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. Dr. Jude Grosser has assumed responsibility for completing the project, following the resignation of Dr. Barrett Gruber. Ms. Amy Dubois is the OPS assistant taking care of the trees at the IRREC, she will continue in this roll. The inventory of recovered 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. Additionally, we now plan to include selected new cybrid grapefruit clones (Flame, Ruby somaclone N11-11, and White Marsh) that contain cytoplasm from ‘Meiwa’ kumquat as scions. These new grapefruit clones have shown significantly improved canker tolerance in greenhouse assays as compared to traditional grapefruit clones, and they are not GMO. Having them in the field on improved rootstocks will expedite determining their true canker resistance, as well as their response to HLB. Dr. Ahmad Omar will assist with grafting. 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
During this reporting period (October, November, and December, 2015), 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. These plants are continuing to be propagated at both Ft. Pierce and Penn State. We now have propagated each line at Penn State with about 10 propagated trees rooted per transgenic line. In addition, we used genomic DNA analysis (Southern blotting) to confirm the presence of the anti-HLB gene in the genome of the grapefruit trees. Control plants that have been through the transformation process were also generated during the reporting period. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. These control plants will be sent to Penn State from Lake Alfred during the next reporting period. We call these the “transformation control” trees. Our 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’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, continued during the reporting period. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State soon. During the reporting period, Dr. McNellis applied for and was granted USDA permits to move sweet orange, rootstock, and “transformation control” trees to Penn State. This will set us up well for tests on these new trees.
This quarter, using Cas9m4, with conjugated activation or repression domains, we intended to modify the expression of citrus proteins responsible for regulating flowering, namely TERMINAL FLOWER-1 (TFL), in order to reduce juvenility. TFL is a repressor of flowering and has been shown to inhibit flowering when overexpressed and to increase flowering when enhanced in Arabidopsis thaliana. We want to down-regulate TFL transiently, so we intend to decrease maturation times and do so without the use of transgenic insertion that is deemed unfavorable. For this quarter, we have gotten our early real-time PCR results. Using an activator construct pCAMBIA-2201-Cas9m4-VP16-EcR along with a sgRNA construct, pCAMBIA-1302-TFL-sgRNA-968 for one experiment, we have generated data from two different experiments. Statistical analysis awaits, but the early data suggest that instead of up-regulating TFL as predicted, we have slightly down-regulated the gene, suggesting that targeting the 5 UTR of the TFL gene does not allow the transient CRISPR machinery to work. Our follow up experiment is using a repressor, pCAMBIA-2201-Cas9m4-KRAB, to verify the extent to which we can down-regulate the gene and hopefully cause early flowering.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we performed concentration gradient experiments to determine the lowest concentration of the natural SAR inducer, which is sufficient for canker resistance. We used 0, 0.25, 0.5, 0.75, and 1 mM of the SAR inducer, as we have found that 1 mM of the SAR inducer was able to induce strong canker resistance. We used both infiltration and soil drench to treat citrus plants with the SAR inducer. For infiltration, treated leaves were inoculated 24 hours later and for soil drench, leaves on the treated plants were inoculated 7 days later. As in the previous experiments, 5 plants were used for each treatment; three leaves on each plant were inoculated; 6 inoculations on each leaf were carried out, and a total of 90 inoculations were used for each treatment. Results showed that, for both infiltration and soil drench, the strength of canker resistance is concentration dependent in the range between 0 to 1 mM. Therefore, 1 mM is likely the concentration that should be used for inducing canker resistance. We will confirm this result in the coming season. Moreover, we found that treatment of citrus plants with the SAR inducer produced systemic residual resistance to canker. We cut back previously treated plants and tested canker resistance on leaves on the new flushes. Canker disease symptom development was significantly attenuated on the leaves on previously treated plants. We will confirm this interesting observation in the coming season. Meanwhile, we are designing experiment to determine whether the systemic residual resistance is conferred by the SAR inducer residue or products induced by the inducer. For objective 2, among 49 independent transgenic lines expressing the Arabidopsis nonhost resistance genes, 20 lines showed increased resistance to citrus canker. We have propagated 10 lines that exhibited good canker resistance in the first test. The progeny plants are growing in the greenhouse and will be tested when they are ready.
