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.
Transgenic studies have proceeded, with several hundred plants containing various combinations of natural or synthetic genes and promoters produced, many currently in greenhouse testing and field trial locations. Additional transgenic plants were propagated for new hot psyllid greenhouse tests, and for field planting. The sweet orange citrus genome sequence was mined to identify genes controlling anthocyanin expression, in an effort to develop visual and citrus-derived markers for genetic transformation; several candidates have been identified for further experiments. More than 875 transgenic plants have now been planted with a collaborator in Martin County, and these are being monitored regularly, along with a second site in Indian River County. The plant materials growing out include sweet oranges, grapefruit and mandarin hybrids. Several new rootstock trials with more than 15,000 trees were planted throughout Florida in the last year, to assess their adaptation to evolving advanced citrus production systems; these trials have been monitored regularly, and data has been collected on their early performance. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests; rootstock hybrids are showing diverse responses when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance; four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one was symptom-free up to 22 months ). We initiated a program to rotate new germplasm (rootstock and transgenic) through a ‘hot psyllid’ house (in collaboration with Dr. Stelinski) to ensure HLB inoculation prior to approved field planting; two groups of 50 trees have been rotated through so far, scheduled for planting at a collaborators field site, under permit from DPI. Rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made 2010 were just planted in the field. Previous work to develop rootstocks against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) continues, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valquarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release were developed for 7 new cultivars, and these were approved for release and commercialization in January 2011 by the UF-IFAS Cultivar Release Committee.
More than two thousand transgenic lines we have produced thus far, and are planted in field trials (2 locations under permit) or are in greenhouse tests at the CREC and with grower-collaborators; we continue to monitor the HLB and canker resistance or tolerance of these lines over time. These transgenic lines contain various combinations of natural or synthetic genes and promoters. New candidate genes continue to be identified by genome mining as well as from other disease resistance plant research. Citrus-specific promoters, transcription factors, and other genetic elements are being identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests, including some containing a broad spectrum, ancient disease resistance gene from rice; the latter are being propagated for HLB challenge. Data are being collected on the early performance of new advanced selections in trials planted to assess adaptation to advanced citrus production systems. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests. Diverse responses of rootstocks are being noted when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance. Greenhouse experiments are being continued examining interactions of rootstock and nutrients in severity of HLB symptom expression. Hot psyllid greenhouse facilities are now being used routinely to assess performance of transgenic citrus (representing our most advanced constructs with phloem-limited promoters and previously proven genes), as well as hybrids between Citrus and Poncirus, for responses to psyllid feeding and HLB development. More than 150 new rootstock candidates preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora, and have been used to produce new trees that were planted into new rootstock trials, or held for pending trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. Additional seedless pummelo-grapefruit hybrids have been identified during the 2011-12 season, some showing field tolerance to canker, good fruit quality, and FC-free, potentially producing grapefruit cultivars that address canker and marketing issues of ordinary grapefruit. Trees were propagated onto 30 new sour orange-like hybrid rootstocks, some already shown to be tolerant of CTV quick decline, and planted in a new field trial. A second demonstration planting of advanced sweet orange selections and newly-released cultivars, selected for high yields and superior juice quality, was established to assess to demonstrate their performance and utility in commercial processing, in collaboration with a major juice processor; these trials allow comparisons to be made between different production regions with the same sweet orange candidate selections. A well-attended field day was held in mid-November at the large CREC field experiment at the St. Helena block in Dundee; this featured Vernia and Valquarius orange trees grown on a number advanced rootstock selections, and highlighted early performance (yield and HLB effects).
