As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. A new 8 acre site has been bedded, supplied with irrigation, and a ground cover established. Several acres in the far NE corner have been prepared for Dr. Dawson’s proposed field test of modified CTV expression vectors designed to produce anti-microbial peptides in citrus host plants. APHIS specified that Dr. Dawson’s site be as far from existing commercial citrus groves as possible, and recommended the NE corner of the Picos Farm. There has been no recent word on the progress of APHIS approval for this project Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Trees were sprayed with microsprinklers throughout the winter freeze, and trees are unscathed. USHRL has a permit pending with APHIS to conduct field trials of their transgenic plants at this site. An MTA is now in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Alphascents has agreed to provide an experimental pheromone attract/kill product Malex to disrupt citrus leaf miner (CLM). Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Because of delays in getting funding and initiating this research infrastructure project, a no-cost extension has been requested and approved.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate transformants of rootstock and scion genotypes. Thousands of putatively transformed shoots have been developed to produce citrus resistant to HLB and CBC or citrus psyllid. Many hundreds have been micrografted. D35S/D4E1 transformed rootstocks have been challenged with HLB and CBC. Initial trials on CBC resistance were inconclusive. HLB-inoculated transformed plants grew significantly better than controls but displayed Las development and show HLB symptoms. More active promoters have been identified and used in recent transformation. A wide series of promoters driving a reporter gene are being tested in transformed citrus. Tests of garlic-lectin transformed citrus are underway to determine effect on psyllid feeding and development. Liberibacter sequence data are being used to develop a transgenic solution for HLB-resistance, targeting a transmembrane transporter. Peptide has been made corresponding to the extra-membrane sequence and a phage display array system is being used to identify structures which are specific to this epitope, with the final steps underway. When identified, transgenics will be constructed and challenged with Las. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data to permit transformation and resistance using citrus-only sequences. Border sequences and promoters from citrus are now in a construct driving GUS and will soon be inserted into citrus. 39 antimicrobial peptides (AMPs) have been assessed in-vitro for activity in suppressing growth of the bacteria causing CBC and two bacteria related to Liberibacter. In the initial studies, the synthetic AMPs D4E1 and D2A21 were among the most active, along with the Tachyplesin (which is among the most effective AMPs in Dr. Dawson’s CTV expression vector study), with minimum inhibitory concentrations at 1 ‘M or less across all test bacteria. An additional 20 synthetic AMPs were assessed, revealing several AMPs that were highly active against all test species, with negligible hemolytic activity, and some of these were constructed using key functional elements from the horseshoe crab-derived Tachyplesin. Transformation constructs will be prepared to produce citrus with these AMP transgenes, having completed an agreement with entities who posses the rights to these AMPs. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. After 1 year in the field, the first trial shows similar levels of infection across all three methods of Liberibacter transfer. The complete experiment is being repeated and planted in February 2010. the greenhouse complement to this study is showing earlier symptom development than field trees, especially from graft-inoculation. High-throughput CBC screening methods are being compared, with the hope that CBC-resistance will be correlated with HLB resistance in transgenics driven by constitutive promoters. Very sensitive methods have been derived and leaf extracts with and without spiked AMP have been tested. A material transfer agreement has been established with Texas A&M University and we have received their spinach defensin AMPs for in-vitro analysis. Since this material is well down the regulatory pathway, it makes no sense to move forward with any transformed citrus which is not markedly superior to this benchmark material.
Progress on first year’s objectives: 1) Build a growth room in Florida for growing citrus for mature transformation. The initial project to construct a closed greenhouse with controlled temperature through air-conditioning has been abandoned due to its excessive cost, that almost doubles the budget available. Alternatively, the CREC is offering an space that could be fully adapted to construct a growth room with all the requirements needed to grow an maintain citrus trees in excellent phytosanitary and physiological conditions. The details of the construction are being provided to the IFAS project manager to guarantee the facility will fulfill the needs to produce plant material for mature citrus transformation. 2) Training of the Florida manager (Dr. Zapata) at IVIA in Spain. Dr. Cecilia Zapata has spent three months at IVIA to learn the technology to transform mature citrus. She has been trained in all tissue culture techniques associated with citrus transformation, from preparation of the source plant material to acclimation of transformants. She has also learnt how to start and maintain a facility to support a mature transformation laboratory. She is initiating her second three-month stay in our lab at IVIA next week. 3) Establishment of genetic transformation systems for mature materials from the most important sweet orange varieties grown in Florida and Carrizo citrange. During Dr. Zapata’s stay in Valencia, several transformation experiments were set up with Valencia, Hamlin and Pineapple sweet oranges and with Carrizo citrange. Screening of putative transformed plants regenerating from the in vitro cultures revealed that Valencia was being readily transformed, at efficiencies slightly lower than those usually get with Pineapple, while Hamlin and Carrizo citrange were more recalcitrant to transgenic regeneration thought some PCR-positive shoots were already obtained. 4) Strategies to improve tree management. We are overexpressing the flowering time genes FT and AP1 from sweet orange in juvenile sweet orange and Carrizo citrange for generating new rootstock and scion orange types putatively more compact and productive. In general, overexpression of either transgene leads to early flowering in vitro and poor regeneration. However, some of the transformants do not flower in vitro but show a compact and branched phenotype. They have been transferred to the greenhouse and are being characterized at the phenotypical and molecular level.
