The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we continued propagating the transgenic lines that overexpress Arabidopsis defense genes and inoculated the previously generated progenies. The new progeny plants are growing in the greenhouse. The progenies obtained in the last quarter have been inoculated with Las-infected psyllids for two months and moved back to the greenhouse for symptom development. HLB symptoms on the plants have been carefully monitored and recorded. For objective 2, we performed the second round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results indicated that several lines of the HLB-resitant/tolerant transgenic lines are not chimeric. If these lines are confirmed to be HLB-resitant/tolerant in objective 1, they will be able to be propagated by grafting for industry use. For objective 3, we continued propagating the gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB and inoculated previously generated progenies. The new progeny plants are growing in the greenhouse. As for the transgenic progenies, those obtained earlier were inoculated with Las-infected psyllids and are currently in the greenhouse for symptom development.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,most of the transgenic lines produced have been confirmed for gene integration by conventional PCR and analyzed for gene expression using qPCR. 40% of the lines tested have been determined to be high expressers while the rest were medium to low in expression. Cuttings from the larger lines have been made and are being rooted in the mist bed for future challange with Diaprepes. A number of other potential root specific promoters have been identified from the phytozome database. qPCR gene expression analyses on non-transgenic leaves, flowers, fruit, phloem, seeds and roots have identified some that can potentially be used in the future for root specific gene expression. Results from some of these studies will be presented in the World Congress on In vitro Biology in the summer.
Our project is focused on the following objectives: 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. 2. Breeding of HLB tolerant/resistant processing sweet orange-like hybrids. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. The project began on 1 November 2015. In the late autumn, and through the winter and spring, we collected data from several ongoing field trials of rootstock cultivars throughout the state. Trees were assessed for HLB incidence and severity through all plantings, and yields and fruit quality was determined in selected replicated trials. Hybrid families planted in the field were evaluated to identify selections producing fruit that resembled sweet orange in appearance, and exhibiting few or no HLB symptoms. Fruit from the best of these were tested for juice quality and a few were also analyzed for volatile components and compared with standard sweet orange. Fruit samples were provided to a major juice company for processing and assessment of the juice for flavor. The entire collection of raw material produced by the breeding program and growing in groves at several different locations throughout the growing areas of Florida has been assessed for HLB symptoms, to identify individuals displaying few or no symptoms of HLB, regardless of parentage; ~4.3% of all trees were characterized as tolerant based on tree health and overall appearance. Specific highlights in the reporting period are below. The GFC/Indiantown project has been terminated and removed. A narrative with figures will be prepared on posted on the CREC website under Varieties and Rootstocks. Yield data and HLB ratings were obtained from the Wheeler multi-scion/rootstock cooperative trial. Difference among rootstocks and scion-rootstock interactions were apparent even among these 3 year old trees. Yield data were obtained from 3 year old trees on various rootstocks planted 8 x 15 in a CPI cooperative project. The CREC plant breeding team s collective field evaluation records have been reviewed, consolidated and organized for more efficient record keeping. A 3.5 year old planting of 220 OLL seedlings was assessed for HLB; two HLB-free seedling trees were identified and propagated for further study. 2016 crosses included several crosses of HLB-tolerant mandarins with sweet orange-like juice profiles with OLL oranges in efforts to develop seedless HLB-tolerant sweet orange-like hybrids for processing and fresh market. 2016 rootstock crosses (diploid and tetraploid) featured HLB tolerant parents, and included crosses of LB8-9 with Phytophthora resistant parents. Two more sets of gauntlet rootstocks were rotated into a hot psyllid house, approximately 100 trees were prepared for field planting (this quarter). Gauntlet trees at Picos Farm were assessed; 10 selections 3-4 years old are promising and being further monitored. Yield and fruit quality data were collected again from 6 and 7.5 year old trees in the St. Helena Project. A public Field Day was held to update interested growers on progress with the trial and selected UFRs.
