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. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. 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. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol may need to be optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. 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. A construct targeting a site overlapping the two conserved leucines has 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. A replacement recessive bs5 allele will be added, and this construct will be prioritized for transformation into Carrizo citrange for proof of concept. Resulting plants with biallelic mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
New rootstocks are appropriate for large-scale grower use when outstanding performance and yield have been documented by multiple statistically replicated trials over multiple years. Outstanding performance has been documented for US-802 and US-942 rootstocks over multiple years in trials affected by HLB, and these rootstocks are available in large numbers through commercial nurseries. Other released new USDA rootstocks with outstanding performance documented at fewer sites and harvests are also commercially available, and should be used for smaller scale plantings until there is more experience with those varieties. It is anticipated that at least one of the best new SuperSour rootstocks will be released for commercial use within 3 years, based on outstanding performance. Field performance information is being collected on more than 400 new rootstocks in 17 different replicated field trials, include tree growth, tree health, fruit yield, fruit quality, and tolerance or resistance to HLB and other diseases. During this quarter, data collection was focused on tree size, health, and PCR evaluation of infection by Las. Data was collected from a replicated greenhouse trial to compare Valencia tree performance on the most HLB-tolerant rootstocks under optimum management conditions. The study will also follow field performance of trees on the most HLB-tolerant rootstocks in the first 1-3 years after they become infected with Las. Focused study in this trial will help to more clearly measure the ways in which tree performance is affected by HLB and estimate the economic viability of commercial production on the most tolerant rootstocks. 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 2017. New trials in propagation continue to focus on sweet orange scion, but include some plantings to assess performance with new scions that have better tolerance to HLB. 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 is underway with Dr. Ute Albrecht (UF, Immolakee), Agromillora and Rucks Citrus Nursery 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. A multi-year collaborative grant proposal was developed with the UF citrus breeding team and other UF and University of California researchers, and submitted to USDA NIFA to help fund expanded rootstock research and development efforts. 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. Extensive information was provided, and cooperative planning for continuing rootstock testing and trials was begun with Catherine Hatcher. An invited presentation on tolerance to HLB in rootstocks was made at the International Citrus Congress in Brazil. A new paper that was developed to provide a comprehensive comparison of field performance and nursery characteristics for USDA rootstocks with other standard rootstocks was accepted, and should appear in the October issue of the journal HortScience. This publication will be a valuable reference for use by growers and nurseries in planning for which rootstocks to use in new plantings.
Good progress was made in the validation of the effectiveness of metabolite profiles for selection of HLB tolerant rootstocks. Focused studies were continued using plant material established in the USHRL greenhouse and field during the previous year. Trees that were inoculated with Las for controlled greenhouse and field metabolic studies were sampled and tested by PCR for Las infection, to identify those suitable for the next group of metabolic samples. Metabolomics data was received from the first set of grafted and non-grafted rootstocks that was sent to the West Coast Metabolomics Center (WCMC) at UC Davis earlier this year for GC-TOF-MS analysis. The research team at the Southwest Florida Research and Education Center (SWFREC)/UF began working with this first data set, in which over 500 metabolites were detected (30% of known chemical structure and 70% of unknown chemical structure) in four standard rootstocks (Cleopatra mandarin, Swingle citrumelo, Ridge Pineapple, and Sour orange), which were grown as seedlings and as grafted plants under greenhouse and under field conditions. Analysis of this first data set is currently being conducted to 1) identify metabolites associated with rootstock traits, 2) decipher tissue-specific (roots and leaves) metabolite profiles and their value for rootstock characterization, 3) identify rootstock effects on scion, and 4) identify significance and impact of environmental factors on metabolite profiles. Preparation of a manuscript for publication of preliminary data is in progress. The results from the second data set, for which samples were submitted to WCMC in June and July, and which include rootstocks with well-characterized responses to HLB, are required to focus in detail on the discovery of metabolites associated with tolerance to HLB and other positive rootstock attributes. The team is in regular contact with WCMC and was informed that results from this data set will be available in the coming weeks. Research results from a previous data set on leaf metabolite profiles of HLB tolerant and susceptible rootstocks were presented at the International Citrus Congress in Brazil in September 2016. A presentation of results from metabolomic studies is anticipated at the 5th International Research Conference on Huanglongbing, to be held in Orlando next spring.
