The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the HLB gauntlet : Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The original PI of this project Dr. Gruber has resigned, so Co-PI Dr. Jude Grosser is assuming responsibility for completing the project. Ms. Amy Dubois is the OPS assistant taking care of the trees at the IRREC, she will continue in this roll. All of the recovered cuttings and seedlings were evaluated and the inventories of liners that could make a good field tree are provided below. These will be grown to grafting size, grafted with selected scions (including a dark red grapefruit somaclone N11-15 showing possible tolerance/resistance to HLB) and planted in IR field trials. Viable cutting inventory: 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: 1. 46×20-04-S22 86 2. 46×20-04-42 94 3. 46×20-04-48 78 4. 46×20-04-S13 86
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 by grafting 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 the lower Las tilter(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. All root samples will be checked at 9 month for Las titer. 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 transferred to soil cones. 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. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. 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. Las titer will be tested periodically. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand 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 expressed gene with a nuclear-localization sequence has been identified and studied, including creating transgenic citrus that express this p235 gene. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR amplification, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product binds to citrus chloroplasts. 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.
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 three 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 will initiate more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs are being created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. 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. Three CRISPR constructs are being created in the Staskawicz lab: 1) A construct targeting two sites that will produce a deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); 2) A construct targeting a site overlapping the two conserved leucines, containing a bs5 repair template for Carrizo that will not be cut; and 3) a construct targeting the same site, with a repair template for Duncan grapefruit.
Chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) are being tested and are among the most promising transgenics we have created, along with thionin transgenics. Trees transformed with a chimera AMP showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated by grafting for HLB challenge. Transgenic Hamlin lines expressing thionin were grafted onto Carrizo for HLB challenge. Replicated transgenic Transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon. Promising resistance to HLB was observed based on plant growth and phenotype. Las titer is being checked from root and new flush rough lemon leaves. Two new chimeral peptides from citrus genes only were developed and used to produce many Carrizo plants and Hamlin shoots which will be tested soon as part of the next generation of this project. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. 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. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. 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. Transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 had increased resistance to canker by detached leaf assay but do not appear as potent as some other AMPs. Transgenic Carrizo and Hamlin with peach dormancy genes show no evidence of enhanced or accelerated dormancy A Las expressed gene with a nuclear-localization sequence has been identified and studied, including creating transgenic citrus that express this p235 gene. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR amplification, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product binds to citrus chloroplasts. 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.
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. Data on the growth rate, disease severity, and Candidatus Liberibacter asiaticus (CLas) titer levels have been collected since April 2012. During the 5-year period, there were significant differences in disease severity, stem diameter, and at times CLas levels among the varieties. 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. 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. As of December 2014, the first HLB symptoms were apparent. At 2 years since exposure, only a small proportion of trees of any genotype (including sweet orange), have marked HLB symptoms. Progress continues on the antibiotic treatment of HLB infected bud-wood to compare growth at different levels of CLas infection. HLB-infected budwood was treated with various concentrations of antibiotics and grafted on sour orange rootstock using 3 fairly HLB-resistant ( Temple , GnarlyGlo , and Nova ) 3 tolerant ( Jackson , FF-5-51-2, and Ftp 6-17-48), and 3 susceptible ( Flame , Valencia , and Murcott ) genotypes. Standard growth measurements and disease severity are evaluated and leaves sampled for qPCR analysis on a quarterly basis. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. One hundred and fifty etiolated seedlings of the trifoliate Rubidoux and the sweet orange Hamlin have been approach grafted together. Generation of new chimeras has been difficult. Several adventitious buds have emerged from the treated graft region, with several appearing to be chimeral. o increase the success rate, additional plants will be grafted over the next twelve months. An existing periclinal chimera (Satsuma and Poncirus) has been imported,has been with DPI two years, but has not yet been released 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 detetcable 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.. 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 and Eremocitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, and are germinating for field testing of HLB resistance.
