In this quarter, three new field trials were planted at the Whitmore Farm in Lake County to study inheritance of fruit quality factors in sweet orange-type material from populations of hybrids between high quality pummelo and mandarin parents. One of these was planted on trellis, to also examine the effect of tree manipulations on the length of time for transition from juvenility to maturity. Tree performance information was collected from existing USDA rootstock field trials. Greenhouse trees were inoculated for assessment of new Supersour rootstock hybrids for tolerance to CTV. Cuttings were made from new Supersour rootstock hybrids to propagate trees for field trials. Seedling and cutting liners of rootstock selections in the greenhouse were budded with Valencia, Hamlin, Ray Ruby, and Minneola to prepare trees for field trials. Studies continue to assess rootstock and scion tolerance of Huanglongbing (HLB) under field conditions and in the greenhouse. Some rootstocks and scions appear more tolerant to HLB infection than others. Some new hybrids that were studied remain healthy and vigorous despite HLB infection and have been selected for further field and greenhouse testing. US-897 rootstock has exhibited strong tolerance to HLB infection and preliminary information suggests it confers increased HLB tolerance on trees with susceptible scions. Experiments were conducted to assess the effectiveness of different methods for testing new cultivars for resistance to Asian citrus psyllid and HLB disease. A field experiment continued to identify rootstocks with resistance to the Phytophthora-Diaprepes Complex. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial and insect resistance genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker (CBC). Selected transgenic rootstocks from this effort were challenged with HLB and CBC to assess resistance potential. Some transgenics were found to have potential resistance or tolerance. Research is continuing to follow leads generated by the HLB gene expression study completed last year, including cloning of a selected promoter strongly expressed in response to HLB infection, and combining that promoter with selected resistance genes to produce transgenic citrus with a rapid resistance response. An early flowering gene, FT, was transformed into superior citrus breeding material to facilitate rapid introgression of favorable traits, such as disease resistance, into new cultivars and increase early productivity of scion cultivars. Four new hybrid rootstocks, US-1235, US-1239, US-1225, and US-1241, were identified as especially promising for expanded field trials in the coming year.
Two trees have been found growing in HLB-ravaged orchards in Guangdong and one other in Guangxi province, that appeared to be free of HLB symptoms, while all other trees planted at the same time were either dead or declining, and replants likewise were afflicted. The trees from Guangdong were propagated at the Guangdong Institute of Fruit Tree Research facilities, and are being grown to conduct new tests of their reaction to HLB following deliberate inoculations. These trees have been tested twice after propagation using standard RT-PCR protocols, and they remain PCR negative for HLB. Two propagations of one of the selections have been replanted in an infected orchard location. The tree in Guangxi has been transplanted to a protected location in Guilin, at the Guangxi Citrus Research Institute. A valuable side benefit of this project has been the opportunity in our search for “survivors” to survey regions where HLB devastation is severe and quite widespread, and in doing so we have also visited orchards that appear to be nearly completely unaffected by HLB though surrounded by severely declining orchards. These surprising locations have been visited both in Guangxi and Guangdong. We have been investigating the nature of their management programs that has enabled them to survive to eight years of age or more in apparently good health. We interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy orchards. We have reported on our experiences and the answers to our questions in recent editions of “Citrus Industry”. Although located in different provinces several hundred miles apart, the key elements outlined to us were the same. These include critically timed pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition. Our impressions have been presented likewise through talks given at various grower meetings in Florida.
At this point, we have collected and germinated seeds from several of the candidate categories listed in the proposal, including pummelos, intergeneric hybrids with Poncirus, and others; seedlings are being grown in DPI-certified greenhouses at the CREC to provide budwood for topworking and young trees to plant directly in the field. We have not been able to acquire seed of all the selections we intend to test, because the project approval came some time after the season for seed acquisition. We had an agreement with one grower in Florida to plant out the range of genetic diversity we hoped to test, but that agreement has been terminated, and we are currently looking for other options within Florida. We are working on the list of materials that can be sent to our collaborators in China, without compromising UF-IFAS intellectual property rights.
