Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 30 putative transgenic plants with citGRP1 were transferred to soil. They will soon be ready for RNA isolation and RT-PCR to check gene expression. About 10 transgenic Hamlin shoots with citGRP2 are in the rooting medium for rooting. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. In addition numerous putative transformants are present on the selective media. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is finally completed. Sequence information was confirmed and used to transform Hamlin. Some kanamycin-resistant shoots have already been obtained. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and will be use to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and queued for testing with no-choice ACP and then free-flying ACP infection.
Oral uptake of dsRNA targeting specific Asian citrus psyllid genes can induce psyllid mortality and reduce Liberibacter titer in infected psyllids. Research has shown that Asian citrus psyllid (ACP) mortality can be induced when adults feed on leaves from citrus that have been infected with a Citrus tristeza virus (CTV) expression vector modified to produce dsRNAs targeting specific ACP genes. The ACP mortality was shown to be directly associated with dsRNA abundance within the leaves. Also, when ACP infected Candidatus Liberibacter asiaticus (CLas) are fed on these plants for 15 days, the remaining live psyllids do not contain detectable CLas. Furthermore, none of the emergent adults that developed on these plants, from eggs laid by the CLas carrying ACP, contained detectable CLas. This is in contrast to what was observed in adults obtained from plants producing dsRNAs targeting the jellyfish green fluorescent protein (GFP) used as a control. The Clas bacterium could still be detected in adult ACP that fed on GFP-dsRNA expressing citrus and also in some adults that developed and emerged on these plants. Studies continue on comparing the effect of multiple ACP gene targeted dsRNA molecules (individually and in combination) that are fed to psyllids either in diets or produced in citrus using the CTV vector. As part of this work, a method was developed that allows complete life cycle development of the ACP on excised citrus flush thus improving efficiency of studies of dsRNA feeding on ACP nymphal stages.
During this period we have analyzed the response of ‘Duncan’ grapefruit (considered susceptible to HLB) and ‘Sun Chu Sha’ mandarin (considered moderately tolerant to HLB) after inoculation with flagellin 22 (flg22) from Liberibacter using comparative Ct real time PCR. The expression of 16 defense-associated genes was analyzed. In ‘Sun Chu Sha’ we observed that PBS1, NDR1, RAR1, SGT1, EDS1, EDR1, PAL1, AZI1, NPR2, NPR3 and RdRp were significantly induced compared to water controls. However, in ‘Duncan’ only PR1 was significantly induced compared to water controls. These results were presented during the 3d International Research Conference on HLB held in Orlando, Florida on February 4-8 of 2013.
The long-term goal of this project is to develop a novel and robust measure to protect citrus from high-priority diseases that threaten the industry and economy. The novelty stems from the fact that we are enhancing the plant defense (called innate immunity) by introducing a protein chimera of two domains: one for recognizing the pathogen that causes the disease and the other for lysing the pathogen. The synergy of the pathogen recognition and lysis makes the chimera a robust therapeutic agent for clearing the pathogen before it can cause infection. In this three-year project, our goal is to demonstrate that a protein chimera shows broad-spectrum activity on three citrus pathogens, namely ‘Candidatus Liberibacter’, Xylella fastidiosa subsp. pauca, and Xanthomonas citri subsp. citri that respectively cause HLB, CVC, and canker. Our initial focus is to design a chimera that recognizes and kills Liberibacter from the citrus phloem thereby providing protection against HLB. Toward this end, we have chosen thionin from vascular plants such as citrus. Thionins are small antimicrobial peptides that can attach to the membrane LPS of gram-negative bacteria such as Liberibacter. Thionins can also lyse the membrane. We have attached another small helical antimicrobial peptide (discovered by our collaborator, Ed Stover, USDA-ARS, Ft. Pierce, FL) to thionin using a flexible linker. The idea is to exploit the synergy of membrane recognition and lysis to direct rapid clearance of Liberibacter by the chimera. We constructed a chimera gene with an upstream signal sequence and a constitutive promoter. The signal sequence is attached for phloem delivery whereas the constitutive promoter is attached to ensure high level of expression. We have successfully performed Agrobacterium mediated transformation of citrus carrizo. The full grown trees will be ready for Liberibacter challenge and efficacy testing in 9 months.
