The seeds of Murraya paniculata were procured from the USDA-ARS National Clonal Germplasm Repository for Citrus and Dates, as well as from the local sources in Florida. Seeds have been available only periodically for our experiments. These seeds were germinated in vitro and used to establish cultures for experiments designed first to improve the efficiency of plantlet regeneration from epicotyl explants through adventitious organogenesis. 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 pCAMBIA2301 and pGreen0029 harbored in Agrobacterium tumefaciens strain AGL-1 and pTLAB21 harbored in Agrobacterium tumefaciens strain EHA101. 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 and despite substantial efforts, we were not successful in recovering transgenic plantlets from any of these experimental treatments. To overcome this regeneration barrier, consequently, we have pursued in parallel the regeneration of Murraya directly from axillary buds obtained from in vitro grown seedlings. A variety of conditions were screened including those relating to the amount of growth regulators, basal medium and carbon source; as a consequence, conditions suitable for micropropagation of axillary buds, as well as induction of multiple shoot regeneration from axillary buds, have been standardized. Prior to genetic transformation experiments, the amount of antibiotics required to inhibit micropropagation culture growth has been determined using non-transformed axillary bud cultures. To study the response of the explants to wounding, wounded (nicked and/or pricked) non-transformed axillary buds have also been cultured. The intact, nicked and/or pricked axillary buds have been co-cultivated with plasmids pCAMBIA2301 and pGreen0029 harbored in Agrobacterium tumefaciens strain AGL-1 and pTLAB21 harbored in Agrobacterium tumefaciens strain EHA101. An experiment was performed using two different sizes of explants while the OD600 of Agrobacterium cultures, duration of co-cultivation and amount of antibiotic were kept constant. The size of the intact as well as the wounded explants influenced their survival. Putative regenerants will be tested for the presence of marker genes. Depending on the response of these axillary buds, various treatments such as OD600 of Agrobacterium cultures, duration of incubation time, duration of co-cultivation and amount of antibiotic used will be carried out to define the requirements for successful genetic transformation of Murraya.
Funding is now in place from the International Citrus Genome Consortium partners (US, Brazil, Spain, France, and Italy), and the project to sequence a citrus genome is well underway. This will be THE reference genome sequence for citrus; it will be of the highest quality technically possible, by virtue of its haploid condition and the use of Sanger sequencing technology. DNA samples 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, and sequencing is ongoing at Genoscope and IGA. Unfortunately, library preparations at JGI failed, so new DNA preps have been sent to JGI recently; a backup plan has been established for timely delivery of new samples, should current samples prove inadequate. The HudsonAlpha Institute in Alabama, a JGI affiliate, will sequence the Brazilian part of the collaboration; this brings Drs. J Schmutz and J. Grimwood, two world authorities in sequencing, to the project. A meeting was held last December at Genoscope (Paris) with representatives from the partner nations, to revisit plans and progress on the genetic linkage map, and haploid transcriptome 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. The sweet orange genome sequencing project is collaboration between UF, JGI, and Roche/454 using their next-gen sequencing platform. Sequencing runs have been completed, yielding 30x sweet orange coverage. In addition, 1.2 million ESTs were produced from an RNA library from leaf tissue, and several additional libraries for sequencing are being constructed with RNAs from 16 different biotic and abiotic conditions, to broaden gene coverage to aid subsequent assembly, gene prediction, and annotation. Six preliminary genome sequence assemblies were attempted using various versions of 454’s assembly program, Newbler, yielding fragmented assemblies upon which gene prediction models could not work. Roche/454 continued their efforts, and a 7th and useful assembly has been produced; currently gene prediction and annotation have begun. 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. 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. Two sets of plants of sweet orange and rough lemon, representing 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 RT-PCR. RNA samples were prepared from all plants at regular intervals, from inoculation through symptom development, to be used in microarray experiments. Hybridization with Affymetrix and Agilent citrus chips (the latter developed by us at UF) has been completed and data sets have been generated; these are under analysis to compare expression profiles over time; some genes clearly up-or-down regulated differentially have already been confirmed by RT-PCR. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from colleagues in Brazil and Japan, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs. We are working with our collaborator in Spain on experiments to examine tissue-specific gene expression. New experiments have been designed and are being implemented.
