texas and California strains cluster together, while Florida strains do not. This approach can now be used to analyze Las strain diversity in important growing regions to identify the most prevalent strains. Our plan is to sequence Las genomes of isolates from four TX and six FL groves. We have propagated the strains to be sequenced by grafting infected trees from selected groves. These grafted plants will also allow us to systematically examine SDE expression profiles in the next reporting period. Objective 2 - Antibody development targeting a core group of SDEs for HLB detection. The goal of this objective is to develop antibodies that detect individual core SDEs for HLB detection. Previously, we have generated one polyclonal antibody targeting the core effector SDE1 (Pagliaccia et al., 2017). In the current reporting period, we generated a monoclonal antibody targeting SDE1 and two polyclonal antibodies targeting another core effector SDE2. The polyclonal antibodies have been evaluated using an indirect competitive enzyme-linked immunosorbent assay (ELISA) protocol that has been developed in the PD's laboratory. We have also started to develop a Sandwich ELISA procedure using the monoclonal antibody. All these protocols have been extensively used to examine field samples from Florida, Texas and California. Objective 3 - Identification of SDEs that contribute to HLB pathogenesis. The goal of this objective is to examine the virulence activity of core Las SDEs and identify those that affect citrus physiology and potentially promotes HLB. Transgenic citrus plants are being generated expressing individual core SDEs in order to determine if any can phenocopy disease symptoms, suppress defense responses, and facilitate Las proliferation. Targets of selected SDEs in citrus are also identified. In the current reporting period, we have been focusing on two core Las SDEs (SDE1 and SDE5), which are both core effectors produced by all Las isolates. A major task was to identify their target proteins in citrus. In the current reporting period, we have completed the characterization of SDE1, which directly targets the papain-like cysteine proteases (PLCPs) in citrus. PLCPs are secreted into the vascular system in citrus. Some PLCPs are induced during Las infection, indicating a positive role in citrus defense. We show that SDE1 inhibits the protease activity of PLCPs in vitro and in planta. Our data suggest that SDE1 functions as a virulence factor to promote bacterial infection by suppressing the activity of PLCPs in citrus (Clark and Franco et al., manuscript under review). Expression of SDE5 in citrus leads to dwarfism, which resembles HLB symptom. The host target(s) of SDE5 have also been identified and their interactions are characterized. Objective 4 - Development of genome-edited citrus with HLB resistance. The goal of this objective is to modify SDE targets in citrus using CRISPR-based genome editing for HLB resistance. Results from Objective 3 revealed that PLCPs, as the virulence targets of SDE1, are promising targets for genome editing. We have successfully developed CRISPR-based genome editing in citrus (Jia et al., 2017), and have initiated experiments to generate edited citrus plants on PLCPs that exhibit dynamic changes during HLB development. We are also generating transgenic lines that over-express PLCPs. These genome-edited plants will be tested for HLB resistance. Objective 5 - Sociological analyses of consumer responses to genome-edited citrus. The purpose of this objective is to understand public responses to genetically modified citrus, especially under the current threat from HLB. This objective started from the current reporting period and the analysis is still on going. In the past reporting period, we conducted a voluntary survey to citrus growers during an event in Texas in order to understand their concerns on transgenic modification of citrus. Two of the most important concerns to growers in order to consider planting genetically modified citrus in the future is the consumer acceptance of the fruit and juice, and to see yield improvement in the new varieties. In general, growers responded positively on using edited citrus without incorporating foreign genes as one of the best strategies to combat HLB. Objective 6 - Extension and outreach. Together, the PD and co-PDs attended and made presentations at numerous (>20) extension and outreach events. Through these venues and one-on-one interactions with individual citrus growers, the project, as well as technologies used and are being developed through this project have been introduced to the community. Information of the project is also presented on the website http://citrushlb.org. One article (Franco et al., 2017) was published in the citrus industry magazine "Citrograph". <br><br><b>Publications</b><br>
Target Audience
The PD and co-PDs are from four research institutions in California, Florida and Texas, the three largest citrus-producing states. Three PD/co-PDs are affiliated with minority-serving universities. Therefore, this project reaches a large, diverse body of students and trainees through the teaching activities of the PD and co-PDs. Science-based knowledge has been delivered to a broad audience including researchers and citrus growers through presentations in scientific conferences (local, national and international) and grower meetings, as well as extension and outreach activities with individual growers and the citrus commodity.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided excellent training opportunities to four postdoc, two Ph.D students, and one undergraduate student in all participating institutions. The students have been exposed to experiments using interdisciplinary approaches, and gained experience on grant writing, manuscript preparation, and research presentation. The trainees also got the unusual opportunities to work with a multi-institutional team and directly communicate with growers.
