Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and six PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for confirmation of transformants; selection is underway. In addition, we will add the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs have been created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Constructs have also been created in which Bs2 is co-expressed with other R genes that may serve as accessory factors for Bs2. Constructs with tagged Bs2 have been confirmed to function in transient assays, and protein expression has been confirmed by immunoblot. These constructs have also been transformed into Arabidopsis for analysis, and two constructs have been provided to the Lake Alfred transformation facility, Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Four CRISPR constructs are being created in the Staskawicz lab: C1) A construct targeting two sites that will produce a 100 bp deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); C2) A construct targeting a site overlapping the two conserved leucines; C3) C2 with the addition of a bs5 repair template for Carrizo that will not be cut; and C4) C2 with a similar repair template for Duncan grapefruit. The constructs have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. To aid in the selection of positive transgenics, we are currently adding a GFP reporter into each CRISPR construct.
Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance has revealed potentially valuable tolerance and indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. In August 2010, the plants were established at Pico’s farm in Ft. Pierce FL. Despite the high incidence of mottle in ‘SugarBelle’/SourOrange, it had the greatest overall increase in diameter. ‘SugarBelle’ and ‘Tango’ (which were not on the same stock as ‘Hamlin’ and so results should be viewed as comparing cultivar/rootstock combinations) were the healthiest in overall appearance in 10/15 and had the most fruit (88 per tree). All cultivars except sweet oranges and grapefruit are progressing in production, but production was compromised in all varieties by the severe HLB pressure at this site, and commercial value of the observed tolerance remains uncertain. A mapping population of Fortune x Fairchild has been planted (collaborating Roose and Gmitter) along with related material, in an effort to identify genes associated with tolerance in the mandarin phenotypic group. The citrus relatives planting (85 seed source genotypes from the gene bank) has been assessed for growth and apparent HLB tolerance. Most trees containing citron in their pedigree have markedly greater canopy densities and greater tree size than other accessions in the Genus citrus. One alleged standard sour orange looks much healthier and is much larger than other sour oranges. Chemical, morphological and transcriptome characteristics are being assessed to determine what factors are associated with observed tolerance, so they can be used in early screening and possibly directed transgenesis. A paper describing HLB resistance in this population has just been published in Plant Disease. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce FL. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. Generation of new chimeras has been difficult. An existing periclinal chimera (Satsuma and Poncirus) has been imported,has been with DPI two years, and agreement has been reached to release this to us for testing. A method for the rapid identification of potential sources of HLB resistance is being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are easily detectable in most plants at 3 weeks Seedlings of Hamlin and Dancy show marked CLas proliferation and systemic movement from 3-6 weeks after exposure to ACP. By nine weeks after exposure, susceptible genotypes can be clearly distinguished from reported resistant material by higher CLas levels in roots. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types were propagated on x639. Replicated trials with standards have been established, in cooperation with G. McCollum. Six locations each of all sweet orange-like together and 4 with all mandarins were established in replicated block plantings with 6-8 trees of each cultivar at each site (in Ridge, IR and Gulf coast). Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, and are being grown for field testing of HLB resistance.
Citrus trees transformed with a chimera AMP (thionin-D4E1) and the thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Replicated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Las titer was checked from new flush rough lemon leaves at six month after grafting. Las titer from 18.6-36.5 was detected in 90% of transgenics expressing the chimera. Some transgenic lines expressing thonin had lower Las titer(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. Root samples from control plants and transgenic Carrizo expressing chimera and thionin were taken nine months after grating inoculation. Our results showed transgenic Carrizo expressing thionin significantly inhibited Las growth (0.5% of control level) compared to control and transgenic Carrizo expressing chimera. Antibody against thionin will be produced for Western detection. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic Carrizo plants carrying second generation AMPs were obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA will be isolated and Las titer will be tested. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las titer will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We are currently analyzing the genome modified plants using transgenic approaches including off-targets. To generate non-transgenic DNA free canker resistant citrus, Cas9 containing nucleus localization signal was overexpressed and purified. The purified Cas9 showed activity in cutting target sequence and will be used to generate canker resistant plants.
