Production of transgenic Citrus plants in the Core Citrus Transformation Facility (CREC) continues to be at the rate of about 100 plants per three months. Orders are being serviced for clients based both in Gainesville and in Lake Alfred. The demand for transgenic material is holding steady. Additional four orders were taken to produce transgenic grapefruit carrying genes harbored in following vectors: pWG22-1; pWG24-13; and pWG25-13, and pWG27-3. However, most of the activities of the facility are directed towards completion of previously placed orders. New orders are being serviced according to the order they were placed. The list of transgenic plants that were delivered within the last quarter includes those concerned with resistance to both bacterial diseases and CTV. Canker and HLB: 1) N1* gene: one Duncan plants; 2) NPR1: three Flame plants and superNPR1-six Flame plants; 3) AS7 gene: two Duncan plants and A13* gene: four Duncan plants; 4) pMKK7 vector: 20 Duncan plants; 5) pMOD1 vector: seven Duncan plants; 6) pNAC1 vector: one Duncan plants; 7) pSuc-NPR1 vector: three Duncan plants. CTV: 1) Gene in p33 vector: 26 Mexican limes, 16 C. macrophylla, and five Hamlin plants. CCTF also produced and delivered eight more Mexican lime plants for the pHK vector order. Change in genotype of these plants is not involved in response to plant pathogens. There are about twenty soil-adapted plants that will be tested by the PCR to confirm the presence of gene of interest in their tissue. Publication supported by this grant: Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266
1- The growth room construction started on October 22nd, 2010, projected finish date is February 11th, 2011. The construction is already one week behind according to the schedule. They are approximately half way done with the wall insulation, the ceiling insulation has not yet begun. We set up a meeting to discuss disposal of the waste stream for the grow room. The director of UF/IFAS Pesticide Information Office, the coordinator of the UF facilities planning and operations and a representative of the EPA were involved in the discussions. A final list of pesticides and chemicals to be used in the grow room was finalized in order to comply with all environmental regulations. 2 – All in vitro clean shoot tips (Hamlin 1-4-1, Valencia 1-14-19 and Pineapple F-60-3) to establish the mother plants were released from Dr. Peggy Sieburth lab, from the Department of Agriculture, Winter Haven. They are still in test tube conditions. The shoot tips are already 3-month old and they are ready for grafting onto rootstocks grown in pots. Since the growth room is not ready, we have transferred them to fresh medium to keep them alive until our growth room is completed. A second transfer of the shoot tips to fresh media is scheduled for January. As mentioned in earlier reports, this material is needed to be grafted on rootstocks in pots at approximately 2 months of growth. Another factor we are worried about is the current rootstock growth conditions. In the lab where they are developing, the lack of appropriate light, temperature, and space to grow them is jeopardizing 6 months worth of work. They are growing but with extreme difficulty. The initial planting material was discarded since it was getting too old and new batches are already growing but until we don’t have a better place where growing them on clean conditions we will continue to struggle. Even if we can occupy the new growth room the last week of January, we are still going to be concerned about achieving our desirable results. 3 – The growth room technician was finally hired and will start working the first week of January. He will go to Spain for training next Spring. 4 – The lab is 80% set up and, after removal of all plants to the growth room, we will clean and finish setting up the lab for full in vitro culture purposes. Supplies and equipment for the growth room will be purchased once it is completed.