The citrus relatives planting (85 seed source genotypes from the gene bank) has been assessed for growth and apparent HLB tolerance. Within the genus Citrus, measures of tolerance msuch as canopy density, health, and tree size, correlate positively with % citron in pedigree, with r2 of 0.3-0.6. A manuscript describing apparent tolerance to HLB in citrus and citrus-related germplasm has been submitted. Chemical, morphological and transcriptome characteristics are being assessed to determine what factors are associated with observed tolerance (three distinct projects), 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. These data suggest that citron-derived germplasm should be used as part of a portfolio of citrus cultivar improvement efforts for Florida production. 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. Data taken in the next quarter should show marked distinctions between genotypes in HLB-tolerance. 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 and is now being grown at USHRL 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 By nine weeks after exposure, susceptible genotypes can be clearly distinguished from reported resistant material by higher CLas levels in roots. Averaged across genotypes and tissues, total CLas per tree was 5 billion in week 3 after ACP exposure and doubled every 3 weeks through week 12. This should be especially useful for screening anti-Las transgenics. 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). 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). 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. 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 and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Propagated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Twelve months after graft inoculation, Las titer was examined and compared in old leaves (most with HLB symptom), young expanded leaves (with or without HLB symptom) and fibrous roots of transgenic and control plants. Our results showed again that transgenic citrus expressing Mthionin has lower Las titer (200-1800X lower) compared to control and transgenic plant expressing chimera. These data suggest transgenic plants expressing thionin are promising for HLB resistance ( published in Frontiers in Plant Biology). Antibody against thionin has been produced for investigating the correlation of thionin expression and HLB resistance. 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. Furthermore, the third generation chimeral peptides were designed based on citrus thionins, the vector construction were finished and citrus transformation are underway. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 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 was isolated and Las titer was tested. Our preliminary results showed that transgenic trees expressing NbFLS2 can reduced Las titer. 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 tilter 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.
This project (Hall-15-016) is an extension of a project that came to a close last summer (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. CRDF funds for the inoculation program cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. Update: Two technicians funded by the grant have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of October 12, 2016, a total of 9,309 plants have passed through inoculation process. A total of 293,895 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. As reported in the last progress report and reiterated here, research recently showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant). Research results will soon be available in which we are comparing success rates using ACP colonies on lemon versus citron, and using ACP colonies from greenhouses versus walk-in chambers.
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. T 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 have confirmed and analyzed the genome modified plants including off-targets. No side effect was observe. Publications from this project 1. Jia H, Zhang Y, Orbovic V, Xu J, White F, Jones J, Wang N. (2016) Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnol J. doi: 10.1111/pbi.12677. 2. Jia, H., Orbovic, V., Jones, J.B. and Wang, N. 2016 Modification of the PthA4 effector binding elements in Type I CsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating Xcc.pthA4:dCsLOB1.3 infection. Plant Biotechnol. J., 14: 1291 1301. doi: 10.1111/pbi.12495. 3. Jia H, Wang N. 2014 Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One. 9(4):e93806. doi: 10.1371/journal.pone.0093806
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 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 have confirmed and analyzed the genome modified plants including off-targets. No side effect was observe. The data has been summarized into one manuscript and submitted. We are currently focusing on generating EBE mutated plants in both alleles and generating plants which do not contain cas9 and sgRNA in the plant chromosome.
Core Citrus Transformation Facility (CCTF) moved to temporary location in the last week of July. The staff tried to minimize the impact of moving on the operation of the lab. We ended up canceling only half week worth of experiments. However, the location of the lab itself presented a challenge and has negatively affected productivity. Within the last two months, the death rate of micro-grafted positive transgenic shoots more than doubled. Some of previously grafted and established plants also exhibited signs of poor growth and few of them died. I have instructed the staff to do grafting in lab in different building and keep grafted shoots out of CCTF. We also consulted with the member of Mature Tissue Transformation who does grafting and got some advice from him. The orders continued to come to CCTF in high numbers. Within this quarter we have received 12 more orders. Altogether, we received 50 orders since the beginning of the year. Resistance to canker and HLB continue to dominate the orders placed at CCTF. CCTF produced 60 plants within the last quarter. Out of those plants, five were Carrizo citranges, three Swingle citrumelos, two Pineapple sweet orange, and 50 Duncan grapefruits. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. Those plants are very tall now and will require additional care soon. If they are supposed to be propagated by cuttings, that should be done very soon, since this type of propagation is not very successful when done during the months of November, December, and January.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,cuttings have been initiated for selected transgenic lines. Molecular analyses has been completed and a wide variation in transgene response is being observed. Due to yet unexplained reasons, transgenic lines expressing the ASAL transgene are performing better horticulturally than lines with expressing the APA transgene. Rooted cuttings are expected to be available in the fall.