A test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for almost six years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation, with the last samples collected this week), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
For the last three months, Core Citrus Transformation Facility (CCTF) continued to provide its service for production of transgenic Citrus plants. Within this period of time, there were only two new orders placed but three clients requested work on three previously placed orders bringing total number to five. Probably as a result of recent Knowledge Mapping meeting organized by CRDF there is an increased interest for transgenic plants and CCTF was already informed of eight new incoming orders. That does not include additional orders from another client with whom CCTF manager communicated for the last three months. Forty plants were produced during the last quarter which represents lower productivity than usual. Partially, this is the result of work on group of three orders that have not yielded any plants. Another reason is the low quality of seeds used to obtain seedlings as starting material for experiments. High majority of produced plants belong to eight orders placed within the last 12-15 months. Five plants belong to three older orders. Most of the transgenic plants are Duncan grapefruit and Valencia orange and one of them is Carrizo citrange. The work has begun on determination of level of expression of AtNPR1 gene in rootstock plants produced for CRDF. Once all plants produced by CCTF and Mature tissue lab are processed, those with the highest expression levels will be selected for further use and propagated.
Mature plant production continues with various genetic constructs with reporter genes from Drs. Dutt, McNellis and Wang. Additional scientists have expressed interest in our services. Transformation efficiencies have significantly increased with reporter genes. We are also trying to further increase Agrobacterium transformation efficiencies of mature citrus by incorporating vacuum infiltration and sonication treatments. These treatments significantly increased transformation efficiencies in immature citrus. In addition to Agrobacterium, we can now supplement plant production with plants produced using biolistics. Scions and rootstocks have been micropropagated (budded scions and rooted cuttings) into three replicates for one location. It remains to be determined whether we will continue micropropagation for replicates at additional locations. We have almost concluded screening Dr. Orbovic’s putative transgenics with qPCR to identify high-expressing lines. This work should be finished by the end of this month. I am switching to citrus pots in the growth room, which can be planted to higher densities than current planting densities. Recently we discovered that mature scion grows significantly faster after budding if the rootstock is not cut-off after the buds break.
Citrus Huanglongbing (HLB) poses the greatest threat to the survival of the Florida and US citrus industry. Research to incorporate HLB resistance/tolerance into citrus has been recommended by the National Research Council as one of the top priority topics for addressing the HLB threat. A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Identification, characterization and validation of candidate genes responsible for HLB-resistance are of the remarkable value to develop new HLR-resistant/tolerant citrus varieties. More than 70 different accessions of Citrus species and relatives have been observed in a field planting in Florida (with cooperation of Dr. E. Stover, USDA). Leaf samples were collected from about 450 trees. The titer of CLas in the HLB-infected plants was determined by TaqMan probe-based qPCR, and the HLB symptom severity on the plants was evaluated by experienced researchers. These accessions showed diverse responses (susceptible, tolerant and resistant) against HLB. Many HLB-tolerant citrus relatives were revealed, including Citrus latipes, Poncirus trifoliata, Severinia buxifolia and Microcitrus australis. The genomes of 15 citrus accessions have been sequenced to about 25x coverage (9 to 11 Gb/genome) and the genome of one Poncirus trifoliata accession has been sequenced to about 50x coverage (~21 Gb). Initial analysis indicated good quality of these sequence reads. We have begun a more detailed analysis of sequence polymorphisms and structural variations within candidate genes. We are also validating the expression of our shortlisted candidate genes by real time PCR among susceptible and resistant citrus accessions.
This project (Hall-15-016) is an extension of a project that recently came to a close (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 were hired during August and are being trained on how to establish and maintain colonies of infected psyllids, how to conduct qPCR assays on plant and psyllid samples, and how to run the inoculations. As of March 31, 2015, a total of 7,448 plants have passed through inoculation process. A total of 148,960 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations.
Most commonly grown citrus cultivars are sensitive to Huanglongbing (HLB). However, some citrus species and related genera are substantially more tolerant, such as Citrus jambhiri (rough lemon), Poncirus trifoliata and Microcitrus australis. Genome analysis will improve our understanding of the HLB tolerance mechanisms. Nuclear DNA from Citrus jambhiri was used to generate more than 235 million paired-end reads (2 X 100 nt) of the rough lemon genome. A reference-guided method was used to assemble the rough lemon genome. Based on analysis of the SNPs identified, rough lemon was found to have originated from the interspecific hybridization of mandarin, citron and pummelo, and its chloroplast is probably derived from mandarin. RNA-sequencing data was used for gene annotation, and some differentially expressed (DE) genes were identified. Most of DE genes were up-regulated in HLB affected trees, compared with non-affected trees. These DE genes were mainly involved in response to stress, carbohydrate metabolic process, response to abiotic stimulus, cell wall organization or biogenesis, ion transport and signaling. Based on our meta-analysis and co-expression network analysis of previously published gene expression data, we have shortlisted 2,000 HLB-responsive candidate genes in citrus (Du et al., 2015; Rawat et al., 2015). To identify sequence polymorphisms and validate the expression patterns of candidate genes, we have isolated genomic DNA and RNA from 20 citrus accessions. The DNA and RNA preps are being evaluated for quality and are to be sent to a commercial DNA sequencing company for large-scale sequencing on the HiSeq 2500. The sequencing data of these samples will be analyzed with the focus on 2000 candidate genes. The genomic indel variations and SNPs will be identified within these sequenced accessions for the selected candidate genes.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, several transgenic lines expressing each of our test genes have been regenerated and many of them have rooted. The rooted material has been successfully acclimatized to the greenhouse for growth and further testing. We are in the process of confirming the gene expression levels in these transgenic lines to isolated lines that have excellent gene expression. These selected lines will be propagated for subsequent evaluation with Diaprepes neonates.