We continue to monitor HLB and canker resistance or tolerance among the more than two thousand transgenic lines we have produced thus far, in field trials (2 locations under permit) and in greenhouse tests at the CREC and with grower-collaborators. These transgenic lines contain various combinations of natural or synthetic genes and promoters. Currently, 54 independent transgenic events represented by 162 individual plants have shown resistance to HLB after 10 months in a ‘hot psyllid’ greenhouse structure, showing no symptoms and negative results following qPCR diagnostics; these results are based on all 3 replicates of each transgenic line showing the same results. Among these plants are commercial cultivars of orange, grapefruit, as well as new sweet oranges and promising new rootstocks from our breeding program. Genetic constructs include both antimicrobial peptides and native citrus defense genes. We continue genome mining to identify and test new candidate genes, citrus-specific promoters, transcription factors, and other genetic elements. More than 875 transgenic plants in a collaborators field site in Martin County are being monitored regularly, along with a second site in Indian River County; apparently healthy trees in these trials are being tested by qPCR for the presence of Liberibacter. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests, including some containing a broad spectrum, ancient disease resistance gene from rice; the latter have been tested tree times for canker responses, and continue to exhibit phenotypes that are much less severe than standard control. Data collection continues on the early performance of new advanced selections in trials planted to assess adaptation to advanced citrus production systems. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests, and identified several that appear to overcome infection, with PCR positive results coming to PCR negative; these are being propagated for new field trials to be planted in 2013. Greenhouse experiments examining interactions of rootstock and nutrients in severity of symptom expression have shown clear differences among rootstocks in response to various nutrient regimes and symptom expression. More than 150 new rootstock candidates preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora have been used to produce new trees that were planted into new rootstock trials, or held for pending trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. Several field trials are revealing very obvious differences among experimental rootstocks for their ability to tolerate HLB infection as well as the rate at which they are becoming infected, suggesting that rootstocks may provide an important protective benefit even in infected trees; we are focusing significant efforts now to carefully monitor disease ingress and severity in the dozens of rootstock trials we have statewide Two demonstration planting of advanced sweet orange selections and newly-released cultivars, selected for high yields and superior juice quality, have been carefully monitored for HLB and canker resistance; these trials are grown using ACPS techniques, and trees are growing off very well. Finally, genetic mechanisms underlying HLB tolerance lemon have been determined. Microscopy and fluorescent compound uptake have shown that phloem is regenerated and is functional in lemon, even in symptomatic leaves. Expression of genes controlling cell wall biosynthesis is upregulated in lemon, in support on phloem regeneration. Further, defense associated genes are slightly upregulated early in disease in lemon, but are highly upregulated later in orange, utilizing all reserves rapidly and accelerating disease progression and decline.
The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, has finally been validated and made publicly available in USDOE’s JGI Phytozome v 9.0 in December 2012. This assembly is a vast improvement over the first version (v 0.9) that was made available to the research community in January 2011. The genome is organized into 9 super-scaffolds, representing the basic nine chromosomes of citrus. This was made possible by the ICGC collaborative genetic mapping effort, in which are lab was a primary contributor. (See Ollitrault et al. BMC Genomics, 2012, 13:593 DOI:10.1186/1471-2164-13-593). New citrus sequences were generated by the Machado lab in Brazil (Ponkan mandarin, 454 and Illumina), the Gmitter lab and UF-ICBR (low-acid pummelo, Illumina), and Illumina datasets for Willowleaf/Avana mandarin, W. Murcott, Chandler pummelo, and Seville sour orange have been provided (Morgante, IGA-Italy; Talon, IVIA-Spain; and M. Roose-UCR). These have been further analyzed to the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. Surprisingly, we have identified the male parent that gave rise to sweet orange. The fine-scale characterization of citrus genotypes opens the possibility that ancient C. reticulata/C. maxima admixtures (such as sweet and sour orange) can be recreated by conventional breeding guided by a set of genome-wide markers, enabling incorporation of specific, limited genomic regions from other citrus or relatives to confer disease resistance, yet retaining the essence of marketable fruit phenotypes. Additionally, we have been able to understand better the evolutionary relationships of ancestral citrus species, and their diversification over time. A manuscript based on these results has been expanded and prepared for submission. Work proceeds on the other objectives of this project. New experiments with new vectors have been initiated to attempt transient gene silencing of HLB and citrus canker-associated genetic targets identified from our microarray studies. We have also initiated preliminary yeast-2-hybrid experiments, as an alternative approach to identifying the effects of target genes on plant phenotypes and disease resposnse. We have used the GoldenGate assay platform for hi-throughput genotyping of DNA from >150 individuals of a large mapping family, and a second family is currently being mapped. Plans have been made for collaboration with the Dvorak lab to anchor the sweet orange genome sequence to the linkage map, thus substantially improving the quality and utility of the previously produced assembly. The genotyping by sequencing (GBS) project is proceeding, once proof of concept was provided, and mapping is underway in a large segregating Citrus x Poncirus family. The RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus has proceeded. RNA samples have been prepared from appropriate times in the disease process, and libraries have been recreated to test, prior to the full sequencing effort. Preliminary runs have enabled us to multiplex libraries and these are ready for sequencing once we have lanes available on the instrument.