The seeds of Murraya paniculata were procured from the USDA-ARS National Clonal Germplasm Repository for Citrus and Dates, as well as from local sources in Florida. Seeds have been available only periodically for our experiments. These seeds have been germinated in vitro and used to establish cultures for experiments designed first to improve the efficiency of plantlet regeneration from epicotyl explants, which is a prerequisite for experiments to develop successful genetic transformation protocols for Murraya. Following this step, we conducted a first round of transformation experiments using the plant regeneration protocol that was developed. A tissue culture medium designated as M10 (Murashige and Skoog’s (MS) standard medium supplemented with predetermined levels of BA and NAA) was used as the regeneration medium for all the transformation experiments. In efforts to standardize the genetic transformation protocol, experiments were carried out with plasmids previously used successfully for citrus genetic transformation in our lab; specifically we used pCAMBIA2301 and pGreen0029 harbored in Agrobacterium tumefaciens strain AGL-1. Various factors were tested in efforts to develop a standard protocol for transformation, such as varying OD values of the Agrobacterium cultures, the duration of explant incubation time, duration of co-cultivation, and the amount of antibiotic used for selection of transgenic shoots and for Agrobacterium removal. Unfortunately, we were not successful in recovering transgenic plantlets from any of these experimental treatments. Currently, we have designed and implemented new experiments using a number of different Agrobacterium strains to which Murraya may be more susceptible, as well as other plasmid vectors that may be more compatible with this system. Our original intention was to test methods and materials that are routinely used successfully for citrus transformation, to avoid additional and possibly lengthy experiments to assess various other Agrobacterium strains and plasmid constructs. We did so, according to plans, but found that the assessment of other strains and plasmids is unavoidable.
Funding is now in place among all the partners of the International Citrus Genome Consortium (US, Brazil, Spain, France, and Italy) to move forward with the project to sequence a haploid citrus genome. DNA samples for sequencing have been prepared, and the strict quality control standards required by the sequencing centers (JGI in the US, Genoscope in France, and IGA in Italy) have been met. DNA samples have been shipped only to Genoscope and IGA at this point. The University of Florida and Brazilian interests remain in negotiations with JGI over contract language, before JGI can proceed with their portion of the sequencing project. Meanwhile, work has proceeded at the UF-CREC to produce sample materials needed for the microarray experiments planned, using Affymetrix GeneChips, a new array platform developed by the co-PIs at UF using Agilent technology, and for the cDNA platform available through our co-PI in Spain. To this end, two sets of plants of sweet orange, rough lemon, and Volkamer lemon, representing the more susceptible and more tolerant types respectively, have been inoculated with budwood from HLB-infected Carrizo citrange (Carrizo is resistant to CTV, so viral interaction complications will be avoided) in an environmentally controlled greenhouse. Samples of RNA have been prepared from all of the plants at regular intervals, to be used in microarray experiments. Plants have been observed for symptoms, and qPCR has identified some that were successfully infected. The HarvEST Citrus EST database is in process of being updated, to provide an improved database for gene expressions studies. The EST sequences from our colleagues in Brazil and Japan have all been downloaded and reassembled, increasing the number of publicly available citrus ESTs to more than 465,000. The plans to exploit genome sequence information for a better understanding of the interactions of citrus plants with the pathogen causing HLB are ultimately most dependent on having the genome assembled and annotated; for this reason, our main focus will be on accomplishing that goal, while continuing to establish the experiments and collecting the samples that will be used for subsequent microarray analyses and deep transcriptome sequencing.
