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
Our progresses we have made for this project: 1) We have used the Kn1 gene to drastically improves shoot regeneration efficiently from transgenic cells of citrus. We have successfully used a maize knotted1 (KN1) gene to enhance genetic transformation efficiencies of juvenile tissues of six citrus varieties, Pineapple, Hamlin, Sucarri, Valencia, Carrizo and Eureka lemon via Agrobacterium-mediated infection. Our results demonstrate that expression of the KN1 gene improved transformation efficiencies from 3- to 15-fold compared to a control vector, 3- to 11-fold relative to the highest transformation efficiencies previously reported for the same citrus varieties. Stable incorporations of T-DNA into our transgenic plants have been confirmed with both histochemical staining of GUS activity and molecular analyses. The majority of KN1 over-expressing citrus plants grow and develop normally at young seedling stages, similar to those of the wild type plants. With all six genotypes of citrus tested including Eureka lemon, a cultivar difficult to transform, we have demonstrated that the kn1 gene can be an effective molecular tool for enhancing the genetic transformation of juvenile citrus tissues. Using mature shoot segments of Valencia and other cultivars as explants, we also found that the KN1 gene can improve transformation efficiencies compared to the control vector BUT an increase in efficiency is lower than what has been observed in juvenile citrus tissues. 2) We have demonstrated that manipulation of auxin transport can significantly enhances shoot regeneration of citrus. We have observed that the apical ends of epicotyl segments regenerated more shoots than the basal ends, and we therefore hypothesized that auxin transport and/or endogenous auxin concentration may play a key role in shoot regeneration of citrus explants. We tested some auxin transport modulators and identify one modulator that improved shoot regeneration. However, when the modulator was included in the transformation experiment, the transformation efficiency did not improve (i.e., number of transgenic shoots produced per explant). We hypothesize that the auxin modulator may inhibit Agrobacterium infection or T-DNA integration. 3) We have shown that an epigenetic modulator may be used to enhance shoot regeneration and transformation of mature citrus tissue. When we used an epigenetic modulator in transformation experiments with mature tissues, we observed increases in transformation efficiency of several citrus cultivars including Valencia and Washington Navel oranges. We have further demonstrated that the epigenetic modulator can lead to increases in shoot regeneration efficiency of mature citrus tissues when compared to the controls. 4) We have demonstrated that low Agrobacterium infection and T-DNA integration efficiencies are limiting factors for mature citrus transformation. As described above, we have developed some tools for enhancing shoot regeneration from mature citrus tissues. However, when these tools were used in mature citrus tissue transformation, the increase in transformation efficiency was lower than in juvenile tissues. We have further shown that the Agrobacterium infection and DNA integration are a major factor limiting transformation efficiency of mature citrus tissues, which provides a basis for our future experimentation to improve transformation efficiency of mature citrus tissues. We have published one manuscript reporting that Kn1 can drastically improve genetic transformation efficiencies of six citrus cultivars including a lemon cultivar: Hu et al (2016): Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant cell, Tissue and Organ Culture. 125: 81-91. The second manuscript reporting the effects of poplar transport of endogenous auxin and an auxin transport modulator on citrus regeneration and transformation will be submitted in 2-3 weeks. The third one is currently under preparation.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,all the transgenic lines produced have been confirmed for gene integration by conventional PCR and analyzed for gene expression using qPCR. 35% of the lines tested have been determined to be high expressers while the rest were medium to low in expression. Cuttings from all the better performing lines have been made and are being rooted in the mist bed for future challenge with Diaprepes. A number of other potential root specific promoters are being evaluated. Several have been cloned and transformation vectors are being produced. Results from our studies have been presented in the World Congress on In vitro Biology.