This project aims to assess a wide range of citrus germplasm, including some relatives, for tolerance or resistance to HLB, through both greenhouse assays and field tests; these germplasm resources wherein tolerance/resistance might actually be found were selected on the basis of research and observations in Asia and Florida. Funding for the project was made available later than anticipated, however we did perform some of the preliminary tasks of collecting germplasm resources in advance of the decision to support the project; once funded, we expanded the effort substantially. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some derived hybrids 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. These were grown in a DPI-certified structure as budwood sources for propagations (just underway), some of which will be tested in protected structures at the CREC by graft inoculation, and for direct planting to the field for HLB challenge. Further, we have explored planting out the Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test, but as these were maintained previously in non-certified structures we are waiting on resolution of the matter with DPI, to be granted conditions and permission to plant these at the Picos Road Farm with USDA-ARS, near Ft. Pierce. This population is of significant interest as it appears that the trifoliate orange and some of its hybrids are very tolerant (though not truly resistant) to HLB. This family provides an opportunity to map the genetic components responsible for the tolerance, if in fact tolerance is found to be segregating in the population. Numerous somatic hybrids of citrus with related genera are also being prepared for inclusion in the plantings. We are in the process of acquiring additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal. Finally, we are making a number of crosses in spring 2010 to produce segregating families of several purported tolerant and susceptible types to begin searching for evidence of genetic control of HLB tolerance/resistance within the citrus gene pool. Our collaborators in China have sought domestic funding support but have not yet been successful, so no materials have been sent there at this time; we remain in communication with them, to explore other options. One company in Florida that initially offered land for the project has since withdrawn its offer, a consequence of an upper level management decision. We have a tentative agreement with another grower on the east coast of Florida to plant out the range of genetic diversity we hoped to test, both as seedlings and as top-worked trees, including some apparently tolerant types we have identified in an HLB-devastated grove in Florida. We are currently exploring other options within Florida, to be followed up with agreements to move ahead; these represent locations where growers have decided not to remove HLB-infected trees, so we expect there to be opportunities to challenge our replicated materials. 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.
Transgenic citrus trees (900 containing 15 potential HLB/canker resistant constructs) were planted at 2 locations with severe pressure. At least 1500 transformations with various new constructs and promoters, mostly using sweet orange selections, have been produced for greenhouse disease tests; many of these will be planted in fall 2010. Our transgenic field trial in Gainesville, testing disease resistance transgenes, survived the brutal winter. We have verified 4 tissue-specific promoters that target gene expression only to phloem. Testing of previously produced transgenics indicates several thus far with delayed, reduced or no symptoms following HLB-inoculation. Transgenic Carrizo plants were produced with insecticidal genes; preliminary feeding tests showed one gene tested killed aphids and psyllids. Several transgenic and hybrid plants have been identified with tolerance to canker. New disease resistance genes were cloned from grape and tested in tobacco for efficacy; preliminary results identified two potential candidates for citrus. A newly developed grape-derived anthocyanin marker allows visual selection of transformants, and will be tested in citrus, an alternative to non- plant markers used in most transformations. Experiments on transient expression of gene constructs are underway; when optimized, this will allow more rapid testing of promising disease resistance genes. Two microarray platforms (our Agilent chip containing disease resistant genes and the Affymetrix Citrus GeneChip) are being used to analyze citrus gene expression over time in response to canker and HLB. Comparisons of sugar and starch metabolism in HLB-infected and healthy plants revealed that starch, sucrose, and glucose accumulated in infected leaves, maltose decreased and fructose levels were unchanged; studies are underway comparing activity of cell-wall bound invertase, a critical enzyme involved in sucrose metabolism and plant defenses. Using the iTRAQ technique, proteomic analysis was used to compare healthy and HLB-diseased mature leaves of sweet orange; 19 proteins were differentially expressed, out of which 9 proteins involved in stress and defense responses were highly up-regulated. Previously, microarray experiments highlighted canker defensive genes in kumquat; real-time PCR confirmed roles of one kumquat R-gene, two kinases, and one transcription factor, new targets for transformation. Cybrid grapefruit plants were produced with kumquat, and preliminary results show that some behave like kumquat in challenges. New DNA samples from a genetic population are being genotyped to contribute to the ICGC sequencing project. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses made 2009 are growing off. Preliminary results of an experiment to study experimental rootstock effects on the growth of HLB-infected budwood revealed significant differences in disease expression and growth. Previous work to develop rootstocks against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) continues, as we collected data from replicated trials and plantings. 15,000 trees were propagated from > 100 advanced UF-CREC rootstock selections, for several planting density/open hydroponic system trials located throughout Florida, to evaluate their potential in closely-spaced groves under intensive cultural management. > 200 candidate rootstock hybrid seedlings were selected following a screen for Phytophthora tolerance /hi-pH soil adaptation. New pummelo-grapefruit seedless hybrids were created, and others available were found to be more tolerant of canker than grapefruit. New grapefruit-type candidates have been found this season, with selection based on fruit quality attributes. Sugar Belle, UF’s 1st fresh release, was licensed to NVDMC, and the first crop was successfully marketed. Six new seedling orange selections were released, as unpatented and unlicensed varieties; 5 are early maturing with exceptional quality, and one with reported canker tolerance. Our first UF-bred orange cultivars, Valquarius (SF14W-62) and Valenfresh (N7-3), are being licensed, to provide 6-8 week earlier and later maturity.
Objective II: Website creation and development. The Citrus Greening/HLB Genome Resources Website has been created and can be accessed at http://www.citrusgreening.org/. Significant developments related to this objective include the following: A. Development of a policy for data sharing. In collaboration with the program manager, a policy was developed whereby the genome resources web site would provide a venue for deposition of preliminary data, with access limited to registered users. B. Website organization. The web site in question includes multiple open-access web pages listing general information and links on the disease, guidelines and links for genome analysis tools such as BLAST and the Artemis genome viewer, and pages summarizing the variety of Ca. Liberibacter sequences that have been deposited at NCBI. With the exception of a 1.2 Mb draft genome sequence for Ca. L. asiaticus, all of the Ca. Liberibacter sequences currently available at NCBI fall into one of three gene clusters, diagrams of which have been posted on the web site with hyperlinks to the corresponding records at NCBI. Users wanting access to restricted pages are required to enter contact information and agree to the terms of the data sharing policy. 45 users are currently registered. The PI has made use of the registration-restricted pages for posting analyses of the initial Ca. L. asiaticus psy62 draft genome sequence and the restricted section continues to be available for deposition of sequence data. The PI is presently posting her own group’s unpublished analyses in the non-restricted section to maximize visibility to the larger community. Future plans for web site development include installation of a more directly interactive genome viewer such as GBrowse. Not only will this provide easier access to genome features and comparative data, but will also provide a platform for progressive incorporation of links between individual gene loci and protein structural predictions, metabolic pathway assignments, and additional data generated by other groups working on this bacterium. The PI has attended meetings during the past year to both publicize online resources and interact with other researchers involved in genome analysis. Objective III: Bioinformatic analysis of Ca. L. asiaticus sequence data Using the genome sequence data deposited for Ca. L. asiaticus psy62, multiple analyses have been conducted including screening the sequence for compositional variation suggestive of horizontal transfer and for repetitive elements with potential diagnostic application. Results of these and other analyses are posted on the ‘Ca. Liberibacter genome resources’ page of the web site. Gene regulation is of particular interest given its role in mediating adaptation of Las to its plant host and insect vector. Relatively few regulators of gene expression are encoded by the Las genome but two of particular interest are RpoH, associated with induction of genes linked to survival at elevated temperatures, and RirA, a protein regulating uptake and metabolism of the critically important nutrient, iron. Using models for binding sites derived from experimentally characterized sequences in related bacteria, RpoH and RirA regulated genes can be predicted from genome sequence data. A manuscript currently in preparation describes sets of genes predicted to be regulated and outlines a method to aid researchers in distinguishing biologically significant predictions from those arising by chance when analyzing experimentally uncharacterized genome sequences. Future plans for bioinformatic analysis include comparison of the Ca. L. asiaticus genome sequence with those of related alpha-proteobacteria and other strains of Ca. Liberibacter as they become available.