The first quarter of 2016 was very strenuous for Core Citrus Transformation Facility (CCTF). Two out of five employees left the facility in January and were eventually replaced by the new members of staff. CREC Center Director informed CCTF of possible move of the lab to the new site in March. Although March 24th was anticipated date for the move, that did not happen and CCTF still operates from its present location. CCTF received unprecedented number of orders (26) within the last quarter. Such a high volume of incoming orders called for an additional increase in production capacity of CCTF. I have purchased necessary consumables and tools for this transition and have taken steps to gradually ramp-up the input of starting material for experiments. However, uncertainty associated with possible move to new location prevented search for additional employees. I expect new recruit to begin working in the month of April. Partial increase in work load in March was little overwhelming for the present labor force and resulted in loss of some transgenic shoots and plants. I hope that when all aspects of CCTF functioning stabilize, so will the level of our production. The newly announced date for the move to new location is June 17th and I will try to organize it in such a way so that it will affect productivity of CCTF to the least possible extent. Between January and April, CCTF produced 57 plants. These plants belong to newer orders placed within the last 12-15 months. Four of the produced plants were Valencia oranges, three were Pineapple sweet oranges, eight were Carrizo citrange, and the rest were Duncan grapefruit. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. They are at the stage when they could easily be propagated by cuttings. I am awaiting further instructions on what to do with these plants.
This project (Hall-15-016) is an extension of a project that came to a close last summer (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. CRDF funds for the inoculation program cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. Update: Two technicians funded by the grant have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of March 17, 2016, a total of 8,169 plants have passed through inoculation process. A total of 160,395 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. 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. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful 75 to 95% of the time when approximately 75% of ACP placed on a plant test positive for CLas and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant).
We continue to produce transgenic, mature citrus trees and transfer them to scientists (Drs. Dutt, Louzada, McNellis, Mou, Wang) as soon the primary or secondary grafts heal. Mature scion transformation efficiencies have increased to 7.6%, and micrografting efficiencies have improved to 77%. Approximately 154 transgenics (primary transgenics and vegetative progeny) have been transferred to Dr. Dawson’s lab for additional testing, and another ~50 will be transferred next month. For out-of-state transport of transgenics, USDA APHIS permits were obtained by scientists prior to shipping. Shipping certification was also obtained through UF. Transgenic, mature citrus has been shipped to Dr. McNellis at Penn State. A manuscript was submitted to a scientific journal describing biolistic transformation of immature citrus rootstock, never previously reported in the literature. Biolistic transformation to produce transgenics will augment those produced with Agrobacterium. A rapid, high throughput, nondestructive MUG assay is being developed to screen whole putative transgenic citrus shoots for GUS expression. It is quantifiable and more sensitive than using X-Gluc as substrate. Fluorescence can be quantified on a plate reader, or visualized on a gel doc with known controls. It is anticipated that GUS expression will correlate to copy number similar to NPTII expression. It remains to be determined whether the shoots will survive immersion in the MUG substrate and subsequent micrografting, but minimal exposure to the substrate, followed by rinsing, might not be harmful. Data are being collected describing the method for potential publication. We are still optimizing the PMI selectable marker using biolistics and Agrobacterium transformations in immature and mature citrus transformation. The results so far look promising and shoot growth doesn’t appear to be negatively impacted like shoot growth on kanamycin medium. Sour orange and Volkameriana seed have been purchased for the growth room because seed of our preferred rootstock varieties have been sold-out. It remains to be determined if these varieties perform well in the growth room.
Three Valencia-type clones (N7-11, B7R9T35 and B7R9T36) with excellent fruit quality were discovered in our collection (older trees) that have no HLB symptoms and are PCR-negative for Liberibacter. Trees of these clones have been propagated for further study. Juice quality, as assessed by a large citrus juice company in Florida, was found to be excellent. We continued to monitor performance, assess HLB severity, tree growth and yields of late maturing sweet oranges at different field sites throughout Florida. Also, we received seeds from collaborators in Sicily of rootstock candidates that showed good preliminary performance at St. Helena, as well as some new candidates of identical parentage for future trials. Seeds were planted to produce larger numbers of trees for extended trials. Gauntlet rootstock screening: Approximaly 75 HLB+ Valencia budstick-grafted hybrid rootstocks already passed through the ‘hot psyllid’ house were planted at Picos Farm in Ft. Pierce. Three parental combinations from 2013 crosses produced numerous hybrids that are growing off beautiful trees from grafted HLB+ Valencia budsticks: C2-5-12 pummelo x C. latipes (papeda); Amblycarpa+HBPummelo x Sour orange+rangpur; and Amblycarpa+HBPummelo x White #1. Stick-grafting and production of rooted cuttings from 2013 gauntlet crosses has been completed. The two early-maturing (December) Valencia somaclones identified, B7-70 and SF14W-65, have been cleaned up and a few pathogen-free trees were provided by the Parent Tree Program (DPI). We are following our plans for rapid pathogen-free budwood increase. We are moving quickly to have a substantial population of budwood increase trees available for nurseries at the time of the official release announcement. These clones have been put up for official release approval this summer, 2015.