Dissemination Streams
Knowledge obtained from this project has been widely disseminated to a broad audience. Last year, we published one research article, presented our project and unpublished findings in numerous national and international conferences, as well as grower meetings. The results have also been disseminated through seminars given by the PD/co-PDs and the participating researchers in universities and research institutions in the US and abroad. The PD also talked to the public about the current status of HLB through her interviews by local newspapers and national broadcast venues.
Next Reporting Steps
Objective 1 We will determine the genome sequences of 30 field isolates from 10 groves in FL and TX. The isolates have been maintained in greenhouse after graft inoculation. We will amplify the Las titer by applying the parasitic plant dodder and sequence Las from dodder infected plants. These grafted plants will also allow us to systematically examine the expression profiles of core SDEs and compare SDE expression across different citrus varieties in different states. Objective 2 We will continue evaluating the current ELISA protocol using a large number of field and greenhouse samples. We will determine a cut-off value with reasonable specificity and sensitivity using statistical analysis. A patent application will be filed. If other good biomarker(s) are identified from core SDE analysis, we will generate antibodies with the hope of adding them into our current ELISA protocol. Objective 3 We will generate transgenic citrus plants expressing SDE1 under the control of an inducible promoter. Transgenic plants expressing the rest of core SDEs will also be generated. These plants will be monitored for development and disease resistance phenotypes. Objective 4 We will examine the inhibitory effect of SDE1 and SDE5 on the protease activity of their targets. The molecular details of the SDE-protease interaction will be characterized and this information will be used to generate genome edited citrus expressing effector-resistant proteases. Transgenic citrus with altered expression levels of the proteases will be tested for HLB resistance. Objective 5 We will conduct sociological analysis on consumer and grower responses to genome-edited citrus products. The information obtained from these analyses will be used to develop educational materials to fill the gaps in knowledge between researchers, growers and consumers. Objective 6 We will continue our extension and outreach activities to advocate citrus HLB to a broad audience, communicate with growers on our new findings and technologies, and pursue applications of the newly developed ELISA-based HLB detection methods. <br><br>
<br>What was accomplished under these goals? IMPACT: Huanglongbing (HLB, aka citrus greening disease) is the most devastating disease that is currently threating the entire citrus industry in the US and worldwide. In the US, HLB is associated with an insect-transmitted bacterium called Candidatus Liberibacter asiaticus (Las), which specifically colonizes the citrus phloem (a vascular tissue). Las infection has caused enormous loss (billions of dollars and thousands of jobs) in Florida and the wholesale orange price has increased almost three times due to this disease. Importantly, there is no effective treatment to cure the trees after they are infected. Therefore, it is extremely important to: 1) develop robust detection methods in order to remove infected trees promptly, thereby restricting the further spread of the disease; 2) enhance HLB resistance through genetic engineering. This project addresses these two urgent needs by studying the Sec-delivered effectors (SDEs) of Las. Antibody-based detection methods using SDEs as the biomarker will support field surveys and tree removal. Understanding the molecular mechanisms by which SDEs contribute to HLB not only provides key information of Las pathogenesis but also identify targets for genetic manipulation in order to control HLB in a sustainable manner. Our sociological analysis and outreach activities will fill the gaps of knowledge between researchers, growers and consumers and help growers make informed decisions during the development of HLB resistant varieties. In this project, six objectives were proposed. In the past year (first year of the five-year project), we have made significant progress on objectives 1, 2, 3, 4 and 6 and also initiated objective 5. Specific results for each objective are listed below. Objective 1 - Systematic analysis of Sec-delivered effectors from various Las isolates. The goal of this objective is to identify core Las SDEs using genome sequencing and analyze their expression pattern in different