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene (with an enhanced, inducible defense response) have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. During the past year we finalized the propagation and grafting of the transgenic plants necessary for this experiment. The experimental plants are transgenic ‘Carrizo’ citrange (lines: 854, 857, 859 and 884) transformed with the AtNPR1. Previous tests showed that lines 854 and 857 overexpressed the endogenous marker gene PR1 (considered a marker of SAR). On the other hand, lines 859 and 884 did not express the AtNPR1 transgenic gene and did not show overexpression of the endogenous PR1 gene, hence were considered as negative controls. Subsequently we grafted a number of these plants with wild type (WT) ‘Duncan’ grapefruit. We also grafted WT ‘Carrizo’ plants with ‘Duncan’ grapefruit as controls. In total, we have about 50 plants including WT and transgenic controls with replicates. We also treated these plants with either salicylic acid (SA) or water (as negative control) and compared their response using TaqMan Real Time PCR. In preparation for the real time experiments we sequenced a number of genes of interest (NPR1, NPR3 and PR1) from both ‘Carrizo’ and ‘Duncan’ to guarantee that the target probe/primer sequences within the genes were identical and that any observed differences in expression were not due to differential efficiency in annealing of the probes and/or amplification. This year we also standardized and validated the Real Time reactions for the transgene AtNPR1 and the endogenous citrus genes PR1, NPR1, NPR3 and EDS5, all of them key SAR genes. Additionally, we identified a previously undescribed gene that is part of the NPR1/NPR3 family of genes in citrus and that our experiments show is also induced by SA. This family of genes has been shown to be central in the regulation of SAR in other species. We are in the process of validating the sequences from ‘Duncan’ and ‘Carrizo’ and developing the necessary primers/probe for the Real Time PCR assay. All of this work will allow us to analyze the response of the plants as proposed in Objective 1. Using Real time PCR (Comparative Ct experiment) we confirmed the expression of AtNPR1 in lines 854 and 857. The expression was about twice as high in the SA-treated plants compared to the water treated plants. Plants from these two lines also exhibited levels of PR1 expression up to 200 times higher than those of transgenic controls or wild types, confirming our previous results. We will repeat the SA treatment experiment to confirm the results and analyze the expression of more genes as stated in our objectives. In addition the same group of plants will subsequently be analyzed as proposed in objectives 2 and 3 for their response to HLB infection. For this purpose we have propagated HLB-infected material and standardized the Real Time PCR detection of the pathogen.
The goal of this project is to transform the citrus and Arabidopsis NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac. (3) Inoculation of the transgenic plants with the HLB pathogen and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. During the first nine months of studies, we have identified three transgenic lines overexpressing CtNPR1 and AtNPR1, respectively, by using Northern blot analysis. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants, suggesting that both CtNPR1 and AtNPR1 are functional in citrus resistance to canker disease. Establishment of resistance to Xac by CtNPR1 is particularly significant for engineering resistance in citrus in the future. Preparation of a manuscript describing these findings is in progress. We have also inoculated the transgenic plants expressing a truncated XIN31 and the preliminary data showed resistance to Xac306. We will further characterize these plants in year 2 of this project. To prepare for greening inoculation, we have grafted the six NPR1 (three each of CtNPR1 and AtNPR1) overexpression lines and the control onto more root stocks to propagate the transgenic population. A total of 7-15 individuals have been produced for each of the transgenic lines. All these plants are currently maintained in green-houses located at the Citrus Research and Education Center in Lake Alfred, and will be used for greening inoculations in year 2 of this project. Finally, we have finished the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. This construct may increase the expression of CtNPR1 in citrus phloem thereby maximizing the opportunity for resistance to greening. Citrus transformation of this construct is in progress. In summary, we have achieved most of the goals for year 1, which establishes a firm foundation for the research in next year. The delay for objective 3 in year 1 is largely due to the fact that greening inoculation requires the transgenic plants growing to relative bigger sizes. To continue our research, we request funds for the second year to achieve the following goals as proposed originally: (1) Inoculation of the characterized NPR1 transgenic plants with the HLB pathogen and monitoring of the bacterium in planta with quantitative PCR; (2) Characterization of transgenic plants expressing the truncated XIN31; (3) Transformation of SUC2::CtNPR1 into citrus; (4) Microarray analysis of the CtNPR1 plants in response to Xac or greening inoculations; (5) Examination of changes in hormone (abscisic acid, auxin, jasmonic acids and salicylic acids) levels in the CtNPR1 plants infected with Xac or HLB; (6) Plant maintenance. Accomplishment of these goals will very likely generate transgenic citrus plants with resistance to the HLB pathogen and advance our understanding of how citrus responds to these two diseases and could lead to new tools and strategies for the control of these two important diseases in Florida.