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. These greenhouse grown trees have now been inoculated with HLB-infected budwood, but no symptoms have yet been observed. Several 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; these are being closely monitored for symptoms and by RT-PCR. Recently propagations from Guangxi were shared with our colleagues in Guangdong and planted in a field challenge to assess the tolerance/resistance 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 join us in the project. We are currently planning another visit to China in October-November 2010, to assess the progress of the work underway, to visit the original trees, 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 observations have been presented likewise through talks given at various grower meetings in Florida and California.
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 year 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. A set of grafted NPR1 plants were recently inoculated with greening. We are currently monitoring disease development. 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.
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. The first year of this project concludes May 1, 2010 and the second year of funding is requested at the current level.
Researchers at the USDA Ft. Pierce have established an in vitro system for growing mature tissue buds in vitro as a tissue source for transformation. One severe problem that they ran into was leaf drop. They have been able to reduce leaf drop substantially (though not eliminate it entirely) and are preparing a manuscript to document this work. The other area where they have made progress is shoot regeneration from mature tissue derived from greenhouse trees. We are preparing a manuscript on this as well. We have started transformation experiments but have run into some Agrobacterium overgrowth issues and are looking into that now. However, given the relatively high levels of shoot regeneration (Valencia, Ruby Red grapefruit, Calamondin, and US-942) that they are seeing they are anticipating that transformation should be pretty straightforward. Their best shoot regeneration is from greenhouse trees. Though they get shoot regeneration of in vitro derived shoots they do not believe that it is quite sufficient for routine transformation (Note: They are still working this part out, so it is not yet clear if in vitro tissue is a viable source of tissue for transformation). At the CREC in the Gmitter lab, work in continuing on the use of Thin Cell Layers (TCLs) as explants for mature tissue transformation. Experiments were done this quarter to induce regeneration in the TCLs by manipulating the amount of growth regulators, carbon source and also by pre-treating the TCLs with BA but regeneration is still problematic from these explants. Therefore they have recently additionally initiated experiments with internodal segments from greenhouse grown plants and are also planning to do preliminary experiments to establish axillary bud transformation. In the Grosser lab at the CREC, transformation experiments were tried this quarter with secondary citrus flushes. These were unsuccessful. Since they obtained positive results with first primary flush tissues, the results suggest that primary flush works better (which is in agreement with what the Spanish group has reported). In the Moore laboratory in Gainesville, experiments continued on using small peptides as vehicles to deliver cargos to plant tissues. If these techniques could be worked out they would have a number of applications for citrus transformation, perhaps even eventually allowing the transfer of genes or gene products to existing trees. Experiments this quarter demonstrated that enzyme (in this case GUS) could be delivered into plant tissues, including whole alfalfa seedlings, mung bean roots and citrus suspension cultures. Work is now underway to determine whether DNA uptake can be achieved.
In this quarter, seed from last spring’s new crosses to develop rootstocks and scions was planted in the greenhouse. New crosses were completed this spring with more than forty different genetic combinations. Fruit quality, yield, and tree size data were collected from four rootstock field trials. Propagations from supersour rootstock hybrids were prepared for budding to produce trees for disease testing and field trials. Rootstock liners were budded with scions to prepare trees for rootstock and scion field trials. Two new rootstock field trials were planted into the field. Studies continue to assess citrus germplasm tolerance to HLB, CTV, and Phytophthora/Diaprepes in the greenhouse and under field conditions. More than fifty citrus genotypes and citrus relatives have been challenged by natural inoculation with Liberibacter in the field, and data are being collected on HLB symptoms and Liberibacter titer by PCR. Detailed information is being collected on HLB tolerance and tree performance in four rootstock field trials. Some hybrids with mandarin or trifoliate orange ancestry appear to be resistant or tolerant to HLB and/or the psyllid vector. All citrus germplasm and cultivars become infected with HLB when inoculated, but different germplasm responds to HLB infection at different rates and with different symptom severity. Some hybrid selections resembling mandarin, grapefruit, and sweet orange appear to exhibit some tolerance to HLB. Greenhouse and field studies are continuing to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Transformed trees containing three new anti-bacterial genes were prepared for greenhouse testing with HLB. Genetic transformation was used to introduce the citrus FT gene for induction of early flowering into citrus scion and rootstock germplasm. Manipulation of this gene with inducible promoters will drastically accelerate the pace of cultivar development (shortening the generation time from 6-15 years to 1 year) and can also be used to increase early cropping of commercial trees. To date, the early flowering gene has been introduced into Hamlin, Ray Ruby, US-812, and US-942. A field day was held at the Ft. Pierce USDA farm to highlight progress in development of new cultivars, and performance information from several rootstock trials was highlighted. Summaries were prepared from ten different rootstock field trials and distributed to citrus growers. The release notice for US-942 rootstock was prepared.