Three Valencia-type clones (N7-11, B7R9T35 and B7R9T36) with excellent fruit quality were discovered in our collection (older trees) that have no HLB symptoms and are PCR-negative for Liberibacter. Trees of these clones have been propagated for further study. Juice quality, as assessed by a large citrus juice company in Florida, was found to be excellent. We continued to monitor performance, assess HLB severity, tree growth and yields of late maturing sweet oranges at different field sites throughout Florida. Also, we received seeds from collaborators in Sicily of rootstock candidates that showed good preliminary performance at St. Helena, as well as some new candidates of identical parentage for future trials. Seeds were planted to produce larger numbers of trees for extended trials. Gauntlet rootstock screening: Approximaly 75 HLB+ Valencia budstick-grafted hybrid rootstocks already passed through the ‘hot psyllid’ house were planted at Picos Farm in Ft. Pierce. Three parental combinations from 2013 crosses produced numerous hybrids that are growing off beautiful trees from grafted HLB+ Valencia budsticks: C2-5-12 pummelo x C. latipes (papeda); Amblycarpa+HBPummelo x Sour orange+rangpur; and Amblycarpa+HBPummelo x White #1. Stick-grafting and production of rooted cuttings from 2013 gauntlet crosses has been completed. The two early-maturing (December) Valencia somaclones identified, B7-70 and SF14W-65, have been cleaned up and a few pathogen-free trees were provided by the Parent Tree Program (DPI). We are following our plans for rapid pathogen-free budwood increase. We are moving quickly to have a substantial population of budwood increase trees available for nurseries at the time of the official release announcement. These clones have been put up for official release approval this summer, 2015.
The UF/CREC Citrus Breeding and Genetics Program is currently focused on 4 compelling and critical 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. This project is a continuation of previous citrus breeding and genetics efforts, with a heightened and renewed emphasis on developing cultivar options for Florida citrus growers that can enable a greater likelihood of sustained viability and profitability in the face of endemic HLB. The project began on 1 November 2015. In the late autumn and early winter, we collected data from several ongoing field trials of rootstock cultivars throughout the state. Trees were assessed for HLB incidence and severity through all plantings, and yields and fruit quality was determined in selected replicated trials. Hybrid families planted in the field were evaluated to identify selections producing fruit that resembled sweet orange in appearance, and exhibiting few or no HLB symptoms. Fruit from the best of these were tested for juice quality and a few were also analyzed for volatile components and compared with standard sweet orange. Fruit samples were provided to a major juice company for processing and assessment of the juice for flavor. The entire collection of raw material produced by the breeding program and growing in groves at several different locations throughout the growing areas of Florida were assessed for HLB symptoms, to identify a subset of individuals displaying few or no symptoms of HLB, regardless of parentage. A new large field trial with more than 20,000 trees on over 100 previously identified and new rootstock candidates was prepared and planted.
The goal of this project was to determine the feasibility of developing a high-throughput screen of citrus seedlings to identify naturally resistant plants based on optical sensing. Although the project initially focused on fluorescent imaging, the use of polarized light proved more advantageous. The buildup of starch in the leaves is a hallmark of the disease, and starch grains rotate reflected light having a wavelength around 595 nm by 90 . The polarized images were acquired using a conventional DLS camera. The strategy was to infect seedling by exposure to a population of psyllids that were positive for CLas (Candidatus Liberibacter asiaticus). Employing the natural method of inoculation has the advantage of detecting seedling resistance at all stages of the process, from alterations in psyllid feeding behavior to resistance to the bacterial pathogen. Seedlings of sweet orange were exposed to infected psyllids for eight weeks and allowed to recover for either two months or four months before image acquisition and analysis of HLB status by quantitative PCR. Image analysis was conducted by manually segmenting the images and analyzing pixel distributions. A total of seven different texture feature sets were identified and assessed in pairwise comparisons using a support vector machine (SVM) approach to predict the HLB infection status. In comparisons among seedlings that had all been exposed to infected psyllids, the accuracies ranged from 59.79% (moderately vs. strongly infected) to 98.06% (questionable vs. weakly infected) depending on the comparison. Previous studies employed clearly symptomatic leaves; whereas, the current study included pre-symptomatic leaves which is a more difficult task. The present study imaged leaves on seedlings where angles and distances to the camera are variable, which is challenging compared to studies using individually mounted leaves. In future studies, increased prediction accuracies seem likely if distances of the leaves to the camera lens are incorporated to normalize image texture. Also, our preliminary results with fluorescence imaging and recent literature suggest that prediction accuracies could be significantly improved by combining fluorescence and polarization imaging to create an index of infection.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, 30 additional transgenic lines were analyzed for gene expression using qPCR. Of them, 10 were determined to be high expressers while the rest were medium to low in expression. Most lines tested negative for the transgene in the aerial leaf and stem while there was gene expression in the roots. In addition, a number of additional lines have been transferred from in vitro medium and acclimated to greenhouse conditions. We expect to begin propagation of the larger plants in the next quarter for challenge with Diaprepes neonates.