The funding that was received allowed the Core Citrus Transformation Facility (CCTF) to have best year since it opened. As such, CCTF has continued to serve research community by providing reliable service through the production of transgenic citrus plants, primarily for NAS/FCPRAC funded projects of other researchers that do not have citrus transformation capacity. The initial objective of this project has been met in a very short period of time. The employment of the additional OPS technician allowed for the increase in the amount of work that is being performed. In the induction phases of transformation process, increment was adjusted to the maximum available capacity of the laboratory. The number of explants that is being processed has increased from 2000 per week to about 2800. However, major improvement achieved due to addition of new employee is that there are no back-logs in the selection phase of the experiments. Number of shoots or soil-adapted seedlings that can be inspected/assayed for the presence of reporter gene or used in the PCR reaction is about 200 per week. Increased capacity to inspect shoots and soil-adapted plants resulted in higher numbers of detected plants. Most recently, new method for the detection of foreign DNA in plants that requires minute amounts of tissue as a source of template was adopted by the facility. Application of this method has further facilitated our rate of detection of transgenic plants because screening can be done on a small shoots before they get micro-grafted on the rootstock plants. This is very important for those orders where binary vectors have no reporter gene. Also, labor that was earlier invested into ‘shotgun’ grafting of many shoots where at least some would be positive is now diverted to care of confirmed transgenic plants. As a consequence, the delivery time for production of PCR-confirmed transgenic plants is made shorter than in the past. The work performed within last 12 months included production of plants transformed with 21 binary vectors. They are: pCL2, pAF1, p6Cass, p pSUC-LIMA1, pLIMA, pLIMA-Sn, pNPR1, pPiTA, pCIT1070, pCIT108, pCIT108p, pCIT108p3, pCIT108p17, pN1*, pC5*, pF3*, pCN1, pCAMBIA2301, pTLAB21, pTLAB32, and pSuperNPR1. Out of these 21 vectors, 17 carry genes associated with improvement of citrus resistance against pathogens. Three vectors (so-called ’empty’) were used to produce transgenic plants without the gene of interest. These plants represent obligatory control plants for comparisons between transgenic material and wild-type plants. Only one binary vector included gene that can affect citrus development. Transgenic plants that were produced belong to seven cultivars: sweet oranges-Hamlin and Valencia, grapefruits-Duncan and Flame, rootstocks-sour orange and Carrizo, and Mexican lime. All together, CCTF produced more than 450 transgenic plants during the first year funding cycle. Relevance of this project to the overall effort to break the HLB cycle and fight against canker remains high. Transgenic plants produced within last 12 months are from orders placed by seven different clients. Six out of seven clients are faculty presently involved in research projects associated with NAS/FCPRAC funded efforts to produce Citrus plants resistant/tolerant to huanglongbing (HLB) or canker. Some of transgenic Duncan grapefruit plants that CCTF produced for one of the recent orders may represent a breakthrough in the fight against Citrus canker. In a challenge experiments with canker-inducing bacteria, these plants exhibited significant increase in resistance to this disease. Within the last three and-a-half months, the CCTF facility received a large number of new orders. Therefore, CCTF continues to be an irreplaceable element in the fight against Citrus diseases and especially HLB. Activities of the CCTF on a few orders that have to do with improvement of Citrus not associated with disease resistance are continuing as well.
