Despite the short time period from the initial funding of our FCATP grant, we have made progress on all areas of the project: 1. Isolate TAL effectors: We have obtained one of the primary Xc TAL effector genes, PthAw,and it has been subcloned into an expression vector. Additional Xc TAL effectors are in the process of being isolated by PCR and subcloned into expression vectors. 2. Identify TAL effector binding sites: A binding site determination has been made for PthAw. Additional binding sites will be determined as the TAL effectors become available. 3. Construction of modified Bs3 promoter constructs: The binding site for PthAw has been introduced into the Bs3 promoter, upstream of the native UPA box which mediates induction by AvrBs3. 4. Rapid testing for promoter function: The new construct was tested with the GUS reporter gene in the standard N. benthamiana assay and was found to be inducible by PthAw in planta. Importantly, the modified promoter was completely inactive in the absence of PthAw. Furthermore, much work has been done to develop a transient assay system in citrus leaves. Conditions have been established that allow the assessment of the function of individual Xc effectors upon delivery into citrus leaves by Agrobacterium. This method is now being used to test Bs3 constructs. One issue that has come to light is that the native pepper strain, Xanthomonas euvesicatoria 85-10, causes a non-specific hypersensitive response, and so delivery of TAL effectors to citrus must come from other bacterial strains, such as Agrobacterium. Additional transient assays are in progress in citrus cell cultures and protoplasts, and Bs3 constructs using the GFP reporter gene will be tested for both GFP and Bs3 activity. The determination of Bs3 activity in citrus is our main objective for the present time. 5. Stable citrus transformation: Initial work on this has been initiated with test constructs. Until constructs with Xc TAL effector binding sites are available, stable transformants are being tested using AvrBs3-inducible constructs. A first project meeting was convened in Gainesville on June 15th with PI’s Horvath, Jones, Stall and Moore. We discussed the initial data reported here and reviewed the goals and milestones for this period. The emphasis on determining the activity of BS3 in citrus was highlighted and experiments planned. An additional meeting will be convened in July in Quebec with PI’s Horvath, Lahaye, and Jones’ lab postdoc Figuieredo.
Objective II: Website creation and development. A Citrus Greening/HLB Genome Resources Website has been created and can be accessed at http://www.citrusgreening.org/. Significant aspects of this objective achieved during this quarter include the following: A. Development of a policy for data sharing. Concerns about publication priority, reluctance to release data that might be in error, and the slow pace at which sequence is generated have all contributed to a scarcity of publicly available genome data. In collaboration with the program manager, a policy was developed whereby the genome resources website would provide a venue where preliminary data and analyses generated from these data could be deposited, with access restricted to registered users. Prior to viewing the restricted data, users would be required to acknowledge the preliminary nature of the data and agree not to publish analyses based on these data without contacting the depositors. B. Website organization. The http://www.citrusgreening.org/ website has been designed to accommodate both information with unrestricted access and data limited to registered users. Web pages with unrestricted access include those containing general information and links on the disease, pages providing guidelines and links for commonly used analytical tools such as BLAST analysis and genome viewing with the Artemis genome viewer, and pages summarizing the range of Ca. Liberibacter sequences that have been deposited at NCBI. With the exception of a 1.2 Mb draft genome sequence for Ca. L. asiaticus, all of the Ca. Liberibacter sequences currently available at NCBI fall into one of three gene clusters, diagrams of which have been posted on the website together with hyperlinks to their component accession records at NCBI. In order to restrict access to pages containing preliminary data, a registration page has been set up requiring users, pending acceptance of the data sharing policy, to enter contact information and choose a user name and password. Once reviewed by the PI, the registrants are granted access to the restricted pages and their contact information added to the list of registered users. Information on data submission is included on the pages with unrestricted access. While it is expected that the site will grow significantly as more data and analyses become available, the basic website structure has been designed to accommodate future growth. Objective III: Bioinformatic analysis. Using the draft genome sequence for Ca. L. asiaticus deposited at NCBI, a single pseudomolecule has been generated from the deposited contigs to aid users in viewing the 34 contigs in their totality (draft sequences are deposited at NCBI as independently numbered and annotated contigs). Various feature files have been produced for the pseudomolecule, so that users can view annotations generated by the MG-RAST server as well as BLAST hits independent of gene calls. Functional roles predicted by the MG-RAST server have been mapped to KEGG metabolic maps and the results compared to maps generated for two fastidious plant pathogenic bacteria and four insect endosymbiotic bacteria. Comparison reveals that the deposited Ca. L. asiaticus sequence encodes only a small percentage of enzymes involved in amino acid, carbohydrate, energy, glycan, lipid, and nucleotide metabolism relative to these other organisms. This limited metabolic capability suggests that the deposited sequence may not represent a complete genome. Objective I: Assess community needs. Various members of the citrus greening community have provided input during website development. Further conversations between the PI and community members about data deposition and desired analyses are expected at the special session on citrus greening to be held at the meeting of the American Phytopathological Society (August 1-5).