The main accomplishments during this quarter: We repeated and confirmed the effects of K and I genes on genetic transformation for cultivars Valencia and Washington oranges and observed drastic increases in transformation efficiencies if compared to a conventional Ti-plasmid vector containing no K or I gene. We have confirmed that the K and I genes can drastically enhance transformation efficiencies of juvenile explants of 5 different citrus cultivars. We started test the effects of the K gene on transformation efficiency of a lemon cultivar. Lemon is difficult for genetic transformation. Our major efforts have been in testing the effects of the K and I genes and other factors on mature tissues. We used K and I genes to do genetic transformation of mature Pineapple orange. The K gene resulted in about two fold increases in transformation efficiency while the I gene produced about three fold increases in efficiency compared to control vector. We have also started testing effects of other factors on transformation of mature tissues in combination with the K gene. We have made some significant progress but microbial contaminations of adult tissues harvested from greenhouse grown trees have sometimes caused problems for us. One example is that we have repeated and confirmed the effects of the transport of an endogenous plant hormone in explants on shoot regeneration efficiency. We observed that manipulating that process improves shoot regeneration and transformation efficiency of juvenile citrus explants. We are testing the effects of the same manipulation on transformation efficiency of adult tissues of citrus.
The overall objective of this project was to use the PAMP receptors EFR and XA21 to engineer citrus plants resistant to both HLB (causal agent Candidatus Liberibacter asiaticus, CLas) and citrus canker (Xanthomonas axonopodis pv citri, Xac). Since neither receptor recognizes a PAMP from CLas, the first objective was to engineer a variant of EFR (EFR+) to recognize the elf18 peptide from CLas. This novel receptor would then be combined with XA21 or an XA21-EFR chimera that recognizes a PAMP from Xac. A number of strategies to engineer an EFR+ variant that recognized elf18-Clas were tested, but none were successful. These included PCR mutagenesis, screening of natural variants in an extensive Arabidopsis accession collection, creating targeted mutations based on the modeled interactions among elf18, EFR, and BAK1, and testing high-throughput strategies such as phage display and FACS. However, we were able to successfully create a functional XA21-EFR chimera. Although we did not generate an EFR+ variant that recognized elf18-CLas, expression of EFR and XA21 may still provide significant protection against citrus canker. Therefore, we transformed three constructs into citrus: EFR alone, EFR with XA21, and EFR with the XA21-EFR chimera described above. The latter two constructs have the potential of providing stronger and more durable resistance than EFR alone. Some transgenic events have been obtained in Duncan grapefruit and sweet orange, and these have been transferred to Dr. Jeff Jones lab at the University of Florida for testing with citrus canker.
The transgenic plants to be developed for this project are now growing in 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. The plants at both locations are growing well. At Penn State, all the transgenic lines have been successfully propagated as vegetative cuttings. All of the lines growing at Penn State have been found to express the FLT-antiNodT fusion protein, with five of the seven lines expressing very high levels of the protein. We must continue to let these plants grow a bit more before starting the HLB resistance tests. We have initiated a collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ rootstock with the FLT-antiNodT expression construct. We have immediately started transformations with the available transformation construct used to transform ‘Duncan’ grapefruit, in plasmid pTLab21. In addition, we are developing an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. In June, Dr. McNellis submitted a Stakeholder Relevance Statement to the USDA Specialty Crop Citrus Disease Research and Extension program to further develop this project. However, a full proposal was not invited. Dr. McNellis will present a poster describing the results of this project to date at the American Phytopathological Society conference in Pasadena, California, August 1-5, 2015.
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