Production from the mature transformation pipeline during the last quarter was interrupted due to the move to the packinghouse while the AC in #103 is repaired. We lost time due to unforeseen delays in connecting utilities & other issues (no hot water, mold, AC issues, electrical problems, & autoclave issues), the physical move itself, & the set-up in the temporary laboratory. Presumably, we will lose some time moving back to #103 at the end of October. We continue to provide transgenics to Drs. Dutt, Grosser, McNellis, Mou & Wang. An additional 12 independent, transgenic mature Valencia and Hamlin scions (some events were duplicated to equal 15) were shipped to Dr. McNellis at Penn State University this quarter. These plants were small & had not undergone the secondary graft, because in vitro plants are easier & cheaper to ship than large citrus trees. Apparently they transplant very well. Ten transgenic Hamlin & Valencia scions (with duplicates & triplicates) were produced for Dr. Mou that also had not undergone secondary grafting. Secondary grafts have been performed on all plants for Drs. Grosser, Dutt, and Wang, to enhance the growth of the transgenics. Six additional transgenics were transferred to Dr. Wang. Approximately 21 transgenics have been produced for Dutt & Grosser since the last report, of which 16 had rooted & were transferred. Dr. Hao Wu presented a talk at the ASHS meetings in Atlanta, GA. His talk was entitled, “Biolistic transformation of citrus”, ASHS Annual Meeting, Atlanta, August 7-11, 2016. Recently we introduced Kurhaski and Glen Navel cultivars for Drs. Grosser and Dutt through shoot-tip grafting (STG). Kurharski is a rootstock similar to Carrizo but it has some nematode tolerance, and Glen Navel sweet orange is pollen sterile, so it will provide a contained system to prevent transgene flow. Some of the budwood from FDACs in Chiefland was contaminated with the yeast endophyte, so it was essential that STGs be conducted on all introduced material prior to tissue culture. Mandarin & pummelo are being introduced for Dr. Wang. Phosmannose isomerase (PMI) selection works well after biolistics in immature citrus & it significantly decreases the number of escapes compared with nptII selection. Using PMI selection after biolistics, we were able to produce an additional 10 immature transgenics while significantly decreasing the number of nontransformed escapes. We are still investigating whether PMI will be useful for mature citrus transformations. Initial observations indicate that mannose is toxic to mature shoot development, but tests are being conducted to determine the effect of mannose after the shoots have formed on sucrose medium.
During this reporting period (April, May and June, 2016), we analyzed over two dozen newly transformed plants from Dr. Janice Zale’s program (University of Florida Mature Citrus Transformation Facility, Lake Alfred, FL). These plants are ‘Hamlin’ sweet orange and ‘Carrizo’ rootstock lines. However, we were not able to detect the FLT-antiNodT fusion protein expressed in any of these lines by protein immunoblotting. This contrasts with our results in ‘Duncan’ grapefruit, where nearly all the independently transformed lines expressed detectable levels of full-length FLT-antiNodT fusion protein. Further molecular analyses during the present funding period indicated that the transgene encoding the FLT-antiNodT fusion protein was not detectable in the ‘Hamlin’ and ‘Carrizo’ lines, although the markers for transformation (kanamycin resistance and green fluorescent protein) were detectable. Unfortunately, these results suggest that these plants are not actually transformed with the FLT-antiNodT fusion protein transgene. However, our original and proposed activity, transforming ‘Duncan’ grapefruit and testing for HLB resistance, was successful and is still in progress. Plants are continuing to be propagated for testing for HLB resistance at in Dr. Tim Gottwalds’ lab at Ft. Pierce. When sufficient plants are ready, these will be transferred into an HLB transmission greenhouse and exposed to Asian citrus psyllids carrying Candidatus Liberibacter asiaticus. During the present funding period, protein samples from the Ft. Pierce trees were analyzed by protein immunoblotting and these trees were found to be expressing moderate to high levels of FLT-antiNodT fusion protein, which is very promising. Although Dr. McNellis had planned to send rooted clones of the seven ‘Duncan’ lines at Penn State to Dr. Gottwald’s lab at Ft. Pierce, the permit for such transfer was not approved. Transporting citrus back to Florida is unlikely to ever be approved. Dr. McNellis then pursued an alternate plan to transfer plants to Ft. Detrick, Maryland, to Dr. Bill Schneider’s lab, for testing for HLB resistance. Dr. Schneider has agreed to participate in this collaboration, and Dr. McNellis has applied for a USDA APHIS permit for this plant transfer. This application is still under review by USDA APHIS.