Two full genome sequences have been assembled and annotated. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI (US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. This genome will soon be released through the Phytozome portal at JGI, as well as Tree Fruit GDR, and will be presented at the coming International Plant and Animal Genome (PAG) Conference in January 2011. The current version, Citrus clementina 0.90, is based on ~6.4x coverage, and is a very preliminary product released to enable citrus research community access. Additional work in 2011 will vastly improve the assembly through inclusion of BAC end sequences and integration with a high-density genetic linkage map, to yield a chromosome-based assembly with improved annotations. Fifteen BAC clones are being sequenced and assembled to compare with the assembled genome for validation of the assembly. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. This genome sequence is based on ~30x depth of sequence coverage and was assembled using Newbler software; it covers 319 Mb spread over 12,574 scaffolds. Half of the genome is accounted for by 236 scaffolds 251 kb or longer. The current gene set (orange1.1) integrates 3.8 million new ESTs (produced this year) with homology and ab initio-based gene predictions; 25,376 protein-coding loci have been predicted generating a total of 46,147 transcripts. The sweet orange genome also will be presented at PAG in January 2011, and can be accessed through the portals indicated above. The 3.8 million sweet orange ESTs came from 17 different libraries that were produced and sequenced using the 454 platform. They represent various biotic/abiotic challenges including psyllid feeding on young seedlings, canker inoculation, and treatment with salicylic acid, among others. Substantial progress has also been made on the other objectives of this project. Studies comparing the time courses of gene expression in two sets of HLB-inoculated sweet orange and rough lemon plants, representing more susceptible and more tolerant types respectively, have been completed using Affymetrix and Agilent citrus chips (the latter developed by us at UF); some differentially expressed genes have been confirmed by RT-PCR. In addition, comparisons of carbohydrate metabolism and anatomical changes associated with gene expression differences in these same plants have been completed; manuscripts are in submission. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from Brazil and Spain, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs.
Our research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. During the first year of the grant’s period peptides, proteins and RNAi moieties were tested by feeding them to psyllids using artificial diets. The diet was optimized by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period and we identified suitable buffers and optimal pH. Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from Diaprepes abbreviatus, the citrus root weevil, were found to be excellent candidates; causing high mortalities when fed to psyllids by artifical diet. Ten psyllids genes representing three gene families of cathepsins (five genes), vacuolar ATPases (four genes), and tubulin (one gene) were targeted and their dsRNA (16 ng/’L) fed to psyllids using artificial diets. Three vacuolar ATPases and three cathepsins (B, L and F) showed significantly higher mortality than the controls. In the first quarter of the second year period our studies continued to characterize the cause of increased psyllid mortality induced by feeding of Double-stranded RNA (dsRNA) molecules targeting specific psyllid genes. Large scale experiments were conducted to harvest sufficient RNA for Northern blot characterization of the integrity and abundance of specific psyllid mRNAs that were targeted and showed enhanced insect morality. The Northern blot analyses although cumbersome and time consuming, are essential for complementing Q-RT-PCR based analyses of targeted transcript abundance. To further support and enhance our RNAi research observations using artificial feeding chambers, we developed a detached leaf assay that supports adult and nymph psyllid survival and allows dsRNA uptake into intact citrus leaves on which the psyllid are naturally feeding. Initial results suggest that transcript specific mortality induced by feeding dsRNA to psyllids in artificial diets can be reproduced using the detached citrus leaf assay. The assay was developed to show that low doses of dsRNA circulating within the phloem can shut down key biological genes in psyllids when ingested, and thus support the possibility that RNAi strategies can be developed to control psyllid feeding on citrus and, therefore, control the spread of HLB. As part of this research a dsRNA virus was also discovered in psyllids and was characterized. This virus is present in natural psyllid populations within Florida, but accumulates to higher levels when the psyllids are maintained in greenhouse colonies. Because it is possible that dsRNA viruses can suppress the RNAi machinery of an insect, we are currently developing dsRNA of virus free psyllid colonies to support future RNAi research in psyllids.