Our objective has been to determine how Asian Citrus Psyllid (ACP) behavior is affected by Ca. Liberibacter asiaticus (Las) infection by comparing ACP response to healthy versus infected citrus trees. In previous experiments, we have determined that ACP adults initially settle on Las-infected plants as compared with uninfected controls. We hypothesized that while the Las-infected plants are initially attractive to ACP, after prolonged feeding, the ACP experiences imbalanced nutrition and therefore leave infected plants to seek a better host (non-infected tree). We have finished conducting our settling experiments to determine how ACP settle in response to choice tests between: control vs. old infection, control vs. new infection, new vs. old infection, old infection vs. nutrient spray, new infection vs. nutrient spray, and control vs. nutrient spray. All plants used in settling experiments were approximately four-year old Hamlin sweet oranges. Control plants were uninfected (healthy) plants. Nutrient sprayed plants were old-HLB infected plants. Infected plants were either newly infected (<5 month since PCR detection) or old infected (>12 months since PCR detection). Our results show that newly infected plants are very attractive to ACP. ACP prefer to settle on these plants over controls and old infected plants. Old infected plants are not as attractive as newly infected plants when compared with controls. ACP initially settle evenly between control and old or nutrient sprayed plants, but then choose to move to the infected plants over seven days. When ACP are given a choice between newly infected plants and nutrient sprayed plants, the nutrient sprayed plants appear to regain some of their attractiveness and ACP settle more evenly between these two plant treatments. In addition, we have conducted a settling experiment between control and newly infected citrus in the presence of high amounts of methyl salicylate released from controlled-release devices. It appears from these experiments, that high levels of methyl salicylate may interfere with ACP’s ability to differentiate between infected and uninfected controls. We are currently exploring this hypothesis by measuring ACP preference to infected vs. uninfected citrus odor in olfactometer assays using methyl salicylate pre-exposed ACP. This may result in a commercial product for disrupting the ACP’s ability of finding infected citrus host plants. ISCA technologies is currently working on a initial proprietary product to disrupt ACP host finding behavior for beta-testing in the field. We are currently in the process of writing a manuscript that summarizes these experiments, which we plan to submit for scientific peer review by early summer.
In the first three months of 2013, Citrus Core Transformation Facility (CCTF) continued to operate at the steady rate without any interruptions in production of transgenic plants. Within this period, CCTF received highest number of orders ever-twelve. Five orders were placed for production of transgenic Duncan grapefruit (pW14, pHGJ1, pHGJ2, pHGJ3, and pHGJ4); four for production of transgenic Mexican lime plants (pOA1, pOA2, pOA3, and pGF1); and three for production of Valencia plants (pOA1, pOA2, and pOA3). All the binary vectors received from clients were already mobilized into appropriate Agrobacterium strains and initial co-incubation experiments were performed. Most of the work done in the CCTF revolved around the recently placed orders. Regarding the production of transgenic plants, CCTF has achieved following results. Transgenic Duncan plants carrying genes from these different vectors were produced: fifteen from the pX4, twenty six from the pX7, thirteen from the pX11, ten from the pX16, three from the pX19, five from the pX28, twelve from pNah, and five from pBI121 vector (to serve as control plants). There were also twelve Duncan plants produced carrying the gene from pMED14 vector and one carrying the gene from pMED16. Despite the high flux of workers in the facility, productivity remained high. Every effort will be made to keep production of transgenic material at satisfactory level considering high volume of incoming orders.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July 2012 on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 Jul 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 Aug. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. About 106 psyllids have been found on sticky cards of which 80% are in no-mulch plots with the majority in plots with trees that do not have receive chemical control . Flush infested with ACP eggs and nymphs have become much more common. These are predominately in no-mulch plots with 85% of the total observed infested flush. Very few infested flush have been found in plots receiving insecticides while no infested flush have been found in plots with both mulch and insecticide applications. The third leaf samples were collected on July 9 for HLB. Tree growth measurements were taken on July 12 for the third time which consisted of trunk diameter and height measurements. Monthly foliar nutrition applications continue to be applied in the first week of each month. Leaf samples for the 3rd nutrient analysis were collected 29 Jan 2013. Normal grove care operations continued. These include one application of Agrmek on July 4 for leafminer, one application of glyphosate for weed control in mulched plots May 30, one application of glyphosate May 30 and Kocide once to control canker on May 30. All trees were desuckered and any low hanging limbs pruned July 2 to reduce the chance of herbicide damage.