citrus varieties. To date, we have analyzed the sequences of ten Las strains and determined their SDE effector repertoire. Eight of the strains were previously sequenced, but most were draft genomes without annotation. We also sequenced two new Las genomes from Florida and China respectively. These ten genomes were assembled and then subjected to comparative genome analysis. Using stringent criteria, 23-35 SDEs were identified per genome. Among them, 13 are considered "core" SDEs because they are present in all the genomes. Our analyses reveal that the SDEs are small in size (less than 25kDa); thus, they should be capable of moving away from the site of primary infection to distal regions in citrus, which allows robust disease detection. The proposed research also includes sequence analysis of 30 new Las genomes from four TX and six FL groves. The Las titer varies throughout the year and can be quite low and variable during summer months. Therefore, we have propagated the 30 strains to be sequenced by grafting infected trees from selected groves. These grafted plants will also allow us to systematically examine SDE expression profiles in the next reporting period. Objective 2 - Antibody development targeting a core group of SDEs for HLB detection. The goal of this objective is to develop antibodies that detect individual core SDEs for HLB detection. To date, we have generated one polyclonal antibody and are in the process of generating two monoclonal antibodies targeting the core effector SDE1. Using the polyclonal antibody, we have developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) protocol, which is currently being evaluated using field and greenhouse samples from Florida, Texas and California. So far, we have got promising results. Objective 3 - Identification of SDEs that contribute to HLB pathogenesis. The goal of this objective is to examine the virulence activity of core Las SDEs and identify those that affect citrus physiology and potentially promotes HLB. Transgenic citrus plants are being generated expressing individual core SDEs in order to determine if any can phenocopy disease symptoms, suppress defense responses, and facilitate Las proliferation. Targets of selected SDEs in citrus are also identified. We are currently focusing our efforts on two core Las SDEs (SDE1 and SDE5) and identified their target proteins in citrus. Remarkably, they both target the same family of secreted cysteine proteases, whose abundances are increased in the phloem of HLB-infected citrus. Expression of SDE1 significantly impairs citrus growth; whereas expression of SDE5 in citrus leads to dwarfism, which resembles HLB symptom. In addition, proteomic analysis suggests that at least some of these proteases exhibit significant changes in abundance upon Las infection. These results suggest that SDE1 and SDE5 may contribute to HLB progression and the proteases are promising targets for genetic manipulation. Objective 4 - Development of genome-edited citrus with HLB resistance. The goal of this objective is to modify SDE targets in citrus using CRISPR-based genome editing for HLB resistance. With potential citrus SDE targets (proteases that are targeted by SDE1 and SDE5) in hand, we have initiated experiments to generate edited citrus plants on PLCPs that exhibit dynamic changes during HLB development. We are also generating transgenic lines that over-express PLCPs. The edited plants will be tested for HLB resistance. Objective 5 - Sociological analyses of consumer responses to genome-edited citrus. This objective will start the next year. Nonetheless, we conducted a voluntary survey to citrus growers during an event in Texas in order to understand their concerns on transgenic modification of citrus. Two of the most important concerns to growers in order to consider planting genetically modified citrus in the future is the consumer acceptance of the fruit and juice, and to see yield improvement in the new varieties. Cost of the new trees was also highly ranked. In general, we are encouraged by a positive attitude from the growers towards using edited citrus without incorporating foreign genes as one of the best strategies to combat HLB. Objective 6 - Extension and outreach. Together, the PD and co-PDs attended and made presentations at more than 10 extension and outreach events. Through these venues and one-on-one interactions with individual citrus growers, the project, as well as technologies used and are being developed through this project have been introduced to the community. Information of the project is also presented on the website http://citrushlb.org. Two articles are published in the citrus industry magazine "Citrograph". <br><br><b>Publications</b><br>