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 have been working on five transgenic lines (HAM 13-3, HAM 13-29, and DUN 57-25, DUN 205-25c, and DUN 207-8) that exhibit tolerance to HLB. HAM 13-3: we had some difficulty with propagation of this particular line. We have repeated the propagation and are waiting for the progenies to become old enough for HLB test. HAM 13-29: we have generated 21 progenies. After HLB inoculation, 11 of these plants have no HLB symptoms and others exhibited mild symptoms. DUN 57-25: four progenies have been made and tested. Three plants have no HLB symptoms and one plant was crappy and discarded. We are continuing propagating this line. DUN 205-25c: this transgenic plant became dark green and the leaves are thick and extremely curly. We are propagating this plant. DUN 207-8: eight progenies have been generated. These plants have been inoculated with HLB. For objective 2, we confirmed the chimerism of the transgenic plants using real-time quantitative PCR. Results showed that HAM 13-3, HAM 13-29, DUN 57-25, DUN 205-25c, and DUN 207-8 are homogeneous (not chimeric). For objective 3, three Ray Ruby grapefruit putative mutants (#3, #6, and #93) showed tolerance to HLB. Seven, 10, and 10 progenies were generated for mutant line 3, line 16, and line 93, respectively. The progenies were tested for HLB resistance/tolerance. Unfortunately, none of these mutated plants exhibited resistance/tolerance to HLB.
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 found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified ~1,200 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a valuable pool of potential candidates from which true HLB tolerance genes can be identified. The original project proposal aimed to identify approximately 1,200 potential candidate genes ~differentially expressed in HLB-tolerant Poncirus and rough lemon, conduct massively parallel sequencing of the ~candidate genes, perform genetic association and linkage analysis to find most likely candidate gene(s) for HLB tolerance, and clone these candidates for function validation. We have identified up-regulated genes in rough lemon in response to CLas inoculation. By combining our new gene expression data with 14 other published data sets, using two gene expression meta-analysis tools, we identified 3,122 probe sets differentially expressed between HLB-tolerant and susceptible varieties or in response to CLas inoculation. These probe sets correspond to 2,147 genes in the Clementine genome. To capture the sequence polymorphisms in these candidate genes for association and linkage analysis and to obtain full-length sequences for gene cloning, we sequenced the genomes of more than 40 Poncirus and Citrus accessions from 25-50x coverage and obtained partial genome sequences of 30 Poncirus and Citrus accessions from our collaborators, resulting in >700 Gb of sequence data. We have assembled the sequence reads into contigs and mapped these to the candidate genes for 48 Poncirus and Citrus accessions. To perform association and linkage analysis, in collaboration with USDA colleagues we collected HLB severity and CLas Ct data from more than 70 Poncirus and Citrus hybrids and accessions planted at the Picos Road Farm (USDA, Ft. Pierce). These accessions were replicated in 8 randomized complete blocks. For approximately 60% of the accessions, data were collected for two growing seasons, in addition to recently published information from the same planting. For the remaining 40%, plants were poorly established initially, and we will continue to collect phenotypic data for one additional growing season on HLB severity and CLas Ct value. Gene cloning efforts have focused on genes expressed at significantly higher levels in HLB-tolerant accessions or after CLas inoculation and with known functions in plant defense or immunity. However, some of the selected genes seemed to encode proteins toxic to E. coli or Agrobacterium cells, resulting in failure in gene cloning. To overcome this, gene expression vectors were modified. Two gene constructs have been developed and are being introduced into citrus to produce transgenic plants. To speed up the cloning of additional genes at full length, we have begun to complete a high-quality reference genome sequence of Poncirus, which currently does not exist. Based on these results, we have published two refereed papers and several abstracts. HLB-tolerant citrus so engineered are expected to be deregulated at much quicker speeds and less expense and be better accepted by consumers. Combining tolerance genes from Poncirus and rough lemon in the same citrus cultivars may result in augmented tolerance, even resistance. The primers and new DNA markers derived from HLB tolerance candidate genes also can be used to screen existing rootstock and scion breeding populations to facilitate development of non-engineered HLB-tolerant/resistant citrus scion or rootstock cultivars by conventional breeding.
Two new replicated field trials with Valencia scion were planted with SuperSour-type rootstocks at two locations, one in the east coast area and one in the west coast area. The west coast trial is on high pH soil and should give good information about the tolerance of the SuperSour rootstocks to high pH, in addition to HLB tolerance. Field performance information is being collected on more than 400 new rootstocks in 17 different replicated field trials. Performance attributes being assessed include tree growth, tree health, fruit yield, fruit quality, and tolerance or resistance to HLB and other diseases. New rootstocks are only appropriate for large-scale grower use when outstanding performance has been documented by statistically replicated trials over multiple years. It is anticipated that at least one of the best new SuperSour rootstocks will be released for commercial use within 3 years. In the meantime, outstanding performance has been documented for US-802 and US-942 rootstocks over multiple years in trials affected by HLB, and these rootstocks are available in large numbers through commercial nurseries. During this quarter, 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 new paper providing a comprehensive comparison of field performance for the new USDA rootstocks with other standard rootstocks, was submitted and accepted for publication in a refereed journal. This publication will be a very valuable reference for use by growers and nurseries making decisions about the best rootstocks to use for new plantings. A new replicated field trial was established to collect detailed information about Valencia tree performance on the most HLB-tolerant rootstocks under optimum management conditions. 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) 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. Cooperative planning continued with UF researchers, to submit grant proposals 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.