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 Jul 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 Aug. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week . About 12 psyllids have been found on sticky card of which 3/4 are in no-mulch plots with more in plots that do not have chemical control than plots that receive insecticides. To date, several flush have been found infested with ACP eggs and nymphs in no-mulch receiving foliar nutrition without insecticides. The first leaf samples were collected on October 17 for HLB testing of which one tested positive but subsequently came up negative following multiple times retesting. A second general leaf sample on 19 Feb all tested negative. Monthly foliar nutrition applications were suspended after the Nov 7 spray in preparation for the winter dormant season and resumed on Mar 7. Leaf samples for the 2nd nutrient analysis were collected 29 Jan 2013. Tree growth measurements of height and trunk area were taken for a second time on 12 Feb 2013. These measurements showed trees grown on mulch were slightly taller than trees in unmulched plots they also had a larger trunk cross sectional area. Trees in the foliar nutrition and insecticide treatments were taller and had larger trunks followed by all other treatments which were similar. Normal grove care operations continued. These include one application of Vendex for spider mite control on Jan 15, one application of glyphosate for weed control in mulched plots Apr 3, one application of glyphosate and Solicam in unmulched plots Apr 3 and Kocide once to control canker on Apr 10. All trees were desuckered and any low hanging limbs pruned to reduce the chance of herbicide damage on Feb 7. Over the nights of Feb 16-18 and March 1-3 a freeze threat was experienced but the flood irrigation plan was executed so that no damage was incurred. New water sensor probes were tested and calibrated for soil moisture data collection.
Obj 1: The GOseq method was incorporated into the PAVE database to aid in identifying candidate effectors to take advantage of the ability to select statistically among significant differentially expressed genes. Predicted ‘effectors’ found in both the Asian citrus psyllid (ACP) and potato psyllid (PoP: study system) transcriptomes have been bioinformatically validated using automated BLAST annotation against the NCBI-GenBank database. PCR primers were designed around conserved nucleotides for each contig and RT-PCR was carried out using PoP cDNA to amplify the corresponding transcript and confirm (in vivo) expression of each transcript. To date, expression of 22 transcripts has been confirmed in PoP and confirmation of all genes in ACP by RT-PCR is in progress. Initially dsRNA screening will employ PoP to test for knockdown of transcripts. Obj 2: Yeast-2 hybrid studies were initiated to study protein-protein interactions important in psyllid-Liberibacter interactions involved in the circulative, propagative pathway. To accomplish this 500 guts from Las-infected ACP and 500 guts and 1000 salivary glands from uninfected ACP were extirpated and used to construct the libraries. Three cDNA libraries: uninfected gut, uninfected salivary gland and infected bacteria have been completed. Furthermore, three Mate & Plate ‘prey’ libraries (uninfected gut, uninfected salivary gland and infected bacteria) have also been completed, with high transformation efficiencies of > 2 million clones for each (50 ml x 10 tubes ea). The titers (cfu/ml) of the libraries are 4.0 x 107 cfu/ml for the uninfected gut, 2.6 x 107 cfu/ml for the uninfected salivary gland and 1.2 x 107 cfu/ml for the infected bacteria, respectively. Bacterial ‘bait’ primers were designed for 4 CLas-specific target genes, amplified each by PCR from infected ACP genomic DNA, and cloned into E. coli using pGBKT7. Transformation of selected candidate ‘baits’ into libraries is in progress. Obj 3: The second biological replicate for proteomic identification of putative effectors is underway. An additional 50 guts and 250 salivary glands of infected and uninfected potato psyllid adults were fractionated by SDS-PAGE (10’14% acrylamide) and digested with trypsin to yield peptides. The peptide mixture was analyzed by nano-LC-MS/MS using an EASY nano-LC (Proxeon, Thermo Fisher Scientific, San Jose, Ca) coupled to an Orbitrap LTQ Velos mass spectrometer. Proteins were identified at 99% confidence with XCorr score cut-offs determined by reversed database search. Mass spectra of peptides were analyzed with SEQUEST and XTandem software. Data were processed and organized using the Scaffold program. A probability threshold of 95% was adopted, and only proteins present in at least two replicates were considered expressed. The PAVE EST database was mined to select optimally sized open reading frames from among the annotated transcripts in the ACP and PoP adult psyllid, gut, and salivary gland databases, and the resultant translated proteins were used for protein identification. Obj 4: Several psyllid and Liberibacter genes were selected based on their potential for involvement as interactors, and tested for knockdown activity in an RNA-interference (RNAi) assay using oral delivery (parafilm membrane). Good quality dsRNA was synthesized (MEGAscript RNAi kit). This approach, together with microinjection of dsRNA, will form the basis for in vitro functional analysis of candidate effectors identified bioinformatically and/or in the yeast dihybrid assays.