Juvenile citrus transformation is at least an order of magnitude more efficient and less cultivar specific than mature tissue transformation. If the juvenile period in subsequent trasngenic plants can be overcome quickly, commercialization could be on a time-frame similar to transgenics from mature tissue transformation. Significant progress was made on all primary objectives, and a new research result from a parallel project was obtained that will impact his project significantly – early flowering (11 months after planting) on several completely juvenile scion hybrids in our breeding program was achieved in the RES (Rapid Evaluation System, funded by NVDMC). Work is in progress (in the RES) to test the horticultural manipulations utilized to achieve this result on commercial sweet orange, grapefruit and mandarin scions most important for our industry, for subsequent use with HLB-resistant transgenic lines. The Agrobacterium-mediated juvenile citrus transformation protocol was improved to increase speed and efficiency: utilizing a combination of improved media, a new anti-oxidant treatment, and a modified micro-grafting technique, we are able to consistently recover and replicate more transgenic citrus plants in half the time. Alternative transformation methodology: A protocol was developed for the direct transformation of embryogenic callus, and numerous transgenic plants were recovered from OLL-8 sweet orange, W. Murcott tangor,(Afourer/Nadercott), and Ponkan tangerine. This technique clearly extends transformation methodology to other important polyembryonic commercial citrus cultivars, particularly those that are recalcitrant to Agro-bacterium mediated transformation (ie. fresh market mandarin types that are important in Calfornia). Transformation of Selected Precocious or Potentially HLB-Avoiding Sweet Oranges: Using the improved protocol, transgenic plants of high quality precocious Vernia sweet orange somaclones C2-1-1, C2-1-2 and C2-2-1, Rhode Red Valencia clones avoiding HLB infection (B4-79 and B10-68) in a heavily HLB-infected Martin County grove, and a high quality processing somaclone OLL#8, containing the LIMA anti-bacterial gene were produced. The OLL somaclones are showing mature tree juice quality in juvenile trees. Non-transgenic seedlings of these clones are being grown for evaluation in the RES to determine the effect of scion genetics on length of juvenility. Rootstock Effect on Length of Juvenility: Juvenile sweet orange scion (OLL-8, a high quality Valencia-type with high solids and enhanced juice color) grafted to 6 selected precocious rootstocks from our breeding program and Carrizo as a control were single-stemmed and planted in the RES. Rootstock effect on the speed of juvenile citrus flowering will be determined. Transfer of genes to induce precocious flowering: We have cloned each of the genomic sequences of ciFT1, ciFT2, and ciFT3 (Arabidopsis Flowering Locus T genes) into a plasmid vector in which their expression is constitutively driven by the 34FMV promoter. Transgenic Carrizo plants have been produced with all of the ciFT constructs, and are being prepared for evaluation. The occurrence of in vitro flowering also suggested that replacement of the constitutive 34FMV promoter with an inducible promoter may provide a better system for controlling precocious flowering, particularly when ciFT3 serves as the transgene. We have obtained vectors for an estradiol inducible system and the experiments to test inducibility in citrus have begun.
Plant material -To get healthy and infected tissue around 60 virus-free plants of sweet were grafted into Rangpur lime, and kept in screen-house until use. Challenge with Candidatus Liberibacter spp -After reached 30 cm tall, a batch of 25 plants were grafted with budwoods infected with Ca. Liberibacter americanus (CLam) and another 25 were grafted with budwoods infected with Ca. Liberibacter asiaticus (CLas). All budwoods were checked by conventional PCR and RT-qPCR by the presence of the bacteria. Ten healthy plants were kept in the same conditions as control of no infected tissue. Every 30 days after grafting samples were collected and evaluated for bacteria. Since the plants were confirmed to be positive for the bacteria they were drastically pruned and transferred to a growth chamber at 22 to 24 oC and photoperiod of 16/8 hours. Total RNA-When the branches were around 15 cm (approximately 40 days after pruning) they were collected, and leaves and barks were separately grinding in liquid nitrogen. RNA was isolated using RNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions. Genomic DNA contamination was removed from the total RNA using the RNase-free DNase (Qiagen) following the manufacturer’s instructions. The concentration of total RNA was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). RNA integrity was verified using a Bioanalyzer 1000 (Agilent, Palo Alto, CA). Synthesis of cDNA and array hybridization: The RNA samples were sent to Roche NimbleGen Systems, where cDNA synthesis and Cy3 labeling was performed. Equal amounts of total RNA for each sample were converted to double stranded cDNA using the SuperScript II cDNA Conversion Kit (Invitrogen, Carlsbad, CA). Because this method uses an oligo dT primer, RNAs lacking polyA tails are likely to be under-represented. Hybridization, scanning, and image analysis of the arrays were performed by Roche NimbleGen Systems according