We received an APHIS permit to plant genetically modified citrus trees in the field. More than 900 transgenic citrus trees, containing 15 different constructs with potential to provide resistance to HLB and/or canker, have been propagated for field planting at 3 locations where HLB/canker pressure is severe. Planting will begin in a few weeks. Several new genetic constructs have been prepared for new transformation experiments, including lima b, a more ‘consumer-friendly’ sister to the lima a that has shown some promise already against HLB. With tests completed proving function two new phloem limited promoters, there are 4 currently available that could be combined with AMPs to target HLB in phloem tissue. Propagation and testing of previously produced transgenic plants continues, with several showing either delayed, reduced or no symptoms of HLB following inoculation. Transgenic plants have also been produced containing insecticidal genes and these are being prepared for tests against aphids and psyllids. Transgenic and citrus hybrid plants have been tested by canker inoculations, and those showing few or no symptoms are being retested. A series of experiments have been established to provide materials needed for research aimed at further characterization of gene expression differences resulting when susceptible citrus are challenged with HLB. RNA and DNA samples have been prepared at regular intervals to follow gene expression changes over time. These experiments may provide genetic targets to induce resistance to HLB, or at least to develop markers for early detection of disease. Studies into the metabolism of sugars and starch in HLB-infected plants have been initiated. Previous research microarray experiments were analyzed and have revealed more information on the defense machinery within kumquat cells. Several of the target genes have been fully cloned for further experiments including genetic transformation, following a more careful study of their gene expression patterns. New DNA samples from a genetic population have been received for genotyping to contribute to the International Citrus Genome Consortium sequencing project in collaboration with colleagues in Spain and France. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses made this spring 2009 have been harvested and planted. Previous work to develop rootstocks tolerant of or resistant to other maladies (such as CTV, blight, Phytophthora/Diaprepes, calcareous soils, etc.) continues, as we collected data from replicated trials and other plantings. New rootstock trials were planned, propagated, and planted, to test the suitability of experimental rootstocks for advanced production technologies; tree size control and broad-spectrum disease resistance and tolerance of adverse environmental conditions are critical to these efforts. Some trials include the recently released UF cultivar Sugar Belle (Tm), but most are with sweet orange. A new greenhouse screen for tolerance to Phytophthora/soil adaptation using tetraploid rootstock candidates has been established. Seeds have been harvested and planted from several dozen newly available rootstock candidates with tree size control and productivity potential, for future rootstock trials particularly aimed at new production technologies and orchard design. New pummelo-grapefruit hybrids, some of which were previously found to be significantly more tolerant of canker than grapefruit, are being propagated for canker challenges. Several of these hybrids also have been found to contain little or no furanocoumarin compounds, suggesting that they might not result in harmful grapefruit-drug interactions; further, some of them produce seedless fruit. New grapefruit-type candidates have been found this season and preliminary selections have been made based on fruit quality and attributes, and several hundred more such hybrids have been produced through crosses made in the spring and subsequent seed collection or embryo rescue, using newly available breeding parents. We are well on our way to meeting project objectives and goals.
Several new sweet oranges have been approved for release by UF-IFAS; these include several from Brazil believed to have canker tolerance, as well as new Valencia types with earlier and later maturity. The former will be generally available, but the latter will be patented and available through license agreements; together, they give industry new options to produce distinctive, high-quality juice. Canker tolerance of the Brazilian selections is currently being assessed under controlled greenhouse conditions, and preliminary results indicate that some of them are, in fact, significantly more canker-tolerant than ordinary sweet oranges. Additional data was collected from other advanced sweet orange selections in replicated field trials, and the best performing of these are being cleaned of pathogens and indexed, to provide certified budwood sources following approval for their release. These include a range of Valencia-types with improved color, yield, and juice quality attributes, as well as some very productive, seedless Midsweet clones; fruit and juice of these selections have been included in several commercial taste panels and assessments, and have been shown to be superior in their quality attributes. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses were made this spring 2009, that utilized germplasm with potential canker and HLB resistance or tolerance. Previous work to develop rootstocks tolerant of or resistant to other maladies (such as CTV, blight, Phytophthora/Diaprepes, calcareous soils, etc.) continues, as we collected data from replicated trials and other plantings. New rootstock trials were planned, with plants being propagated or already planted, to test the suitability of experimental rootstocks for advanced production technologies; tree size control and broad-spectrum disease resistance and tolerance of adverse environmental conditions are critical to these efforts. Some trials include the recently released UF cultivar Sugar Belle (Tm), but most are with sweet orange. New transgenes have been developed or acquired, and experiments have been established to introduce them into HLB and canker susceptible citrus. Previously developed transgenic plants have been inoculated with HLB, and those thus far showing no symptoms and negative qPCR tests (after more than 18 months) are being propagated for further testing. Several newly developed transgenic lines are being propagated for canker inoculations and evaluation, as well. New pummelo-grapefruit hybrids, some of which were previously found to be significantly more tolerant of canker than grapefruit, are being propagated for canker challenges. Several of these hybrids also have been found to contain little or no furanocoumarin compounds, suggesting that they might not result in harmful grapefruit-drug interactions; further, some of them produce seedless fruit. A series of experiments have been established to provide materials needed for research aimed at further characterization of gene expression differences resulting when susceptible citrus are challenged with HLB. RNA and DNA samples have been prepared at regular intervals to follow gene expression changes over time. These experiments may provide genetic targets to induce resistance to HLB, or at least to develop markers for early detection of disease. Previous research revealed several target genes in kumquat associated with its canker resistance (hypersensitivity). Microarray experiments were analyzed and have revealed more information on the defense machinery within kumquat cells. Several of the target genes have been fully cloned for further experiments including genetic transformation, following a more careful study of their gene expression patterns. Publicly available citrus genome resources are being mined for genes that may be used to hasten development of citrus plants resistant to HLB, canker, and other production-limiting diseases. Additional molecular markers have been developed and mapped in collaboration with colleagues in Spain and France, in support of the International Citrus Genome Consortium sequencing project. We are well on our way to meeting project objectives and goals.