Models relating yield to ground and foliar enhanced fertilization in the presence of HLB have been fully developed and explored. Although the results are promising, the amount of data available does not permit a general recommendation. Based on these initial results, several growers have volunteered to provide more data. Efforts are being made to obtain the data.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Mutagenesis of Arabidopsis EFR Random mutagenesis was performed on the extracellular domain of EFR, and a library containing approximately 10^6 clones with an average mutation frequency of 0.4% was produced. From this library 13,000 clones were screened for ROS induction in response to elf18CLas. Unfortunately, No elf18CLas responsive clones were found. It was observed that there was a high frequency of non-functional clones in this library (as assessed by ROS production induced by wild-type elf18), so a further library was produced with a lower mutation rate (0.1%). A further 6,000 clones were screened from this library without isolating any elf18CLas responsive clones. Given the lack of positive results arising from these screens it has been decided to use different approaches to engineer elf18CLas responsiveness to EFR. Firstly, target mutations were produced in EFR at sites which are known to be important for elf18 binding and responsiveness. However none of these produced a response to elf18CLas. Secondly, we have shown that elf18CLas fails to compete well with elf18, in ROS and growth inhibition assays, suggesting that binding of elf18CLas EFR is not occurring. Therefore, a first step toward engineering an EFR variant capable of responding to elf18CLas is to evolve an EFR variant that gains binding to elf18CLas. In order to engineer EFR capable of binding elf18CLas, experiments have been initiated to determine the feasibility of performing a phage display screen to identify mutants of EFR. Initial data indicates that fragments of the EFR extracellular domain can be expressed in E. coli and can bind to biotin-labeled wild-type elf18. Further experiments are underway to determine the minimal region of EFR necessary for binding and the specificity of binding, which will later enable mutagenesis of this region. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Transgenic Arabidopsis plants are being produced with XA21 or XA21-EFR to assess their resistance to Xanthomonas campestris pv. campestris 8004. This work is required to test unambiguously the functionality of XA21 in conferring anti-bacterial disease resistance in dicots. In addition, tomato plants are being transformed with XA21 to determine functionality in this species. These plants will also be crossed with tomato EFR lines to determine the effectiveness of the presence of both genes in bacterial resistance.
This study addresses two general questions: 1) Will our constructs disrupt normal growth and development in citrus, and 2) Will these constructs confer a degree of resistance to infection by Liberibacter asiaticus? We have answers for the first question and seek a one-year extension to address the second. Objective 1. Express R proteins in a phloem-specific manner in Arabidopsis and citrus. It was evident very early from the results of our experiments, and of others, that the Arabidopsis SUC2 promoter was phloem-specific in citrus and, thus, efforts were directed towards the generation of transformed citrus containing wild type and constitutive mutants of the two R genes, SSI4 and SNC1. Our rationale was that by restricting expression to phloem tissues (or to the wounding response) potential negative effects on growth and development would be minimized. From 30-60 transformants of each R gene variant were obtained in Arabidopsis and at least 10 in citrus (Duncan grapefruit). In Arabidopsis, some stunting was observed when transformed with the constitutive ssi4, but not with wild type SSI4, or with wild type or mutant SNC1. A similar result was obtained with citrus; however, the stunted growth (or seedling death) phenotype was much more pronounced. However, as with Arabidopsis, no abnormal phenotype was observed with either variant of SNC1. e triggered by psyllid feeding. This objective is a variation of the first, except the restriction in expression of the potentially harmful R genes was imposed by the wound-inducible PAD4 promoter, a promoter known to be activated by aphid feeding in Arabidopsis. Our rationale was that in case AtSUC2-directed expression resulted in a stunted growth phenotype, the use of an inducible promoter, such as PAD4, would provide a way to evaluate the effectiveness of R protein expression in inhibiting Liberibacter infection. Our expectation was that the Pad4 promoter would not be active, except upon deliberate wounding under controlled conditions. As with the AtSUC2 promoter, the expression pattern of PAD4 was more variable in Arabidopsis as determined using a GUS reporter; however, PAD4/GUS expression in transformed citrus appeared to be strictly wound-inducible. Expression of the R gene variants using the PAD4 promoter gave a result similar to that obtained in Arabidopsis: expression of the SSI4 constitutive mutant was sometimes harmful to the plant; whereas, expression of the constitutive mutant of SNC1 was not. These experiments can be summarized as follows: 1-Restricted expression of the wild type SSI4 and SNC1 genes using either the AtSUC2 or AtPAD4 promoter had no negative impact on growth and development in citrus. 