Objective 1. (Greenhouse experiment): seedlings of the required rootstocks: x639, Swingle, WGFT+50-7, UFR-3 and UFR-15 were moved up to round citripots and moved into the HLB screening greenhouse. Liners are approaching stick-grafting size, and it should be possible to begin grafting in 2-3 months. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). All the pretreatment data have been collected on trees, leaf and soil nutrient analysis were done to obtain a complete pretreatment tree assessment. First fertilizer applications were made at both the field locations and data was collected. All the data and photographs have been recorded for mid-year evaluation. Second round of applications are being made currently. All the micro-nutrient and foliar application products have been collected. Macro-nutrient applications will be made in August. Currently, all the data from pre-treatment and mid-year are being analyzed to confirm any differences among the treatments. Ojective 5. (funded by Orie Lee, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. The second treatment was applied May 30th, 2016. There was no further activity during this period. 1. Control no extra nutrition 2. Harrells St. Helena mix (2lbs per tree) 3. Harrells St. Helena mix (2lbs.)+ 2x TigerSul manganese (90 gm) 4. Harrells St. Helena mix (2lbs.) + 2x Florikan polycoated sodium borate (32 gm) 5. Harrells St. Helena mix (2lbs.) + 2x TigerSul manganese (90 gm) + 2x FL sodium borate (32 gm) 6. 4x TigerSul manganese (180 gm) 7. 4x Florikan polycoated sodium borate (64 gm) 8. 4xTigerSul manganese (180 gm) + 4x Florikan polycoated sodium borate (64 gm) Baseline yield data was taken from each plot in January, 2016; pfd is severe and will impact the 2017 harvest. Donated micronutrient treatments were also applied at the Hughes Post Office block – where there were yield increases this past season, enhanced by specific treatments, especially those containing both boron and manganese. Trees look exceptional at present, and we expect to see a yield increase for the 2nd year in a row since we began the study.
Objective 1. (Greenhouse experiment): seed was extracted, seed coats removed using a chemical-removal procedure and planted of the needed rootstocks: x639, Swingle, WGFT+50-7 and UFR-3. Seeds germinated well and healthy seedlings are now being transferred to larger pots as necessary to begin the treatments/grafting. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). All the pretreatment data have been collected on trees, leaf and soil nutrient analysis are done to obtain a complete pretreatment tree assessment. First fertilizer applications have been made at both the locations and data is being collected. Second round of applications will be made in June 2016. Ojective 5. (funded by Orie Lee, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. The second treatment was recently applied (May 30th, 2016). 1. Control no extra nutrition 2. Harrells St. Helena mix (2lbs per tree) 3. Harrells St. Helena mix (2lbs.)+ 2x TigerSul manganese (90 gm) 4. Harrells St. Helena mix (2lbs.) + 2x Florikan polycoated sodium borate (32 gm) 5. Harrells St. Helena mix (2lbs.) + 2x TigerSul manganese (90 gm) + 2x FL sodium borate (32 gm) 6. 4x TigerSul manganese (180 gm) 7. 4x Florikan polycoated sodium borate (64 gm) 8. 4xTigerSul manganese (180 gm) + 4x Florikan polycoated sodium borate (64 gm) Baseline yield data was taken from each plot in January, 2016; pfd is severe and will impact the 2017 harvest. Donated micronutrient treatments were also applied at the Hughes Post Office block – where there were yield increases this past season, enhanced by specific treatments, especially those containing both boron and manganese.
During this reporting period (January, February, and March, 2016), control plants that have been through the transformation process, but not containing the transgene, were generated and sent to Penn State, and they are growing well at the Penn State location. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. We call these the “transformation control” trees. The transgenic plants being produced for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. Dr. McNellis has applied for and been granted an APHIS BRS permit to send propagated FLT-antiNodT plants to Florida for replicated testing for HLB resistance in Dr. Gottwald’s lab. However, before sending the plants, we must obtain the needed Florida state permit (FDACS 08084), and this is in progress. Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) transformed ‘Hamlin’ sweet orange and the ‘Carrizo’ rootstock with the FLT-antiNodT expression construct, and we received these plants at Penn State in early April, 2016. During the next reporting period, we will test these plants for expression of the FLT-antiNodT anti-HLB protein. Dr. McNellis will also produce rooted cuttings of all these lines for later testing for HLB resistance in Florida.
During this reporting period (January, February, and March, 2016), control plants that have been through the transformation process, but not containing the transgene, were generated and sent to Penn State, and they are growing well at the Penn State location. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. We call these the “transformation control” trees. The transgenic plants being produced for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. Dr. McNellis has applied for and been granted an APHIS BRS permit to send propagated FLT-antiNodT plants to Florida for replicated testing for HLB resistance in Dr. Gottwald’s lab. However, before sending the plants, we must obtain the needed Florida state permit (FDACS 08084), and this is in progress. Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) transformed ‘Hamlin’ sweet orange and the ‘Carrizo’ rootstock with the FLT-antiNodT expression construct, and we received these plants at Penn State in early April, 2016. During the next reporting period, we will test these plants for expression of the FLT-antiNodT anti-HLB protein. Dr. McNellis will also produce rooted cuttings of all these lines for later testing for HLB resistance in Florida.