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. These include Polyamines such as putrecine, spermine and spermidine; and Antioxidants such as lipoic acid, glycine betaine and glutathion. Lipoic acid continues to yield the best results. A carrot suspension culture overlay procedure is also being evaluated. Experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency are also underway, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publication from work on alternative transformation systems: Dutt, M. and J.W. Grosser. 2010. An embryogenic suspension cell culture system for Agrobacterium mediated transformation of citrus. Plant Cell Reports. 29(11): 1251-1260. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ A field trial was established in collaboration with Mr. Orie Lee to evaluate sweet orange seedlings from six selected somaclones of precocious ‘Vernia’ sweet orange under commercial conditions. Juvenile Vernia trees are less thorny than other commercial sweet oranges, and our plan is to girdle the trees to induce early flowering and fruiting once the trees reach adequate size. The goal is to quickly establish a producing grove from juvenile budwood – as necessary to have a system for comparable transgenics. Significant progress was also made to identify rootstocks to enhance early production from juvenile scions, including subsequent transgenics. The 2.5 year old field trial using a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks is showing significant rootstock affects on precocious bearing – the best selections from this trial will be tested with juvenile transgenics, based on yield and fruit quality data to be taken in February. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious ‘Vernia’ sweet orange (including somaclones) were regenerated and successfully micrografted for further study of early flowering and transgene expression. 31 transgenic ‘Vernia’ trees were produced containing four different gene constructs. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
In cloning the three SA genes, EDS1, SID2, and WIN3, we currently confirmed the cloning of the full-length ctEDS1 and are in the process of moving the sequence to the binary vector for plant transformation. We showed in the last progress report that we obtained 3′ end RACE sequence for ctWIN3 and 5′ end RACE sequence for ctSID2. In order to amplify the other ends of the two genes, we tried to design different primers for RT-PCR. We also performed TAIL PCR, in which we used citrus genomic DNA as a template in a series of PCR in order to obtain the missing regions of the two genes. However, these attempts were unsuccessful. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we have been doing bioinformatics analysis and have identified additional SA genes that have citrus homologs with available sequence. We are currently design primers to further amplify these additional SA genes. For ctEDS5/pBINplusARS transformation, we obtained 5 Col-0 and 5 eds5-1 carrying the transgene. We are in the process of screening T0 seeds for additional independent transformants. In the meantime, we planted these 10 transgenic plants for disease resistance assay with Pseudomonas infection. We continue to characterize the transgenic plants overexpressing ctNDR1/pBINplusARS, ctNPR1/pBINplusARS, or ctPAD4/pBINplusARS. We obtained 4 homozygous ndr1 + ctNDR1/pBINplusARS and performed disease resistance assay. The recent data confirmed our earlier report that ctNDR1 complemented Arabidopsis ndr1 mutant. Additional analysis will be conducted to verify this result and to further characterize the defense phenotypes of the transgenic plants. For plants overexpressing ctNPR1/pBINplusARS or ctPAD4/pBINplusARS, we did not observe complementation of npr1 or pad4 mutant with transgenic plants currently obtained. We reason that overexpression of these two genes may be toxic or citrus cDNA clones may not be well expressed in Arabidopsis. We are currently trying to clone the genomic fragments of these two genes. We will repeat npr1 or pad4 complementation once we obtain the genomic clones.
During the 2nd quarter of funding, the Core Citrus Transformation Facility (CREC) continued it’s mission of producing transgenic Citrus plants according to the orders from multiple clients. The demand for genetically transformed citrus plants remains high. Most recently, CCTF received three new orders to produce transgenic grapefruit carrying genes harbored in following vectors: p19-5; p20-7; and p21-1. The bulk of the work presently revolves around orders placed in the previous quarter but work also goes on to complete older orders. Out of presently serviced orders, all except two are concerned with resistance of different citrus cultivars to diseases, primarily HLB and canker. The following transgenic citrus plants were delivered to various researchers: Resistance to bacterial diseases-canker and HLB: 1) N1* gene: two Duncan plants; 2) pCIT108P3 vector: two Flame plants; 3) NPR1: three Flame plants and superNPR1-four Hamlin plants; 4) AS7 gene: eight Duncan plants and A13* gene: four Duncan plants; 5) pMOG800 vector: two Duncan plants. Resistance to CTV: 1) Gene in p33 vector: 18 Mexican limes, 16 C. macrophylla, and seven Hamlin plants. Orders not associated with citrus disease resistance: 1) CL1 gene: one Duncan. 2) pHK vector: 12 Mexican limes. During this quarter, more than forty recovered new transgenic plants were soil-adapted, and are ready for PCR testing to confirm the presence of the trasngene of interest. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