Excellent progress was made in continuing the development of new hybrid rootstocks for the Florida citrus industry. As requested by CRDF, project 15-002 will place highest priority on hybrid rootstocks being considered for release to growers over the next six years, including about 400 Supersour-type rootstocks. It is expected that at least one of these SuperSour rootstocks will be released within three years, with more to be released in the following years as further information is collected from ongoing field trials. During this quarter, six new replicated rootstock field trials were planted, including trials on flatwoods and ridge sites. Emphasis in the new trials was on SuperSour selections, although standard rootstocks and previously released USDA rootstocks were included to allow good comparisons for relative assessment of field performance. The new plantings included two trials with Hamlin scion, two with Valencia scion, and two with specialty scion cultivars. Analysis of results from previously established trials indicates that HLB introduces additional variability to trials that requires more than the previously effective five or six statistical replicates to provide clear evidence of rootstock performance. Based on this observation, new USDA rootstock trials will usually include more than six statistical replications, regardless of the number of trees per replication. Using statistical comparisons is essential to develop reliable information about relative rootstock performance in field trials. During this quarter, trees in field trials were scored for health, HLB symptoms, and samples were collected from some groups for PCR detection of Las infection. Typically, trees in plantings with good ACP control remain nearly free of Las infection for the first 1-2 years, regardless of surrounding tree infection. Because of this, rootstock HLB tolerance can only be assessed when trees in field trials are more than 2-3 years old, or when special steps are taken to ensure trees become infected earlier. During this quarter, yield and fruit quality data were taken from 8 rootstock field trials with early maturing scion varieties. 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 Show. A new publication was prepared, providing a comprehensive comparison of the new USDA rootstocks with other standard rootstocks, and will be submitted for publication in the next quarter. 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. Greenhouse experiments continued to assess rootstock tolerance to HLB, CTV, and high pH. Cooperative 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.
Excellent 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. As requested by CRDF, the project will place highest priority on work with new hybrid rootstocks already created, selected, and included in field trials. As requested by CRDF, other parts of the original proposal that involved integration with the breeding program, recurring selection, and discovery of HLB-associated biomarkers and antimicrobial compounds will not be conducted. In this quarter, data from a preliminary metabolomic study was summarized and written into a manuscript, and will be submitted for publication in the next quarter. This study demonstrates striking metabolic differences between HLB-sensitive and HLB-tolerant genotypes, both with and without Las infection, and will serve as a foundation for continuing work under this grant. Specific studies were initiated to further identify key metabolic compounds and collect the first stage of information to be used in the validation process. For the first year of the project, three greenhouse studies with potted trees, and five field studies (with established trees of different ages) will be conducted, to collect information about metabolite expression in leaf and root tissue, as well as seasonal effects on metabolite profiles. The work will focus on 12 rootstocks where previous studies have identified relative differences in tolerance to Las infection. Field plantings, established under a grant previously funded by CRDF, are available with these 12 rootstocks suitable for the testing needed. In this quarter, trees in the selected field trials were scored for HLB symptoms and tested by PCR for Las infection. Leaf and root samples were collected from selected trees in five field trials and one greenhouse trial, and underwent preliminary processing for metabolomic analysis. A contract was prepared for the metabolomic analysis of this first group of samples, and those first samples will be analyzed in the next quarter. In preparation for additional greenhouse studies, budded trees were propagated in the greenhouse for study in the coming year. Environment may have strong effects on metabolomic data, so field testing, greenhouse testing, and information on seasonal variation will be critical for success in validating the approach. To clarify environmental effects, greenhouse studies will be used, and a greenhouse suitable for the work is being constructed. In this quarter, construction of the new greenhouse began, and is expected to be completed in March 2016. The project is evolving to include a new researcher at University of Florida in Immokalee, Dr. Ute Albrecht, who has experience and special expertise in this research field. It is anticipated this will result in no additional cost, but will significantly increase productivity and effectiveness of the project.