We received an APHIS permit to plant genetically modified citrus trees in the field. More than 900 transgenic citrus trees, containing 15 different constructs with potential to provide resistance to HLB and/or canker, have been propagated for field planting at 3 locations where HLB/canker pressure is severe. Planting will begin in a few weeks. Several new genetic constructs have been prepared for new transformation experiments, including lima b, a more Òconsumer-friendlyÓ sister to the lima a that has shown some promise already against HLB. With tests completed proving function two new phloem limited promoters, there are 4 currently available that could be combined with AMPs to target HLB in phloem tissue. Propagation and testing of previously produced transgenic plants continues, with several showing either delayed, reduced or no symptoms of HLB following inoculation. Transgenic plants have also been produced containing insecticidal genes and these are being prepared for tests against aphids and psyllids. Transgenic and citrus hybrid plants have been tested by canker inoculations, and those showing few or no symptoms are being retested. A series of experiments have been established to provide materials needed for research aimed at further characterization of gene expression differences resulting when susceptible citrus are challenged with HLB. RNA and DNA samples have been prepared at regular intervals to follow gene expression changes over time. These experiments may provide genetic targets to induce resistance to HLB, or at least to develop markers for early detection of disease. Studies into the metabolism of sugars and starch in HLB-infected plants have been initiated. Previous research microarray experiments were analyzed and have revealed more information on the defense machinery within kumquat cells. Several of the target genes have been fully cloned for further experiments including genetic transformation, following a more careful study of their gene expression patterns. New DNA samples from a genetic population have been received for genotyping to contribute to the International Citrus Genome Consortium sequencing project in collaboration with colleagues in Spain and France. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses made this spring 2009 have been harvested and planted. Previous work to develop rootstocks tolerant of or resistant to other maladies (such as CTV, blight, Phytophthora/Diaprepes, calcareous soils, etc.) continues, as we collected data from replicated trials and other plantings. New rootstock trials were planned, propagated, and planted, to test the suitability of experimental rootstocks for advanced production technologies; tree size control and broad-spectrum disease resistance and tolerance of adverse environmental conditions are critical to these efforts. Some trials include the recently released UF cultivar Sugar Belle (Tm), but most are with sweet orange. A new greenhouse screen for tolerance to Phytophthora/soil adaptation using tetraploid rootstock candidates has been established. Seeds have been harvested and planted from several dozen newly available rootstock candidates with tree size control and productivity potential, for future rootstock trials particularly aimed at new production technologies and orchard design. New pummelo-grapefruit hybrids, some of which were previously found to be significantly more tolerant of canker than grapefruit, are being propagated for canker challenges. Several of these hybrids also have been found to contain little or no furanocoumarin compounds, suggesting that they might not result in harmful grapefruit-drug interactions; further, some of them produce seedless fruit. New grapefruit-type candidates have been found this season and preliminary selections have been made based on fruit quality and attributes, and several hundred more such hybrids have been produced through crosses made in the spring and subsequent seed collection or embryo rescue, using newly available breeding parents. We are well on our way to meeting project objectives and goals.