Objective 1: A comparative study of two susceptible hosts, Duncan grapefruit (DG, C. paradisi), and Rough lemon (RL, C. jambhiri) and two resistant cultivars of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami’, evaluated the mechanisms involved in the resistance of kumquat to the citrus canker. MK and Nagami NK developed a hypersensitive response (HR), with necrotic lesions with population of Xanthomonas citri subsp. citri (Xcc) < 5 log units after 168 h in detached leaf and attached leaf assays. Early expression of genes related to programmed cell death associated with HR were identified in MK and NK. The resistance in kumquats has several characteristics associated with HR: 1) Rapid necrosis of leaf tissue in 48-72 h post inoculation in vitro or 72-96h in planta; 2) Disruption of epidermal and mesophyll cells by 72 h; 3) Xcc bacterial ingress limited to few cell layers below the epidermis; 4) Xcc population growth arrested at 72 h coincident with the cellular disruption; 4) Light microscopy and TEM, show death of the cells adjacent to the inoculation site with very few bacteria proliferating; 6) 5) HR-related genes and other putative resistance-related genes expressed early in resistant KN but not in susceptible DG. Behavior of susceptible DG and RL was: 1) No symptoms are detect in susceptible until 72 hr after inoculation and water soaked developes at 168 hr; 2) pustular callus-like lesions erupted through the cuticle by 10-16 days post inoculation (dpi); 4) Xcc populations reach 6 log cfu of Xcc per inoculation site at 168 hr; 5) Xcc population increases up to 15 dpi. Objective 2: Validate the inheritance or resistance for cybrids with susceptible Red grapefruit (RG) and RL with Valencia orange (VO). The putative RL cybrid has been recently been fully analyzed and determined using Single stranded repeat (SSR) analysis to be a cybrid from a mislabeled callus line of Valencia orange and not Meiwa kumquat. Hence the inheritance of resistance is not the HR type but is one of moderate susceptibility compared to high susceptibility in RG. Evidently, there is a definite expression of resistance in the cybrid inherited as a result of presence of the heterologous mitochondrial or chloroplast genome from the VO callus line. Evidence for this is as follows: Intermediate lesion symptoms are observed for RL+VO cybrid in vitro and in-planta. In contrast to development of callus, the inoculated area develops necrosis by 10 dpi. Xcc population plateaus by 10 dpi below the bacteria populations susceptible RL or Red GF. Two types of lesion were observed: necrotic and also callus, suggesting that cell death occurs and arrests the proliferation of Xcc. Expression of HR-related genes is intermediate between MK and RL, further substantiating that some yet to be determined elements of resistance have been inherited in the cybrids. Finally, the current set of Ruby red grapefruit cybrids with VO planted in canker-affected locations on the east coast continue to be more resistant than Red grapefruit trees around them. To expedite and standardize the evaluation of resistance in citrus germplasm, a prototype needle-free device was designed and evaluated for delivery of Xcc into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomates by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand-held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand-held injection. Results from inoculations with the automated device are in accord with the results for the hand-held syringe method.
This is a 3-year project with 2 specific aims: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, the pBI1.4T-AtMKK7 construct has been mobilized into the Agrobacterium strain EHA105. The culture of the Agrobacterium was used for co-incubation with ‘Duncan’ grapefruit explants. About 1700 explants were incubated and 17 shoots were tested with PCR. Nine of these shoots were positive in the initial screen and all of them were grafted onto Carrizo. The transgenic plants are growing. Further confirmation of the presence of the AtMKK7 gene in the transgenic plants by PCR and analysis of the expression levels of AtMKK7 in each transgenic line will be performed. Resistance of the transgenic lines to citrus canker and greening (HLB) will be characterized when the transgenic plants are available. For objective 2, we previously did irradiation for the first batch of ‘Duncan’ grapefruit hypocotyl cuttings with a irradiation dosage of 40G. The irradiated cuttings generated significantly fewer shoots than the control, suggesting that the irradiation caused severe damage to the hypocotyl cuttings. Calli were formed on both irradiated cuttings and the control. The shoots and calli generated on both the irradiated cuttings and the control have been transferred onto selective medium containing 0.2 mM of sodium iodoacetate. We prepared another batch of explants (90 tubes of ‘Duncan’ grapefruit seedlings) for irradiation. A total of 90 plates of hypocotyl cuttings (each plate with 40-50 stem pieces) was irradiated with a dosage of 30G on 06/28/10. The irradiated stem pieces were placed on non-selective shooting medium. Ten plates of hypocotyl cuttings were kept as non-irradiated controls, for future comparison. The plates are kept under 14 hour photoperiod. Shoots generated from the irradiated hypocotyls were transferred onto selective medium with 0.2 mM of sodium iodoacetate. We are also preparing the third batch of hypocotyls for irradiation.