This project has three objectives: 1) gap closure of Ca. Liberibacter asiaticus (Las) found in Florida; 2) complete genomic sequencing to closure of Ca. L. americanus (Lam) strain S’o Paulo from Brazil, and 3) comparative genome analysis of Las and Lam to attempt to determine common factors enabling pathogenicity to citrus. Objective 1 Progress: Within the recently published Las strain psy62 chromosomal genome (Duan et al. 2009), many unique genes of unknown function are found, and several phage related genes are found integrated into the chromosome, but no replicating phage DNA was found. We reported last quarter the finding of two complete circular lytic phage genomes, SC1 and SC2, with the copy number of SC1 replicating an average 10X higher in citrus and 20X higher in periwinkle, than when the phage is integrated into the chromosome in infected psyllids. [Gabriel & Zhang, 2009. Phytopathology 99 (6): S38]. Neither phage were previously reported. Phage SC1 is 40 kb in size. SC2 is 39 kb in size and appears to be a degenerate variant of SC1. Phage particles, 25 nm in size, and likely corresponding with infectious SC1 phage, have now been documented inside Las cells, inside swollen Las cells that appear to be lysing and outside of Las cells by electron microscopy in Las-infected periwinkle phloem cells. Phage particles were not seen in Las-infected citrus. In citrus, the phage are replicating to relatively high copy number but not lysing Las cells, as they are in periwinkle. Since some of the SC-1 and SC-2 phage genes were found by Duan et al (2009) to be integrated into the Las genome, both phage likely have a lysogenic cycle. Lysogenic phage are known to mobilize bacterial genes when they repackage into infectious particles and then horizontally transfer these genes between different bacteria that they infect. Any genes carried on a phage that replicates to a level 10X higher than the host chromosome also replicates the copy number of all genes it carries to a level 10X higher than the rest, dramatically increasing gene expression levels, possibly affecting virulence. Following that lead, several new Las genes were found on SC1 and SC2. Detailed genomic analysis has revealed a total of six outer membrane proteins, five of which are predicted to form beta barrel structures, several of which were not previously reported in the psy62 genome. One of these appears to be an enzyme that is predicted to modify the bacterial outer membrane structure; another is a predicted porin, which form structures through which molecules can passively diffuse. A third, and previously unreported protein is a predicted outer membrane anchored protein with a collagen triple helix repeat. This unusual protein may be capable of forming a fibrous protein matrix, perhaps causing Las aggregation and trapping Las within phloem cells. We observed Las cells within periwinkle phloem by EM to occupy well over 50% of the cell cytoplasm. Objective 2 Progress: In collaboration with Fundecitrus in Brazil, Lam strain ‘S’o Paulo’ DNA samples extracted from citrus and purified on pulsed-field gels by Dr. Nelson Wulff has resulted in 78 kb of partially confirmed Lam genomic sequence obtained by 454 sequencing assembled into contigs greater than 1 kb. Dr. Wulff recently brought new Lam DNA to our lab for additional sequencing and comparison with the Las genome and the yet to be published sequence from the Liberibacter causing “zebra chip” disease of potato (Objective 3). We are on track to meet target deadlines of the original proposal.