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 have been trained to establish and maintain colonies of infected psyllids, conduct qPCR assays on plant and psyllid samples, and run the inoculations. As of December 31, 2015, a total of 7,853 plants have passed through inoculation process. A total of 154,595 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Research concluded during September 2015 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. (in press)
The main accomplishments during this quarter: We have confirmed the K gene overexpression-mediated improvement on transformation efficiency of a lemon cultivar we used. We have tested the effects of the K gene on genetic transformation efficiencies of 6 citrus cultivars and we observed 3-15 fold increases if compared to our control vector, and 3-11 fold increases if compared to the highest transformation efficiencies of the same cultivars previously reported by others. We have observed and confirmed the stimulatory effects of one non-conventional regulator of gene expression on shoot regeneration efficiencies of some citrus cultivars. We have been testing the effects of that factor and other factors in combinations of the K gene on transformation efficiencies of both mature and juvenile citrus explants and our preliminary results suggest that there are significant improvements in transformation efficiency for both juvenile and mature tissues. We are also repeating the effects of endogenous auxin and the auxin transport on efficiencies of shoot regeneration and Agrobacterium-mediated infection of mature tissues of citrus. One manuscript reporting the drastically improvement of six citrus cultivars including a lemon cultivar has been accepted for publication in “Plant Cell, Tissue and Organ Culture”. The article is currently in the production stage and should be out in either February or March issue.
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 five 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. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for identification of transformants. 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. Constructs are also being 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 have been transformed into Arabidopsis. Protein expression will be confirmed by immunoblot. GFP is currently being added to the constructs to facilitate selection of transformants in citrus. 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. C1 and C2 have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. “C1” clearly cut the target sites of both varieties, causing 100-bp deletions. Sequence analysis confirms that “C2” cuts the target site in Carrizo. Considering this site is identical in both Duncan alleles, we expect it to cut Duncan as well. And, considering “C3” and “C4” are built from “C2,” we expect them to target the cut site as well. Sequence analysis is underway to confirm these expectations. In addition, to aid in the selection of positive transgenics, we are currently adding a GFP reporter into each CRISPR construct.
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 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). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree. All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. 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.).
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. 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. 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. 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 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. 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 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). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree. All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. 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.).
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, all transgenic lines as described in the project proposal have been regenerated and most plants have been acclimatized and transferred into the greenhouse for further growth. A few putative transgenic lines have been transferred to fresh in vitro rooting medium in efforts to stimulate root production. Root samples from 21 lines have been analyzed for gene expression using qPCR. Of them, 12 were determined to be high expressers while the rest were medium to low in expression. We are in the process of evaluating the remaining greenhouse acclimated lines for gene expression. The high expresser lines will be propagated for subsequent evaluation with Diaprepes neonates.
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. Grafted plants are currently growing, and will be tested for responsiveness to the elf18 ligand for EFR and for canker resistance. 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 is being 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 were provided to the Lake Alfred transformation facility, but the transformation attempts have so far been unsuccessful, possibly due to negative effects of the constructs in Agrobacterium or in citrus. Troubleshooting of these transformations with a negative control construct 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 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting a site overlapping the two conserved leucines, and one targeting two flanking sites to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
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 are propagating a number of citrus transgenic lines overexpressing Arabidopsis defense genes. Our previous results indicated that these transgenic lines are likely resistant or highly tolerant to HLB. The progeny plants are growing in the greenhouse. For objective 2, we performed on round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results showed some of the lines may be chimeric. We are repeating the qPCR experiment. For objective 3, we are propagating the previously generated gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB. The progeny plants are growing in the greenhouse.
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