About 500 supersour-type (SS) rootstock hybrids have been selected for propagation and further testing. Selected SS rootstocks are being evaluated for tolerance to CTV quick decline and propagated for placement into field trials. Commercial cooperators are being identified who will host early stage trials of some SS rootstocks. Rootstock liners were budded with scions to prepare trees for trials. Budded greenhouse trees for field trials were grown to planting size. A new field trial was planted to assess the interaction of rootstocks and scion cultivars on tree performance under an open hydroponic management system. Data on tree size and HLB titer were collected from several rootstocks trials to assess rootstock effect on tree growth under HLB disease pressure. Data were collected from a trial planted on trellis to examine the effect of tree manipulations on the length of time for transition from juvenility to maturity. This information will be valuable to accelerate the pace of development for new rootstocks and scions. Studies continue to assess citrus germplasm tolerance to Huanglongbing (HLB) and Phytophthora/Diaprepes in the greenhouse and under field conditions. In a new greenhouse study, Poncirus trifoliata, Cleopatra mandarin, and several hybrid selections were inoculated with HLB to further evaluate the apparent HLB tolerance in some trifoliata-type selections revealed in a previously completed greenhouse study. Greenhouse trees inoculated with Citrus tristeza virus (CTV) were tested for virus titer in preparation for CTV-induced decline evaluation of supersour rootstocks. More than fifty citrus genotypes and citrus relatives, as well as thousands of progeny from crosses, 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. All citrus germplasm and cultivars become infected with Liberibacter when inoculated, but different germplasm responds to HLB infection at different rates and with different symptom severity. 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. Preliminary evaluations were completed on gene expression in HLB-susceptible and HLB-tolerant selections to identify differences that can help guide selection from conventional breeding and transgenic efforts. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial, insect resistance, and other genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker. Efforts continue to transform trees with seven different promoters and three new anti-bacterial genes targeted at producing HLB-resistant cultivars. Testing of transgenic plants for HLB resistance continues under the resources provided by this grant. Genetic transformation was used to introduce the citrus FT gene for induction of early flowering into citrus scion and rootstock germplasm. Commercial field plantings of the new seedless mandarin cultivar ‘Early Pride’ were planned with cooperators. The final revision of the release notice for US-942 rootstock was prepared and submitted for official approval.
Although Year 2 of this project officially began on July 1, 2010, funds did not reach any of the researchers until 9/9/10, and at this time, only the UF PI (Moore) and the UF subcontractors (Grosser, Gmitter) received funding. The USDA subcontractors did not get their funds until late in September due to procedures at UF and USDA. Therefore little new research was started in this quarter. Other funds were found to maintain materials and personnel.
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. We recently started to propagate new lines from cuttings of 9 individually transformed plants: lines 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (We had to wait until the plants were large enough to withstand the taking of multiple cuttings). However, we have found that propagation by cuttings is difficult with certain lines and, even when it is possible, it may take several months for new growth on the cuttings. This process is still underway. Concurrently, we have standardized more probes and primers for the detection of SAR-associated citrus genes. Making these primers and probes requires knowledge of nucleotide sequence of the genes. Then the primers and probes must be tested and conditions optimized before experiments can be done. The list of genes we can test now includes: AZI1, BLI, CHI, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, PR1, R13032, R20540, RAR1 and SGT1, in addition to our controls 18S and COX. We chose these 15 genes because they are either important in the early induction and regulation of SAR (AZI1, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, R13032, R20540, RAR1 and SGT1) or are targets of the regulatory SAR pathway (BLI, CHI and PR1). In Objective 1 of this project, we propose to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA), by testing for expression of the above listed genes. This has been accomplished for the first set of lines. We intend to repeat this experiment with the increased number of transgenic lines once they are ready, with the increased number of genes we have identified, and using the commercial version of SA (Actigard, Syngenta Corporation).