During the final quarter of the year, the Core Citrus Transformation Facility (CCTF) maintained its level of performance and produced transgenic citrus plants for many satisfied customers. During the 4th quarter, the CCTF continued to utilize the provided funding to process the orders for transgenic plants, mostly servicing CRDF funded researchers studying transgenes with potential to generate resistance to HLB. Orders processed during this quarter include: pY46-Carrizo; pY102-Carrizo; pY141-Carrizo; pY150-Carrizo; pCitIntra-Duncan; pAZI-Duncan; pAtBI-Duncan; pBCR2-Duncan; pDPR1-Duncan; pLP1-Hamlin; pLP1-C-mac; pLP2-Hamlin; pLP2-C-mac. During the last quarter of this funding year, work was mostly concentrated on recent orders. Fourteen Duncan plants were produced carrying a gene of interest from the p35S-TRX vector and 23 more Duncan plants were produced carrying a gene from the pSucTRX vector. Multiple Duncan plants were produced toward satisfaction of ‘WG’ group of orders: eight-pWG22-1 plants, three-pWG21-1, and four pWG25-13 plant. Also, following Carrizo plants were produced for the ‘Yale’ order: nine plants with the gene from the pY46 vector and 11 plants with the gene from the pY102 vector. Eighteen Duncan plants were produced after treatment with bacteria harboring pBCR2 vector. Three more Duncan plants were produced with the EDS5 gene and six Mexican limes with the P35 gene. Four additional Duncan plants carrying a gene from pSUC-CitNPR1 were produced. The impact of citrus diseases on Florida citriculture is rapidly growing, and our participation in this battle is growing with some positive results already being published. Continued funding for CCTF which is an integral part of this community and contributes greatly towards common goal will allow for the progress to go on by keeping production of transgenic material un-interrupted and at high levels.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. A number of plant derived insecticidal transgenes, each driven by a root specific promoter were incorporated into Carrizo citrange. As part of this project we cloned four insecticidal genes and two plant derived promoters. Constructs were initially tested on N. benthamiana for confirmation of transgene activity. Carrizo citrange was subsequently transformed utilizing the conventional Agrobacterium mediated transformation process. A total of 123 putative transgenic lines were generated. PCR screening identified 74 of these lines to be transgenic. 54 of these transgenic lines had a high level of transgene expression as determined by qPCR. We have also identified a number of putative root specific genes from Citrus clementina by data mining the phytozome database. Of them several putative sequences were selected for characterization using qPCR. RNA was extracted from mature and juvenile roots from non-transgenic Swingle citrange. Each of the identified sequences were characterized in these different tissues. In addition, transcript levels in the leaves were also measured. The Cic1867m gene was determined to be very root specific and a 1.2 kb fragment of its promoter was cloned from Clementine genomic DNA using PCR. Deletion analyses identified a 0.8 kb fragment from that promoter fragment to be sufficient for root specific activity and transgenic plants were produced using this promoter. Cuttings from all the better performing lines have been made and are being rooted in the mist bed. These clones will be sized up for Diaprepes feeding experiments. Clonally propagated plants will be force fed with Diaprepes neonates – when available and root damage / insect mortality evaluated.