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. Annual data collection from several ongoing field trials of rootstocks for the 2015-16 season was completed. Trees were assessed for HLB incidence and severity through all plantings, and yields and fruit quality was determined in selected replicated trials. Mutant selections of Valencia orange, produced and planted 10 years ago were found to be 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. The CREC plant breeding team s collective field evaluation records have been reviewed, consolidated and organized for more efficient record keeping, and new files have been added. Newly released early Valencia cultivars EV-1 and EV-2 ( were propagated at the CREC as potential legal budwood increase or foundation trees and distributed to 20 licensed nurseries to expedite introduction to our industry. These have the potential to replace Hamlin and help improve the quality of Florida juice products. Approximately 1000 liners of gauntlet candidate rootstocks produced from rooted cuttings were grafted with either EV-1 or potentially HLB tolerant OLL seedling lines (four lines being tested) for subsequent field evaluation. Gauntlet HLB Screening: HLB-infected Valencia budstick grafting of new candidate rootstocks continued, approximately 150 new hybrids were grafted during this quarter. Two more sets of gauntlet rootstocks,were rotated into a hot psyllid house and approximately 100 trees were prepared for field planting. Gauntlet rootstock and transgenic trees at Picos Farm were sampled and PCR tested. The large majority of the best gauntlet trees remain PCR+, but Ct values were generally higher than commercial trees, indicating lower bacterial titer in the best performing trees. One healthy tree remains PCR-negative. Several transgenic trees remain PCR-negative or have very high Ct values after 5 years, mostly trees containing NPR-1 or SABP-2, and 11 trees containing Xa21. Two-year old transgenic trees containing the Valencia beta-glucanase gene look quite good. Six super-root ‘sports’ of UFR rootstocks (from UFR 1,3 and 6) being tissue propagated at Ruck’s nursery were identified by Beth Lamb. She provided us the cultures and we grew out liners from these cultures and they were budded with a potentially HLB-tolerant OLL seedling line for field testing. These ‘sports’ grow feeder roots much more quickly than the original clones – which could enhance their performance against HLB. We produced approximately 300 cuttings of rootstock SG-6-50 – recovered from cut-off field trees planted near Clewiston in 1997. This rootstock candidate is a hybrid of Smooth Flat Seville and Swingle, and is showing good HLB tolerance. Original seed trees produced seedy, polyembryonic fruit, but were destroyed by the canker eradication effort. These liners will be utilized in the Grosser/Gmitter/Bowman MAC project.
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 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 have confirmed and analyzed the genome modified plants including off-targets. No side effect was observe. The data has been summarized into one manuscript and submitted. We are currently focusing on generating EBE mutated plants in both alleles and generating plants which do not contain cas9 and sgRNA in the plant chromosome.
The general goal of this project was to rapidly propagate complex citrus rootstock material for field testing in the Indian River citrus production area. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Dr. Jude Grosser assumed responsibility for completing the project, following the resignation of Dr. Barrett Gruber. Ms. Amy Dubois continued as the OPS assistant taking care of the trees at the IRREC, she continues in this roll. The final inventory of recovered liners from the micropropagation effort is provided below. All trees in group B have been grafted, and grafting of Group A is underway (with assistance from Dr. Ahmad Omar). Trees will be planted in field trials as they reach adequate size (this will happen after the ending date of the project). Since we now have new cybrid grapefruit clones showing improved tolerance to canker (10-fold better) in greenhouse screens, we decided to use these clones as the scions (including cybrids of Flame, Ruby somaclone N11-11, and White Marsh). These cybrid grapefruit clones contain cytoplasm from ‘Meiwa’ kumquat. As mentioned, these new grapefruit clones have shown significantly improved canker tolerance in greenhouse assays as compared to traditional grapefruit clones, and they are not considered GMO. Final trees to be tested in the field have potential to solve both the HLB and canker problems for Indian River grapefruit growers. Finishing all the trees and getting the field trial established will be accomplished with funding from other grants. Viable cutting inventory (Group A, tetraploid selections from ‘gauntlet’ screening): Rootstock # of liners recovered 1. A+HBPxCH+50-7-12-14 44 2. 46×31-00-S10x46x31-00-S11-S5 78 3. Orange 10 x Green 7-11-1 52 4. A+VolkxOrange19-11-5 90 5. A+HBJL2BxOrange14-09-7 71 6. A+HBJL2BxOrange19-09-31 14 7. A+HBJL1-09-14 25 8. A+FDxOrange19-11-11 50 Viable seedling inventory (Group B, diploid sour orange-types): 1. 46×20-04-S22 86 2. 46×20-04-42 94 3. 46×20-04-48 78 4. 46×20-04-S13 86
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