The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, will soon be publicly available. New citrus sequences were generated by the Machado lab in Brazil (Ponkan mandarin, 454 and Illumina), the Gmitter lab and UF-ICBR (low-acid pummelo, Illumina), and Illumina datasets for Willowleaf/Avana mandarin, W. Murcott, Chandler pummelo, and Seville sour orange have been provided (Morgante, IGA-Italy; Talon, IVIA-Spain; and M. Roose-UCR). Comparative analysis has elucidated the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. The fine-scale characterization of citrus genotypes opens the possibility that ancient C. reticulata/C. maxima admixtures (such as sweet and sour orange) can be recreated by conventional breeding guided by a set of genome-wide markers, enabling incorporation of specific, limited genomic regions from other citrus or relatives to confer disease resistance, yet retaining the essence of marketable fruit phenotypes. A manuscript based on these results has been expanded and prepared for submission. Work proceeds on the other objectives of this project. We identified miRNAs induced in citrus-pathogen interactions, presumably regulating target genes involved in signaling pathways and metabolic events important for plant resistance. In order to set up protocols to validate microRNA expression in plant-pathogen interactions and identify target genes, we performed a comprehensive analysis of the expression of 7 different citrus miRNAs in the context of 4 different Xanthomonas citri subsp. citri (XC) ‘ Citrus limon interactions, and certain miRNAs appear to be XC strain specific in their responses. We have used the GoldenGate assay platform for hi-throughput genotyping of DNA from >150 individuals of a large mapping family; we have produced a preliminary linkage map that shows excellent coverage and distribution of markers. The genotyping by sequencing (GBS) project has proceeded and currently it appears that it may generate as many as 2000 high-quality SNP markers for mapping a large segregating Citrus x Poncirus family. A large scale RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus, that weren’t seen previously in microarray studies, or to validate those already seen, has progressed. RNA samples have been prepared from appropriate times in the disease process, and libraries have been recreated to test, prior to the full sequencing effort.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and we are collecting information on disease symptom progression. We have increased the numbers of individuals of most accessions, and have begun re-inoculations of previously inoculated seedlings with the same HLB source. We have extracted nucleic acids from most individuals, run RT-PCR on these, and we continue to find very few PCR+ plants. We will continue to monitor these individuals in the greenhouse. The Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test at the Picos Road Farm near Ft. Pierce have been budded and growing off, prior to field planting. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to explore potential tolerance from these sources. Currently, there are at least 8 propagations of a total of 102 individuals from the original CCMP, and we plan to plant these this summer 2011. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have been unable to find a cooperator to plant the same experimental materials in the field as we have been testing in our greenhouse. The exception to this is the CCMP, soon to be planted.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and we are collecting information on disease symptom progression. Additional seedlings that have reached sufficient size have now also been inoculated with the same HLB source. We have extracted nucleic acids from most individuals, run RT-PCR on these, and have found very few PCR+ plants. We will continue to monitor these individuals in the greenhouse. The Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test at the Picos Road Farm near Ft. Pierce have been budded and growing off, prior to field planting. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to explore potential tolerance from these sources. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified. We are still exploring other options within Florida for a field trial, but no secure, long-term commitments have been forthcoming, despite multiple discussions with growers throughout the state. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
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.
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.