Several new sweet oranges have been approved for release by UF-IFAS; these include several from Brazil believed to have canker tolerance, as well as new Valencia types with earlier and later maturity. The former will be generally available, but the latter will be patented and available through license agreements; together, they give industry new options to produce distinctive, high-quality juice. Canker tolerance of the Brazilian selections is currently being assessed under controlled greenhouse conditions, and preliminary results indicate that some of them are, in fact, significantly more canker-tolerant than ordinary sweet oranges. Additional data was collected from other advanced sweet orange selections in replicated field trials, and the best performing of these are being cleaned of pathogens and indexed, to provide certified budwood sources following approval for their release. These include a range of Valencia-types with improved color, yield, and juice quality attributes, as well as some very productive, seedless Midsweet clones; fruit and juice of these selections have been included in several commercial taste panels and assessments, and have been shown to be superior in their quality attributes. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses were made this spring 2009, that utilized germplasm with potential canker and HLB resistance or tolerance. Previous work to develop rootstocks tolerant of or resistant to other maladies (such as CTV, blight, Phytophthora/Diaprepes, calcareous soils, etc.) continues, as we collected data from replicated trials and other plantings. New rootstock trials were planned, with plants being propagated or already planted, to test the suitability of experimental rootstocks for advanced production technologies; tree size control and broad-spectrum disease resistance and tolerance of adverse environmental conditions are critical to these efforts. Some trials include the recently released UF cultivar Sugar Belle (Tm), but most are with sweet orange. New transgenes have been developed or acquired, and experiments have been established to introduce them into HLB and canker susceptible citrus. Previously developed transgenic plants have been inoculated with HLB, and those thus far showing no symptoms and negative qPCR tests (after more than 18 months) are being propagated for further testing. Several newly developed transgenic lines are being propagated for canker inoculations and evaluation, as well. New pummelo-grapefruit hybrids, some of which were previously found to be significantly more tolerant of canker than grapefruit, are being propagated for canker challenges. Several of these hybrids also have been found to contain little or no furanocoumarin compounds, suggesting that they might not result in harmful grapefruit-drug interactions; further, some of them produce seedless fruit. A series of experiments have been established to provide materials needed for research aimed at further characterization of gene expression differences resulting when susceptible citrus are challenged with HLB. RNA and DNA samples have been prepared at regular intervals to follow gene expression changes over time. These experiments may provide genetic targets to induce resistance to HLB, or at least to develop markers for early detection of disease. Previous research revealed several target genes in kumquat associated with its canker resistance (hypersensitivity). Microarray experiments were analyzed and have revealed more information on the defense machinery within kumquat cells. Several of the target genes have been fully cloned for further experiments including genetic transformation, following a more careful study of their gene expression patterns. Publicly available citrus genome resources are being mined for genes that may be used to hasten development of citrus plants resistant to HLB, canker, and other production-limiting diseases. Additional molecular markers have been developed and mapped in collaboration with colleagues in Spain and France, in support of the International Citrus Genome Consortium sequencing project. We are well on our way to meeting project objectives and goals.
Since the funding period started, one laboratory technician was added to the staff of CCTF. He was trained in all procedures employed in the lab and presently is capable of performing duties with very little supervision. Organization of the work in the lab was set to allow for higher influx of orders through different assignment of chores. The list of transgenic plants that were produced and confirmed by the presence of reporter gene and appropriate PCR reaction: LIMA gene: 3 Flame; LIMA(SN) gene: 5 Flame; CIT108 gene: 12 Valencia; CIT108p gene: 7 Flame; CIT108p3 gene: 14 Flame; CIT108p17 gene: 6 Flame; CL1 gene: 1 Valencia; 2 Duncan; CL2 gene: 1 Valencia, 1 Duncan; PITA gene: 40 Duncan, 3 Valencia; C5* gene: 3 Duncan; CN1 gene: 1 Duncan; N1* gene: 2 Duncan; F3* gene: 8 Duncan. Presently, there are about 90 plants in the soil that were picked as positive for presence of reporter gene but not tested by PCR.