Good progress was made in the validation of the effectiveness of metabolite profiles for selection of HLB tolerant rootstocks. Focused studies were continued to identify key metabolic compounds in 12 specific rootstocks of known HLB-tolerance, as well as scions grafted on them, and collect the detailed information to be used in the validation process. In this quarter, the second set of leaf and root samples from greenhouse and field tests with different rootstocks were processed and shipped to the West Coast Metabolomic Center for metabolomic analysis by gas chromatography-time of flight (GC-TOF) mass spectrometry (MS). The GC-TOF-MS analysis for the first cycle of samples will be completed in the next quarter and results available for detailed analysis by our research team in Ft. Pierce and Immokalee. The experimental design of the first and second set of samples collected in the first year of the project will allow comparison of metabolite profiles of the same genotypes in different seasons, in grafted and ungrafted trees, and in different ages and sizes of plants, providing critical information about how metabolite profiles change under different conditions. In preparation for the second year of the project, clean seedlings and budded trees were prepared for greenhouse studies, and budded trees were prepared for field planting. Selected trees within the experiments were inoculated with Las, and tissue was collected to analyze for Las infection by qPCR. The material in this study will provide valuable insight into metabolite changes during the early stages of HLB disease development. Following approval by CRDF, the project was revised to include a new researcher at University of Florida in Immokalee, Dr. Ute Albrecht, who has experience and special expertise in this research field. Grant funds will be provided through a Non-Assistance Cooperative Agreement to support this work in Immokalee. A post doc has been hired in Immokalee to work on the project. The project changes actually reduce the overall cost, but will significantly increase productivity and the opportunity for success. In this quarter, a study by our team of metabolic differences among rootstocks was published, entitled “Metabolic variations in different citrus rootstock cultivars associated with different responses to Huanglongbing”. That study used GC-TOF-MS analysis to compare metabolite profiles of six rootstock cultivars infected and not infected with Las. The study identified numerous chemical compounds that appeared strongly associated with Las tolerance, and offers good opportunities for further study and incorporating the tolerance behavior into other rootstocks. The study demonstrated large metabolic differences between an HLB-sensitive rootstock and the HLB-tolerant rootstocks US-897 and US-942, both with and without Las infection, and will serve as a foundation for continuing work under this grant.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Five putative transgenics have survived and passed a PCR screen, and these will soon be 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. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for confirmation of transformants; selection is underway. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to coexpress 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. Four CRISPR constructs are being created in the Staskawicz lab: C1) A construct targeting two sites that will produce a 100 bp deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); C2) A construct targeting a site overlapping the two conserved leucines; C3) C2 with the addition of a bs5 repair template for Carrizo that will not be cut; and C4) C2 with a similar repair template for Duncan grapefruit. The constructs have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. To aid in the selection of positive transgenics, a GFP reporter has been added into each CRISPR construct. The constructs with GFP will be re-verified in N. benthamiana and sent for transformation.
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 is ready for submission. 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. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce FL. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. Generation of new chimeras has been difficult. An existing periclinal chimera (Satsuma and Poncirus) has been imported 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 ddoubled every 3 weeks through week 12. 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). All cultivars except sweet oranges and grapefruit are progressing in production. 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 compared to control and transgenic plant expressing chimera. These data suggest transgenic plants expressing thionin are promising for HLB resistance (The manuscript for this research will be published in Frontiers in Plant Biology). Antibody against thionin will be 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.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We are currently analyzing the genome modified plants using transgenic approaches including off-targets. To generate non-transgenic DNA free canker resistant citrus, Cas9 containing nucleus localization signal was overexpressed and purified. The purified Cas9 showed activity in cutting target sequence and are being used to generate canker resistant plants. We have conducted multiple tries of genome editing using protoplast. Currently, we are optimizing the condition to conduct genome editing using protoplast. We also tested different sgRNAs to generate deletion in the coding region of CsLOB1.
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