2-Similarily, expression of the constitutive mutant of SNC1 had minimal effect on growth and development. In contrast, expression of the constitutive mutant of SSI4 is sometimes harmful to normal growth and development in both Arabidopsis and in citrus. Additionally, preliminary tests indicate that none of the constructs effected psyllid feeding preferences. In preparation for assessing disease resistance of the transformed citrus, a single leaf assay to monitor the early events in the transfer of Liberibacter from the psyllid to the plant has been developed as outlined in the proposal pending with the CRDF (Nov 2012). In brief, the real time PCR protocol was refined by developing calibration curves for the Las and various plant and psyllid control amplicons so that detection is now reproducible down to 12 copies. In addition, significant improvements have been made in single-leaf cage design that will enable feeding to be restricted to a 6 mm area of leaf.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This new greenhouse is compliant with DPI regulations for propagation facilities. – The initial cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation have been identified and are currently being propagated.
For the last three months of 2012, the Mature Tissue Transformation Laboratory (MTTL) continued to operate in the ‘maintenance’ capacity mode. Level of operation was determined by the amount of plant material available and its quality. The process of increasing the number of rootstock plants is slow and it has been hindered by low germination rate of seeds that are old. Although new seeds were ordered in December, they will not be available until late January/mid February when Swingle citrumelo and C. macrophylla fruit are available. One of the batches of Hamlin buds grafted in early October had low percentage ‘take/success’ rate. The outside provider of grafting services claimed that the buds coming from mother plants were not of the highest quality. In the meantime, this person has left the business and the facility contracted other provider. In couple of experiments, a high percentage of explants that were used in co-incubations with Agrobacterium got contaminated. We are investigating whether those incidences were the result of human error in the steps of transformation taking place in the laboratory, or if plants that served as starting material for explants were infected while in the growth chamber. During these three months, six co-incubation experiments were performed. Four of those experiments were done with Valencia explants and two with Hamlin explants. For the Valencia experiments, we cut 2170 explants and 1030 explants were cut for Hamlin experiments. In order to be able to assess the ability of the lab to process different orders at the same time, two additional Agrobacterium strains were used for co-incubation experiments. One of those harbored a binary vector with the gene for green fluorescent protein (GFP) as a reporter gene. In one of the Hamlin experiments, out of 16 shoots inspected for GFP fluorescence two were positive. Those two shoots were micro-grafted on Carrizo rootstock plants. Some GUS assays were done on shoots obtained from experiments done earlier. Out of 19 shoots, one was positive. Two additional Ray Ruby plants were cleaned of microorganisms and are ready to become source of budding material. One more Hamlin plant was also cleaned.
The four most promising anti-NodT scFv antibodies have been selected for further development. Anti-NodT antibody #1 has been successfully expressed in E. coli. This means that we can generate as much of the antibody as needed. The antibodies are being augmented with two 6xHis epitope tags – one at the amino terminus, and one at the carboxy terminus. The protein can be detedcted with anti-His antibodies. The anti-NodT scFv antibody is soluble, and should be usable for protein immunoblotting and other applications. We experienced some delays in cloning the scFv antibody DNA into the appropriate citrus transformation vector. However, these difficulties have now been solved and we now expect to have the scFv citrus transformation construct completed within a few weeks, and we will commence transformation immediately.
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