to the manufacturer’s recommendations. The oligo-array includes 32,000 unigenes of sweet orange with six replicas of each unigenes with density of 340,000 spots. The preliminary results presented above were from the hybridization of RNA of plants infected with Ca. L. americanus. Other results with Ca. L. asiaticus were not evaluated yet. A set of primers for validation by RT-qPCR were designed. Ca. L. asiaticus and Ca. L. americanus were first detected 90 and 150 days, and the symptoms were observed 120 to 150 and 220 days after grafting, respectively. The rate of infection of Ca. L. asiaticus reached 95 % of the grafted plants whereas Ca. L. americanus was just detected in 30 % of the plants. Comparing two biological replicas of infected and healthy plants about 600 genes were up regulated in plants infected with Ca. L. americanus. Those genes were clustered in unclassified proteins (45%), metabolism (11%), protein fate (6%), classification not yet clear-cut (6%), cellular communication/signal transduction mechanisms (5%), transport facilitation (5%), cell rescue, defense and virulence (5%), transcription (4%), protein with bindind function or cofator requirement ‘ structural or catalytic (3%), subcellular localization (3%), control of cellular organization (2%), cellular transport and transport mechanisms (1%), energy (1%), systemic regulation of/interaction with environment (1%), storage protein ( 1%), and others categories presented inferior percentages to 1 % (cell cycle and DNA processing, development (systemic) and protein activity regulation). 200 genes were down regulated and they grouped into unclassified proteins (43%), metabolism (14%), subcellular localization (8%), transcription (6%), protein with binding function or cofator requirement [structural or catalytic] (7%), transport facilitation (5%), energy (5%), protein fate (4%), cellular communication/signal transduction mechanism (3%), %), control of cellular organization (1%), development (1%) and other categories.
Two phloem-specific promoter constructs comprising the -940 and -690 5′-deletions of the Arabidopsis SUC2 promoter sequences have been cloned into pCAMBIA vectors to drive expression of Arabidopsis resistance proteins (R proteins). The resistance protein coding sequences were cloned from cDNA derived from wild type and mutant lines of Arabidopsis plants. In the initial stages of the project we will evaluate phloem-specific expression of the R proteins in transformed Arabidopsis plants to see if any detrimental effects on plant growth or development occur. Our working hypothesis is that limiting expression of these proteins to the phloem may also limit the harmful effects of expression. At present we have wild type and constitutively active mutant forms of two R proteins, AtSSI4 and AtSNC1, cloned into the pCAMBIA vectors downstream from the two versions of the AtSUC2 promoter. In addition, each of the R proteins has been cloned in these vectors in versions where the leucine-rich repeats (LRR) have been deleted. The effect of removing this region of the R protein has not been well documented, but our rationale is this will uncouple the action of the activated R protein from signaling pathways related to pathogen invasion. Deletion of the LRR region may simplify the regulation of the R protein’s activity. Transformation of these constructs into Arabidopsis is scheduled for the fourth week of January, 2010. We expect to quickly move those constructs that are not lethal in Arabidopsis into citrus plants for evaluation regarding the survival and spread of Liberibacter from grafts with infected plants. It is anticipated that Objective 1 milestones will be met within year 1 from the actual start of funding. These include the evaluation of phloem-specific R protein constructs in Arabidopsis and citrus. The research of team of Dr. Eva Czarnecka and Lance Verner are now working on this project full time. The original proposal specified that Dr. Czarnecka (Co-P. I.) would be assigned 0.65 FTE.
We have collected and germinated seeds from several accessions from within the candidate categories listed in the proposal, including pummelos, intergeneric hybrids with Poncirus, wild citrus species, and various sweet orange lines for which there is emerging anecdotal evidence of differential sensitivity to HLB. 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; currently we have sufficient budwood to begin propagations for replicated field trials. In the 2009-10 fruiting season we collected seeds to increase the number and diversity of accessions that can be tested; these have been planted already, and are growing in our certified greenhouses to provide additional materials for tesing. We have a tentative agreement with one grower on the east coast of Florida to plant out the range of genetic diversity we hoped to test, both as seedlings and as top-worked trees, including some apparently tolerant types we have identified in an HLB-devastated grove in Florida. We are currently exploring other options within Florida, to be followed up with agreements to move ahead; these represent locations where growers have decided not to remove HLB-infected trees, so we expect there to be opportunities to challenge our replicated materials. We are in communications with our collaborators in China, to develop plans for importation of our plant materials there for testing in secure locations in Guangdong province.