Main goal – Analysis of transcriptome of sweet orange Hamlin during the infection of Candidatus Liberibacter spp. Main activities Experiment set up Ð From February to June 60 plants of sweet orange Hamlin were grafted into Rangpur lime and established at screen house. They will be grafted with infected budwoods in vegetative development of the plants and grafting of the budwoods. I have a pure source of Candidatus Liberibacter asiaticus (CLas) kept under screen house but source of Ca. Liberibacter americanus (CLam) has been difficult to keep, because the bacteria have low transmission efficiency with infected budwoods. Infected plants in the field have decreased in the last years, and CLas seems to be more pathogenic than CLam. The experiments will be changed from the original version in order to improve the quality of results. Besides the time course for sampling of infected and health leaves, tissue of bark (enriched with phloem tissue) in the infected branches will also be collected and compared with leaves. A concern in the experiments is regard with time of infection that can really be associated to the interaction plant with the bacteria. Preliminary we consider 0, 2, 6, 12, 18,24, 30, 45, 60, and 90 days after grafting. I am evaluating to consider three main pools. One before the bacteria can be detected by RT-qPCR (0, 2, 6, 12, 18 days); other pool of samples collected at 24, 30, 45 and 60 days. Sample after 90 days will be considered alone, since at that time severe damages caused by the disease could disturb the results. Thus the experiments could be conduced with three pools of leaves + control, three of bark + control, with tissue infected with both bacteria individually. Array design – The arrays and the hybridization will be conduced as service by Nimblegen / Roche. A set of arrays was already tested in other experiment with suitable quality. The database was 32,000 unigenes of sweet orange (CitEST). Each unigene was represented 6 times with oligo probes of 50 to 60 bases. Therefore, an array has a density of 180,000 spots. Procedure for RNA isolation with high quality is established. RT-qPCR Ð Although the experiments with arrays were not finished, we are looking for good candidates as house keeping genes in citrus. Among them .-tubulina, expansin-like B1, and ankirin. Some troubles Ð Although FCPRAC sent a check with part of financial support in Abril 2009, until now the check could not be paid. The bank cannot explain what is going on, but they promised in August the funds would be available. Actually all activities were until now conduced with funds of other projects.
The HLB pathogen is acquired by the Asian citrus psyllid during the feeding process, which indicates that the alimentary canal is the first organ of the transmission cycle. Our first task in this collaboration has been completed by stockpiling this organ for mRNA analysis. 1000 adult, noninfective psyllids were submerged, one at a time, in 50% RNAse Later¨ (Ambion), a general protease that destroys transcript degrading enzymes. The alimentary canals of each were extirpated in this solution and transferred with a pinpoint to 0.5ml of 100% TRIzol¨ (Invitrogen), a powerful phenolic that allows for deep freezing and long-term storage. The same low volume of phenolic and high number of guts has been shown with whiteflies to yield an outstanding 28 nanograms mRNA per microliter final concentration. As an aside, alimentary canals were dissected and pooled from two adult psyllids confined to an infective orchard tree branch for three days, to see if a qPCR signal could be had in so small a sample. Results were a Ct value of 35, where 40 is no signal and 20 is a high-titer signal. This first test run indicates that quantification of titer in individual guts is a highly feasible approach to quality control when stockpiling of organs from infective psyllids commences. The laboratory rearing system is under construction and excellent progress has been made once psyllids began reproducing at high levels (June onward) and some adults and immatures, as well as samples from different portions of the plants on which colonies are being reared have been collected for qPCR analysis (in progress).
The project has two objectives: (1) Over-express MAP kinase kinase 7 (MKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, the Arabidopsis MKK7 gene has been cloned into the CTV-based expression vector and transition expression of MKK7 in citrus leaves is underway. The Arabidopsis MKK7 gene has also been cloned into the plant binary vector pBI1.4T (a pBI121 derivative) and transformed into citrus using the Agrobacterium-mediated approach. Characterization of the MKK7 transgenic citrus plants is underway. For objective 2, citrus cell suspension culture has been established and the concentration of sodium iodoacetate that will be used for the selection has been experimentally determined. Two citrus cell suspension cultures were obtained, one Navel orange cell suspension culture and one Hamlin cell suspension culture. Both cell suspension cultures were established using soft, friable callus derived from nucellar tissues. After subculturing for several generations in the Murashige and Tucker (MT) liquid medium, the cell aggregates in the Navel orange culture formed large clumps and very few single cells could be visualized (data not shown). In contrast, the cell aggregates in the Hamlin culture were dispersed and very small clumps and single cells were equally distributed throughout the liquid media. When placed on the MT solid embryogenic callus-inducing medium, the cells quickly propagated and formed calli. Therefore, the Hamlin suspension cells were used as starting materials for the selection. The Hamlin cell suspension culture has been scaled up in the MT liquid medium. Several flasks of the culture are maintained for subculture. Since different plant species and different starting materials require different concentrations of sodium iodoacetate to completely inhibit their growth (data not shown), the concentration that could completely inhibit the growth of the Hamlin suspension cells was experimentally determined. The Hamlin cells from the suspension culture were grown on the MT medium plates supplemented with different concentrations of sodium iodoacetate ranged from 0 to 0.2 mM. Hamlin suspension cells were found to be highly sensitive to the inhibitor. A concentration of 0.1 mM of sodium iodoacetate could completely arrest their growth. Therefore, 0.1 mM of sodium iodoacetate will be used in the selection.