This project had a single objective to assure un-interrupted production of transgenic citrus plants through the services of Citrus Transformation Facility (CTF). Continued operation of the facility was going to guarantee a foundation regarding this aspect of research for the scientific community involved in fight against huanglongbing (HLB) and citrus canker. By producing transgenic plants and bringing into life the ideas of scientists working in different laboratories, CTF would facilitate the process of search for candidate plants that would be tolerant/resistant to diseases and provide major relief for Florida Citrus Industry. The objective of this project was successfully accomplished. Throughout the funding period of three and-a-half years, CTF operated without major interruptions and provided service in the form of production of transgenic plants. The level of production stayed at expected, satisfactory level and resulted in creation of 781 citrus plants that represent independent transgenic events. These plants belong to eight different cultivars: Duncan grapefruit, Carrizo citrange, Pineapple sweet orange, Mexican lime, Valencia sweet orange, Swingle citrumelo, Kumquat, and Pomelo.During this project, CTF worked on 103 orders that were placed by 10 clients and this group included eight faculty members based at University of Florida, one faculty from University of California and one Foundation. The number of projects for which the plants were produced was higher than the number of clients. For example, Dr. Nian Wang was one of the clients but orders from his lab were the part of at least five different projects done by his post-doctoral associates.All produced transgenic plants were associated only with research that has to do with disease resistance. This is a clear indication of the role CTF plays in the efforts to overcome effects of HLB and citrus canker.CTF supported the projects from Dr. Nian Wang’s lab that resulted in successful application of CRISPR/Cas9-mediated genome editing technology in Citrus. Plants produced this way will be the most market-friendly as they only have minor modifications of their own gene(s) and in the near future will be free of any other inserted DNA. The work done in CTF lead to production of Duncan grapefruit plants that exhibit high degree of resistance to citrus canker. Other commercially important cultivars could be made with improved resistance to canker based on results of this project.As a part of another research project aimed at control of Asian Citrus Psyllid (ACP) in citrus groves, transgenic Indian curry leaf plants were also produced in CTF. There are six such plants that are being tested for the ability to kill ACP after they were allowed to feed on their leaves. All these successes were achieved even though CTF experienced high flux of employees, had to move to temporary location for six months, and was exposed to adverse effects of hurricane Irma.Future opportunities for the CTF are determined by the needs of Florida Citrus Industry. As those needs change from the search for the solution to HLB and canker to a development of specialty fruit with consumer-oriented traits in post-HLB era, CTF will be there to support all these efforts.
Update for this quarter: The Stover team has submitted paperwork to renew their primary BRS permit, which covers the transgenic materials planted by Z. Mou, J. Jones, T. McNellis as well as USDA scientists. A new permit has been approved (AUTH – 0000043619 effective 12/17/2020) for material with “Confidential Business Information” for a project led by R. Shatters. The new BRS permitting system called e-file has used us a first trial and it has been very arduous and time-consuming. Information was provided for the BRS review of the transgenics at Picos Farm for CREC (Dutt, Grosser and Gmitter) who maintain a separate permit. UF collaborators have been permitted into the test site and samples and data have been collected. Data were collected on McNellis trees by USDA. Samples we previously collected have been processed by technicians at home (with APHIS-BRS permission) and are ready for qPCR. A manuscript has been prepared and submitted for canker data collected in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside. Previously Stover analyzed data on canker incidence in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside, from data collected 8/17, 9/19, and 9/20, Most notably: Almost all accessions with lower ACC lesion incidence were hybrids vs. pure trifoliate, though a few pure Poncirus had lower ACC than most. Based on chloroplast genome data from 57K Affymetrix SNP chip, provided by M. Roose, 11 of 33 reported seed parentage for hybrids was inaccurate, convention of female first was not followed. Of 34 hybrids validated, similar numbers had Poncirus, grapefruit, and sweet orange chloroplasts. Chloroplast type did not affect ACC incidence, but in each year accessions with grapefruit chloroplasts had small but statistically higher ACC severity than those with Poncirus chloroplasts. Hybrids of Citrus with Poncirus have markedly reduced ACC sensitivity compared to Poncirus, indicating that this trait is readily overcome in breeding. Seed from fruit harvested for transgenic gene flow experiment coninue to be processed for PCR. Previously established at the site: A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Availability of the test site for planting continues to be announced to researchers. Plantings: 1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit. 2) Under the Stover permit a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project. 5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant. 6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.). 7) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo. 10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls. 11) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type. 12) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State13)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
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