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. Interestingly, S. buxifolia plants did not show severe HLB symptoms yet had relatively low Ct values when assayed for CLas titer. Two general trends observed so far were increased Ct values on the same trees in the summer than in the spring or fall and the inconsistent relationships between stem diameter and shoot growth and HLB severity across a diverse group of Citrus, Poncirus, and distant relatives. To identify sequence polymorphisms in candidate genes and assess their association with HLB tolerance/resistance, 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). We have detected 614 nucleotide-binding site – leucine-rich repeat (NB-LRR) genes with more than 50% coverage in the re-sequenced genomes, and 149 NB-LRR genes with more than 95% coverage in re-sequenced genomes. No NB-LRR genes were lost in the re-sequenced genomes. This superfamiliy of genes represents the largest group of disease resistance genes in plants, conferring resistance to bacterial, fungal, viral and nematode diseases. Most of the NB-LRR genes confer disease resistance in other plants in a race-specific fashion, but some render broad-spectrum and durable quantitative disease resistance. Single nucleotide polymorphisms (SNPs, one of the most common sequence polymorphisms) were found in 330 NB-LRR genes of Poncirus trifoliata DPI-50-7, 98 NB-LRR genes of Poncirus trifoliata Flying Dragon, 156 NB-LRR genes of Poncirus hybrid US-897 and 107 NB-LRR genes of Poncirus US-812. We also detected 72 genes from another two gene families that have more than 90% coverage in the DPI-50-7, Flying Dragon, US-812, and US-897. Real-time PCR was performed with HLB-resistant and HLB-susceptible samples to detect the relative change in the expression of these candidate genes. Results so far showed that several candidate genes were up-regulated with higher expression values in HLB-tolerant Poncirus genotypes when compared with HLB-susceptible citrus genotypes.
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 run two different time course experiments, one with Agrobacterium and the other using cell penetrating peptides (CPPs). The time course experiments were performed on three different citrus varieties: ‘Duncan’ grapefruit for the Agrobacterium experiment and ‘Pineapple’ sweet orange and a trifoliate cultivar ‘812’ for the CPP experiment. For both experiments, they plants were microinjected with the Cas9 repressor of and a sgRNA construct target the 5′ UTR of TFL. Control solutions were included. Sample leaves that had been treated with the experimental or control treatment were removed for a period of up to five days. After which, the leaves were harvested for their RNA and cDNA preparations were made. The real-time analysis run on these samples has been performed and the results are being investigated for accuracy. For the next quarter, we hope to have good, clear results of the experiments above. While perfecting the real-time analysis of the data more experiments will also be run to further test our CRISPR/Cas9 transient expression system in citrus.
Objective 1. (Greenhouse experiment): seed was extracted, seed coats removed using a chemical-removal procedure and planted of the needed rootstocks: x639, Swingle, WGFT+50-7 and UFR-3. Seed germination is underway. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The trial sites have been confirmed at two locations: Peace River Packing groves, Fort Meade and Orange Co, Arcadia. The trial sites have been surveyed and experimental setup has been custom designed for two locations and both the trials (CRF+ Tiger Micronutrient and hybrid fertilizer). The control release fertilizer has been formulated for both the trials and has been acquired from Harrell s and H.J. Bakers. Currently, data is being collected for before treatment time point. Data collected includes tree canopy measurements, leaf area index, leaf and soil nutrient levels, tree health, and HLB qPCR test). First fertilizer application will be made in second week of February at both the locations. Ojective 5. (funded by Orie Lee, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. The first treatment was applied first week of October, 2015. 1. Control no extra nutrition 2. Harrells St. Helena mix (2lbs per tree) 3. Harrells St. Helena mix (2lbs.)+ 2x TigerSul manganese (90 gm) 4. Harrells St. Helena mix (2lbs.) + 2x Florikan polycoated sodium borate (32 gm) 5. Harrells St. Helena mix (2lbs.) + 2x TigerSul manganese (90 gm) + 2x FL sodium borate (32 gm) 6. 4x TigerSul manganese (180 gm) 7. 4x Florikan polycoated sodium borate (64 gm) 8. 4xTigerSul manganese (180 gm) + 4x Florikan polycoated sodium borate (64 gm) Baseline yield data was taken from each plot, just completed (January, 2016).
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