McTeer trial – (3-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): Continued evaluation of this trial shows significant differences in tree health among rootstocks, with Orange #19 showing the healthiest trees. However, HLB has significantly impacted the quality of fruit trees on all rootstocks, even from very healthy looking trees. We will look at these trees one more season to see if continued remediation will improve fruit quality next season. St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 4.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). Full data (yield, fruit quality and HLB infection rates) was presented in our Field Day handout. Big differences have shown up in HLB infection rates per rootstock. Control commercial rootstocks have the highest infection rates, with most >70% infected. The rate of infection on tetraploid rootstocks was half that of diploids, with tetrazyg Orange #15 showing the lowest rate (just 7%). Disease severity of infected trees is also being impacted by rootstock. Good candidate rootstocks for ACPS are emerging. Greenhouse Experiments – Rootstock liners have been grown off for the nutrition and rootstock comparison studies, and have been moved to the HLB house for graft inoculation, to begin this quarter. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. Transgenic tetraploid lines containing an insecticidal Snowdrop lectin gene were regenerated from the tetrazyg selections Green #7 and Orange #4. 12 transgenic lines of Orange #4 and 3 of Green #7 have been successfully micrografted, acclimatized and transferred to the greenhouse. A construct containing the Snowdrop Lectin insecticidal gene combined with the antimicrobial gene CEMA was completed. Transformations are underway. We have codon optimized the Snowdrop Lectin insecticidal gene (GNA) for optimal expression in citrus. Two vectors containing this optimized gene have been produced; a) codon optimized GNA fused with a Tobacco PR1b signal peptide for improved extracellular secretion of the GNA protein by plant cells; b) codon optimized GNA fused with a HDEL C-terminal extension for retention of the GNA protein in in the endoplasmic reticulum. Dual protection against psyllids and Liberibacter: A construct containing the native Snowdrop Lectin insecticidal gene with the antimicrobial gene CEMA have been constructed. Transformation using this vector are being carried out.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks in airpots are growing well in our RES system, with some plants reaching 8 feet in height. Additional transgenics were propagated onto additional new rootstocks expected to reduce juvenility, including the somatic hybrid Amblycarpa + Flying Dragon. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. The first transformation experiments using the two-transgene Gateway based cloned construct combining our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene) were initiated, and so far 30 putative transgenic lines of the sweet orange cultivars Hamlin and Valencia have been regenerated. These plantlets have been micrografted to Carrizo rootstock. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance.A construct containing CEMA gene stacked with the NPR1 gene has been constructed. Also, another vector containing a AttacinE gene stacked with the NPR1 gene is also under construction. Correlation of transgene expression with disease resistance response: More than 150 transgenic lines with different genes have been analyzed using ELISA by either C-myc or LIMA antibody (which also works for CEME) to measure transgene expression. As expected, significant differences were observed in our transgenic plants. Correlations between the data obtained from ELISA and other molecular data with HLB challenge response data are underway. Transgenic lines examined by ELISA include 40 lines with NPR-1, 50 lines with LIMA, and 9 lines with CEME. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): We have finished construction of several parts of the T-DNA region of a pCAMBIA0390 derived binary vector for cre-lox based marker-free selection. A fusion codA-hptII gene driven by the d35S promoter have been constructed and a cre gene driven by a glucocorticoid-responsive elements promoter have also been constructed and cloned into a pUC based vector. We are experiencing problems cloning the glucocorticoid receptor gene driven by a constitutive mirabilis mosaic virus promoter as all sequenced clones have mutations and/or deletions in them. Work is underway to rectify this.
Work has been continuing on the development of a construct using the FT3 cDNA insert and an FMV promoter. This construct will eventually be used to test the efficacy of the FT3 cDNA as compared to the genomic DNA construct currently being used. Over the past several months, extensive testing was conducted to establish a more effective disinfestation technique for use on seeds and other explant tissue. This technique should allow for the continuous use of seed for transformation, even many months after their initial collection. Transformation of Carrizo has picked up following the most recent harvest of seed. These transformants will be used in the experiments examining the effects of GA and day length on FT phenotype. This is month 7 of the in vivo tracking of FT1, FT2, and FT3 and samples are continuing to be collected and processed. These data will be evaluated at the end of the year-long trial to compare month-to-month variations in gene expression. The FT3 protein that was commercially synthesized has finally arrived and experiments with direct application of the protein will be commencing shortly.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 60 different peptides for activity against HLB. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids.
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