Our significant progresses during this reporting time period are: 1) Using mature shoot segments of Valencia and Washington navel, we have demonstrated that the Kn1 gene can improve transformation efficiencies by approximately 2-fold compared to the control vector, which is much lower than those observed in juvenile citrus transformation. 2) We used an epigenetic modulator in our transformation experiments and observed about a 2- to 3-fold increase in overall transformation efficiency in mature tissues of Valencia and Washington navel oranges. We further demonstrated that the epigenetic modulator produced a 10-fold increase in shoot regeneration efficiency of mature citrus with no transformation when compared to the controls. 3) With expression of a 35S::GUS gene containing an intron as an indicator, we examined Agrobacterium infection and T-DNA integration activities in mature citrus using tobacco leaf discs and juvenile citrus tissues as references. Consistent with the fact that tobacco leaf discs can be efficiently transformed with Agrobacterium, we observed very high levels of transient and stable expression of GUS in the cut edges of tobacco discs. When juvenile citrus tissues were used for Agrobacterium infection, we observed reasonable levels of both transient and stable GUS gene expression. Using mature explants of Valencia, however, we observed extremely low levels of transient and stable expression of the GUS gene. As we have shown that although both the Kn1 and Ipt gene dramatically enhanced transformation efficiencies of juvenile citrus via increased shoot regeneration, they were far less effective at improving transformation on mature citrus tissue. Also, in mature Valencia and Washington navel oranges, we found that using an epigenetic modulator led to about 10-fold increase in shoot regeneration, but only a 2- to 3-fold increase in transformation efficiency (i.e., transgenic shoot production). We hypothesized that after improved shoot regeneration, Agrobacterium-mediated T-DNA integration remained the major challenges to improving mature citrus transformation. We are now working to enhance efficiencies of Agrobacterium-mediated stable T-DNA integration. Combining the various molecular tools we have, we would like to develop a ‘vector’ that is highly efficient and genotype-independent for mature citrus transformation. One manuscript reporting the drastically improvement of six citrus cultivars including a lemon cultivar has been published: Hu et al (2016): Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant cell, Tissue and Organ Culture. 125: 81-91. Two manuscripts are under preparation, reporting some of the results summarized above.
New field performance information is being collected on about 400 new SuperSour-type rootstocks in field trials. Performance attributes being assessed include tree growth, tree health, fruit yield, fruit quality, tolerance of high pH soil, and tolerance or resistance to HLB and other diseases. New rootstocks are only appropriate for large-scale grower use when outstanding performance has been documented by statistically replicated trials over multiple years. It is anticipated that at least one of the best new SuperSour rootstocks will be released for commercial use within 3 years. In the meantime, outstanding performance has been documented for US-802, US-897, and US-942 rootstocks over multiple years in trials affected by HLB, and these rootstocks are available in large numbers through commercial nurseries. During this quarter, trees in field trials were measured for tree size and scored for health, HLB symptoms, and samples were collected from some groups for PCR detection of Las infection. During this quarter, yield and fruit quality data were collected from 3 rootstock field trials with late maturing scion varieties. The most outstanding rootstock in a long-term Valencia trial on a flatwoods site in Collier County was US-802 rootstock, which yielded significantly more fruit than every other rootstock in the trial, including the second highest yielding rootstock, US-942. Every tree in this trial is infected with Las, but trees on US-802 still yield much more fruit than trees on any other rootstock. Analysis was completed on data from several established trials to assess relative rootstock performance, rootstock effects on yield, fruit quality, tree size, and HLB symptom development. A comprehensive presentation on standard and new rootstocks was made at the Florida Citrus Growers Institute. A new paper was prepared, providing a comprehensive comparison of field performance for the new USDA rootstocks with other standard rootstocks, and was submitted for publication. Trees in the USDA nursery on a large number of advanced rootstock selections, especially SuperSour-type, were continued in propagation for field trials to be planted in 2016. Nursery experiments were conducted with promising new rootstocks to determine nursery-related traits important for commercial use. Cooperative work continued with commercial nurseries involved with micropropagation, to facilitate more rapid deployment of the best new rootstocks. A cooperative project has been initiated with Dr. Ute Albrecht (UF, Immolakee) to compare trees on rootstocks propagated by seed, cuttings, and micropropagation, so that growers can have confidence that rootstocks propagated by the different methods will have equivalent performance. Greenhouse experiments continued to assess rootstock tolerance to HLB, CTV, and high pH. Research was initiated to more fully study the HLB-tolerance of trees formed by grafting HLB-susceptible scions on HLB-tolerant rootstocks. A better understanding of this behavior will help to more quickly identify new rootstocks with higher and more reliable levels of HLB tolerance in the field. Cooperative planning continued with UF researchers at several locations, to submit grant proposals to USDA NIFA to help fund expanded rootstock research and development efforts. Cooperative grant-funded work continued with UF researchers to establish acid fruit rootstock trials in South Florida. Cooperative grant-funded work continued with UF researchers and a commercial nursery to propagate trees for use in multiple rootstock field trials sponsored by the HLB MAC program. Trees from the commercial nursery are scheduled to be planted into six cooperative field trials in 2016, and six more field trials in 2017.