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found two peptides that appear to effectively protect sweet orange trees from HLB. However, we and other labs continue screening for better genes that more effectively control HLB and can be approved for use in a food crop. In the California lab, we developed methods to rapidly screen anti-bacterial peptides against Ca. L. psyllaurous in tobacco plants. Tobacco plants were either inoculated with Ca. L. psyllaurous by using the tomato psyllid (Bactericerca cockerelli) and challenged one week later with recombinant Tobacco mosaic virus (TMV) expressing the specific peptides, or the plants first were inoculated with recombinant TMV, followed one week later by using B. cockerelli to inoculate Ca. L. psyllaurous. These assays are being analyzed presently. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
This project has three objectives: 1) gap closure of Ca. Liberibacter asiaticus (Las) found in Florida; 2) complete genomic sequencing to closure of Ca. L. americanus (Lam) strain S’o Paulo from Brazil, and 3) comparative genome analysis of Las and Lam to attempt to determine common factors enabling pathogenicity to citrus. Objective 1 Progress: The recently published Ca. Liberibacter asiaticus (Las) strain psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with Las to date. In the present study, shotgun sequencing and a fosmid DNA library of curated Las strain UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in Las and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. The SC-2 phage DNA appeared to stably replicate as an excision plasmid at a level 2-3X higher in planta than in psyllids. Objective 2 Progress: Similar phage DNA sequences (corresponding to both SC1 and SC2 and including putative lysogenic conversion genes) were found in Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected citrus in Brazil in collaboration with Dr. Nelson Wulff at Fundecitrus. We now have approximately 86% of the predicted Lam genome confirmed. Interestingly, both the SC1 and SC2 Las phage were found in Lam, and the gene order of the phage was also highly conserved. The 5 new and potentially pathogenicity related genes found in SC2 were also found on the equivalent Lam phage. This may be further evidence of the importance of these phage in lysogenic conversion of Liberibacter to become more virulent. This Lam genome is about one year from completion.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: We sequenced all of the constructs introduced into Arabidopsis which consisted of the AtSUC2 promoter (940 upstream from ATG) driving expression of the mutated (constitutive) and wild type forms of SSI4 and SNC1 (R proteins). For SSI4, which is derived from the Nossen cultivar of arabidopsis, two closely related genes (MUF8.3 and 8.2) are present in the Columbia cultivar. We cloned both, and the original from the Nossen cultivar, and cloned them behind the AtSUC2 promoter. These wild type versions of SSI4 will be used as controls for non-active forms of the R protein (pathogen activated). The four AtSUC2/R protein constructs (mutant and wild type of each of the two R proteins) were transferred from the pCAMBIA1305.1 vector, which confers hygromycin resistance, into pCAMBIA2301 with kanamycin resistance since the former is detrimental to transformation into citrus. The four constructs were submitted to the UF Citrus Research Facility at Lake Alfred for transformation into citrus. Transformation of these constructs into the Duncan variety of grapefruit is currently in progress at the Lake Alfred Citrus Research and Education Center (Dr. Vladimir Orbovic). Conclusions: Our hypothesis was that phloem-restricted expression of the R protein constitutive mutants would limit potential negative impacts on growth. The Arabidopsis transgenic plants expressing R protein mutants did not seem to be significantly affected in the majority of cases. Approximately 8% of the snc1 transgenics exhibited a stunted phenotype, very similar to the snc1 mutant expressed from its native promoter (not phloem specific). The first series of ssi4 transgenics (construct 5-2) had a point mutation (C>T) in the coding region that generated a premature stop codon and shortened the protein by 78 aa in the C-terminal region, past the leucine rich repeat (LLR). Five percent of these truncated ssi4 transgenics showed phenotypic differences mostly in the rosette appearance and lighter green, splotchy coloring. However, this effect will be investigated again in the new full-length ssi4 transgenic plants.
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