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 original source trees have been tested twice after propagation using standard RT-PCR protocols, and they remain PCR negative for HLB; recent RT-PCR tests on the propagated trees have likewise proven to be HLB-negative. 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; several propagations have been made from this tree and these were inoculated with HLB-infected budwood. Recently propagations of it have been shared with our colleagues in Guangdong and planted there in a field challenge to assess the tolerance/resistance of this selection under natural and high-disease pressure. To expand further our search for survivors, and to continue to learn about Chinese citrus industry adjustments in response to HLB, we have established contact and good communication with a citrus extension specialist in the Fujian Provincial Academy of Agricultural Sciences, Mr. Li, Jian. This contact will provide us access to Fujian, another very seriously HLB-affected region of China. Mr. Li is very familiar with the local industry, and production areas and practices. He is aware of the goals of our collaborative project with scientists in Guangdong and Guangxi, and he is enthusiastically interested to aid us in participation. We are currently planning another visit to China in mid-late spring 2010, to assess the progress of the work underway, to expand our explorations for HLB survivors through the new contacts in Fujian, and to continue to work with our collaborators in Guangdong and Guangxi in search of additional survivors. We will also plan new experiments together at that time, to begin to address the causes and underlying mechanisms of these apparently tolerant selections, using various molecular techniques including gene expression studies, confirmation of genetic identity of the materials, and repeat inoculations in the field. This return visit is central to encouraging the continuation of the collaboration, to participate in planning experiments for a more in-depth analysis of the nature and underlying mechanisms of this phenomenon, and most importantly to confirm that the resistance persists following further propagation and inoculation with HLB. 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.
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. This genome sequence, when completed, will be THE reference genome for citrus, as it will be of the highest quality technically possible. 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 to the three centers, and sequencing has begun at Genoscope. Unfortunately, library preparations at JGI failed to achieve the needed insert size, so new DNA preps will be sent from Spain to JGI in January 2010.The Brazilian group remains in negotiations with JGI over contract language, but resolution is anticipated no later than February 2010. A meeting was held at Genoscope (Paris) with representatives from the sequencing partner nations, to revisit plans and progress. Progress was reported on the genetic linkage map, on haploid transcriptome sequencing, and genome sequencing (GS has completed 2.1x already); most importantly, a new time-line was produced that could lead to availability of gene sets by October 2010. Two sets of plants of sweet orange, rough lemon, and Volkamer lemon, representing the more susceptible and more tolerant types respectively, were inoculated with HLB in an environmentally controlled greenhouse. Plants were monitored for symptom development and for Las by qPCR. RNA samples were prepared from all plants at regular intervals, from inoculation through symptom development to be used in microarray experiments. Hybridization of RNA with Affymetrix and Agilent citrus chips (the latter developed by us at UF) has been completed and data sets have been generated; these are currently being analyzed to compare gene expression profiles over the time course.Though funding to the collaborators at UCR was delayed, they proceeded with their objective to update the HarvEST-Citrus EST database, including EST sequences from colleagues in Brazil and Japan, to provide an improved database for gene expression studies; currently there are more than 465,000 publicly available ESTs. Likewise, the collaborator in Spain was delayed in receipt of funds, but he has worked with us in preliminary experiments to test the feasibility of tissue-specific gene expression analysis that may be conducted. The sweet orange genome sequencing project is a collaboration between UF, JGI, and Roche/454 using next-gen sequencing platforms. Sequencing runs have been completed, and we now have >30x sweet orange genome coverage. In addition, 1.2 million ESTs were produced by one Titanium run of an RNA library from leaf tissue, to aid in subsequent assembly, gene prediction, and annotation. Six preliminary assemblies of the genome sequence have been attempted using various versions of 454’s assembly program, Newbler. To this point, the work has yielded fragmented assemblies upon which gene prediction models had a difficult time to work. Roche/454 will continue their efforts at assembly. Both genome sequences, when assembled and annotated, will be housed in a new database, Tree Fruit GDR, which was funded by an SCRI grant to include citrus. Further, the sequences will be available also through the JGI plant genome portal, and will be deposited with NCBI, as well. 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 conduct experiments using microarray analyses and deep transcriptome sequencing.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