Good progress was made on work to identify metabolite profiles associated with tolerance to HLB and other stresses in advanced rootstock selections, and validate the effectiveness of these metabolite profiles for selection by comparison of existing rootstock selections within the USDA program. The work will focus on 12 rootstocks where previous studies have identified relative differences in tolerance to Las infection. Specific studies were continued to identify key metabolic compounds and collect the first stage of information to be used in the validation process. In this quarter, leaf and root samples from one greenhouse test with different rootstocks and three field tests with different rootstocks were processed and shipped to the West Coast Metabolomic Center for metabolomic analysis by gas chromatography-time of flight (GC-TOF) mass spectrometry (MS). The GC-TOF-MS analysis for the first cycle of samples is expected to be completed in the next quarter and results available for detailed analysis by our research team in Ft. Pierce and Immokalee. In preparation for the second cycle of sample collection in the next quarter (first year), clean seedling and budded trees were prepared in the greenhouse, and samples were collected and analyzed for PCR verification of Las and CTV infection status for trees in two different field trials. The experimental design of the first and second cycle of samples collected in the first year will allow comparison of metabolite profiles of the same genotypes in different seasons, providing critical information about how metabolite profiles change over the growing season. In preparation for sample collection in the second year of the project, clean seedlings and budded trees were prepared for greenhouse studies, and clean seedlings and budded trees were prepared for field planting. At the field site, trees were removed and land prepared for planting of the new trees being used in the study. Material was prepared for controlled Las inoculation of trees to be studied in the greenhouse and the field. The project was revised to include a new researcher at University of Florida in Immokalee, Dr. Ute Albrecht, who has experience and special expertise in this research field. Grant funds will be provided through a Non-Assistance Cooperative Agreement to support this work in Immokalee. The project changes actually reduce the overall cost of the project, but will significantly increase productivity and the opportunity for success. In this quarter, a manuscript on a preliminary metabolomic study by our team was submitted for publication. That study used GC-TOF-MS analysis to compare metabolite profiles of six rootstock cultivars infected and not infected with Las. The study identified numerous chemically unknown compounds that appeared strongly associated with Las tolerance and offer good opportunities for further study and manipulation of the tolerance behavior. The study demonstrated striking metabolic differences between the HLB-sensitive rootstock Cleopatra and the HLB-tolerant rootstocks US-897 and US-942, both with and without Las infection, and will serve as a foundation for continuing work under this grant.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and six PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for confirmation of transformants; selection is underway. In addition, we will add the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs have been created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Constructs have also been created in which Bs2 is co-expressed with other R genes that may serve as accessory factors for Bs2. Constructs with tagged Bs2 have been confirmed to function in transient assays, and protein expression has been confirmed by immunoblot. These constructs have also been transformed into Arabidopsis for analysis, and two constructs have been provided to the Lake Alfred transformation facility, Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Four CRISPR constructs are being created in the Staskawicz lab: C1) A construct targeting two sites that will produce a 100 bp deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); C2) A construct targeting a site overlapping the two conserved leucines; C3) C2 with the addition of a bs5 repair template for Carrizo that will not be cut; and C4) C2 with a similar repair template for Duncan grapefruit. The constructs have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. To aid in the selection of positive transgenics, we are currently adding a GFP reporter into each CRISPR construct.
Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance has revealed potentially valuable tolerance and indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. In August 2010, the plants were established at Pico’s farm in Ft. Pierce FL. Despite the high incidence of mottle in ‘SugarBelle’/SourOrange, it had the greatest overall increase in diameter. ‘SugarBelle’ and ‘Tango’ (which were not on the same stock as ‘Hamlin’ and so results should be viewed as comparing cultivar/rootstock combinations) were the healthiest in overall appearance in 10/15 and had the most fruit (88 per tree). All cultivars except sweet oranges and grapefruit are progressing in production, but production was compromised in all varieties by the severe HLB pressure at this site, and commercial value of the observed tolerance remains uncertain. A mapping population of Fortune x Fairchild has been planted (collaborating Roose and Gmitter) along with related material, in an effort to identify genes associated with tolerance in the mandarin phenotypic group. The citrus relatives planting (85 seed source genotypes from the gene bank) has been assessed for growth and apparent HLB tolerance. Most trees containing citron in their pedigree have markedly greater canopy densities and greater tree size than other accessions in the Genus citrus. One alleged standard sour orange looks much healthier and is much larger than other sour oranges. Chemical, morphological and transcriptome characteristics are being assessed to determine what factors are associated with observed tolerance, so they can be used in early screening and possibly directed transgenesis. A paper describing HLB resistance in this population has just been published in Plant Disease. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce FL. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. Generation of new chimeras has been difficult. An existing periclinal chimera (Satsuma and Poncirus) has been imported,has been with DPI two years, and agreement has been reached to release this to us for testing. A method for the rapid identification of potential sources of HLB resistance is being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are easily detectable in most plants at 3 weeks Seedlings of Hamlin and Dancy show marked CLas proliferation and systemic movement from 3-6 weeks after exposure to ACP. By nine weeks after exposure, susceptible genotypes can be clearly distinguished from reported resistant material by higher CLas levels in roots. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types were propagated on x639. Replicated trials with standards have been established, in cooperation with G. McCollum. Six locations each of all sweet orange-like together and 4 with all mandarins were established in replicated block plantings with 6-8 trees of each cultivar at each site (in Ridge, IR and Gulf coast). Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, and are being grown for field testing of HLB resistance.
Citrus trees transformed with a chimera AMP (thionin-D4E1) and the thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Replicated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Las titer was checked from new flush rough lemon leaves at six month after grafting. Las titer from 18.6-36.5 was detected in 90% of transgenics expressing the chimera. Some transgenic lines expressing thonin had lower Las titer(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. Root samples from control plants and transgenic Carrizo expressing chimera and thionin were taken nine months after grating inoculation. Our results showed transgenic Carrizo expressing thionin significantly inhibited Las growth (0.5% of control level) compared to control and transgenic Carrizo expressing chimera. Antibody against thionin will be produced for Western detection. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic Carrizo plants carrying second generation AMPs were obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA will be isolated and Las titer will be tested. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las titer will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We are currently analyzing the genome modified plants using transgenic approaches including off-targets. To generate non-transgenic DNA free canker resistant citrus, Cas9 containing nucleus localization signal was overexpressed and purified. The purified Cas9 showed activity in cutting target sequence and will be used to generate canker resistant plants.
The first quarter of 2016 was very strenuous for Core Citrus Transformation Facility (CCTF). Two out of five employees left the facility in January and were eventually replaced by the new members of staff. CREC Center Director informed CCTF of possible move of the lab to the new site in March. Although March 24th was anticipated date for the move, that did not happen and CCTF still operates from its present location. CCTF received unprecedented number of orders (26) within the last quarter. Such a high volume of incoming orders called for an additional increase in production capacity of CCTF. I have purchased necessary consumables and tools for this transition and have taken steps to gradually ramp-up the input of starting material for experiments. However, uncertainty associated with possible move to new location prevented search for additional employees. I expect new recruit to begin working in the month of April. Partial increase in work load in March was little overwhelming for the present labor force and resulted in loss of some transgenic shoots and plants. I hope that when all aspects of CCTF functioning stabilize, so will the level of our production. The newly announced date for the move to new location is June 17th and I will try to organize it in such a way so that it will affect productivity of CCTF to the least possible extent. Between January and April, CCTF produced 57 plants. These plants belong to newer orders placed within the last 12-15 months. Four of the produced plants were Valencia oranges, three were Pineapple sweet oranges, eight were Carrizo citrange, and the rest were Duncan grapefruit. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. They are at the stage when they could easily be propagated by cuttings. I am awaiting further instructions on what to do with these plants.
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