This project has three objectives: 1) gap closure of Ca. Liberibacter asiaticus (Las) found in Florida; 2) complete genomic sequencing to closure of Ca. L. americanus (Lam) strain S’o Paulo from Brazil, and 3) comparative genome analysis of Las and Lam to attempt to determine common factors enabling pathogenicity to citrus. This project is complementary and synergistic to work that was recently published (Duan et al. 2009. MPMI 22:1011-1020), in which the nearly complete genomic sequence of Las strain psy62, extracted from infected psyllids, was obtained. Within psy62, many unique genes of unknown function are found and it is difficult to establish which genes among the many unique unknowns might be important to enable pathogenicity on citrus. If the same unknown genes are found in a different species that causes the same disease (Huanglongbin, or HLB) on citrus, this information can considerably focus the research priorities for candidate genes that may be investigated as possible targets for chemical or genetic control of HLB. Our approach is to obtain the complete genomic sequence of a different Liberibacter species, Lam, that also causes HLB, and compare it to psy62. In collaboration with Fundecitrus in Brazil, DNA samples extracted from citrus midribs infected with Lam were purified on pulsed-field gels by Dr. Nelson Wulff and brought to UF. We used multiple displacement amplification (MDA) to obtain sufficient DNA for sequencing. Two trial shotgun libraries were constructed, one from each sample, and tested for quality by 454 sequencing. After bioinformatics filtering, Sample 1 yielded 1.6 Mb of sequence, of which 13 kb (0.8%) was new Lam DNA sequence. Sample 2 yielded 9.2 Mb of sequence, of which 41 kb (0.4%) was new Lam DNA sequence, resulting in 54 kb of new Lam DNA sequence to date. More importantly, the principle contaminating DNA from these trial runs was identified as citrus mitochondrial DNA, which guided modification of our extraction method so that this contaminating DNA has now been greatly reduced from a third DNA sample, which has been sent for full scale sequencing. In anticipation of a draft Lam genome, subsequent Las/Lam genome comparisons, and also to help close remaining gaps in the psy62 genome, two fosmid libraries were constructed using DNA extracted from HLB infected citrus, one from Lam strain S’o Paulo and one from Las strain UF506. Each library consisted of 1100 fosmid clones, both with an average insert size of ca. 31 kb, cloned in fosmid vector pCC2FOS. The Las UF506 library was used to close gaps surrounding 8 unjoined contigs of psy62, all containing phage-related genes. DNA probes were made as PCR products amplified from Las infected citrus and used to probe nylon blots of the UF506 fosmid library. Eight fosmids were identified and partially sequenced to join 7 previously unjoined psy62 contigs to form one complete 40,048 bb circular phage genome (named SC-1) plus one 31 kb contig, potentially representing a highly related but separate phage SC-2. SC-1 has 28 direct repeat regions. Both SC-1 and the SC-2 supercontig were validated by PCR amplification of HLB infected citrus along 89% of their entire lengths.
Due to the recent arrival of the funds on campus, experimental aspects of the project are still in the early stages and limited to preparation of primers and the cloning of the promoters and resistance protein genes that will be evaluated in the project as follows: 1-A series of phloem-specific promoters from Arabidopsis and other plants are in the process of being cloned for evaluation in transformed plants. Initial assessment of the constructs will be evaluated in transient assays and suitable candidate clones will be transformed into Arabidopsis and citrus plants to test for phloem-specific expression. These promoters will be used to express resistance proteins (R proteins) and R protein mutants in an attempt to confer resistance to Liberibacter infection. Due to the potential of R proteins to interfere with normal growth and development of the plant, their expression will be limited to the phloem tissues, the site where the pathogen resides. 2- Several R proteins are being cloned for expression in Arabidopsis and citrus plants. Attenuated mutants of these proteins will be first expressed in the transient assay using Arabidopsis leaf mesophyll protoplasts to evaluate their effects on normal cellular function. Expression of these proteins will be driven by the phloem-specific promoters cloned in objective 1 above.
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