Obj.1. The construction of six cDNA libraries have been completed from biologically relevant psyllid populations. Ca. Liberibacter was found (project #34) to be overwhelmingly present in guts but not in salivary glands, and liberibacter presence by qPCR in immatures and adults suggested that more information would be gained by focusing on whole adult- and immature instar liberibacter interactions, and on guts instead of salivary glands as originally proposed. The libraries were constructed from adult and immature psyllids, and dissected guts from psyllids, reared on HLB infected-infected and uninfected plants. Plants and psyllids from each treatment were tested by qPCR to confirm infection by, or absence of liberibacter (see report #21). Obj. 2. All six cDNA libraries have been constructed and two have been sequenced and annotated. The other four are presently being sequenced using Illumina short base read technology. The PG and PI libraries were assembled with PAVE and consensus sequences were annotated and display on a web-based summary and query-system. The unique transcripts (UniTrans) singletons and contigs were blasted against the UniProt Invertebrate subset (db Jan 4, 2010) and blast hits with e-values of >1e-20 were accepted for annotation. The PAVE query system offers the ability to query the UniTrans db by characteristic (e.g. number of ESTs, Invertebrate UniProt hit, EST library composition, R test statistic, etc.) and to query the UniProt Invertebrate proteins that matched the UniTrans. Each UniTrans is allowed multiple protein hits (1e-20 or better) from different organisms. Multiple proteins from the same organism were matched to a UniTrans and only the top match was accepted from that organism (‘non-redundant) match. Common proteins can be found in the UniTrans query system by asking for UniTrans with a high NRO count, indicating many organisms had a match to that particular UniTrans. The best annotation is defined as a protein matching a UniTrans with an e-value of 1e-40 and over 60% of the ESTs in the UniTrans matching the same protein with an e-value of 1e-10 or better. To assist with assignment of GO/GOSlim functions or pfam descriptions these annotations are added using the invertebrate matches. To search for protein annotation not yet known in UniProt’s invertebrate taxonomic set the assembly is blasted against full UniProt. Because the psyllid assembly contains bacteria, which will not match proteins in the UniProt databases, we blasted contigs against NCBI’s nucleotide database (Feb 10). Bast matches with e-values of 1e-20 or better were used from the full UniProt and from the nt databases. Obj. 3. We proposed a plan that would better and more cost effectively allow us to carry out quantitative analysis of whole immature instars, versus whole adults, guts, and PSG/ASGs from time course exposure (AAPs) to HLB plants within a defined time frame (steady-state qPCR-based titer). This involves extensive direct sequencing of random cDNAs from HLB+/- stages, instars, and organs, and is proposed because the relative cost of sequencing has declined, as the extent of coverage vs. cost has increased. In this way we can more effectively compare expression levels between whole adults and immatures, and adult guts and SGs. To explore the number of guts and salivary glands that would be needed to produce sufficient RNA for RNA-seq quantitative sequencing (4-5 reps each time course AAP) we produced treatments of psyllids and dissected the guts and salivary glands from adult psyllids and isolated the RNA. We determined that 150 salivary glands and 75 guts would each yield about 1 ug of RNA, a sufficient quantity for quantitative analysis, per replicate per treatment. In the remainder of 2010 psyllids will be exposed to Liberibacter-infected citrus over a set of acquisition access periods and subjected to quantitative gene expression using RNA-Seq analysis.
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.
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