Four phloem-specific promoter constructs have been cloned into the pCAMBIA plant transformation vectors. These are -940 and -690 5′-deletions of the Arabidopsis SUC2 promoter driving expression of GUS and GFP reporters. These are will be transformed into Arabidopsis to confirm phloem-specific expression. In parallel, these two constructs of the AtSUC2 promoter will also be used to drive expression of a series of resistance proteins (R-proteins) that have been shown to provide disease resistance when either over-expressed as wild type proteins or expressed as activation mutants. The R-proteins that are being cloned for phloem expression include the following: AtSSI4, and AtSNC1. The AtSSI4 and AtSNC1 proteins will be expressed both in the wild type and mutated forms. The others will be over-expressed in their wild type form.
Experiment set Ð To get healthy and infected material with Candidatus Liberibacter asiaticus (CLas) and Ca. L. americanus (Clam)60 seedlings (pathogen free) of sweet orange were grafted into Rangpur lime, and growth in pots with subtract. When the sprouts were 30 cm big, two infected budwoods with Ca. L. asiaticus or Ca. L. americanus were also grafted into the rootstock. Ten plants were grafted with healthy budwoods as control. Total DNA of all plants were purified and used in RT-qPCR to detect the presence of both bacteria. Five months after grafting with infected budwoods Ca. L. asiaticus was first time detected, but symptoms were observed just 220 days after grafting. On the other hand, Ca. L. americanus could be detected 90 days after grafting and symptoms were observed 120 to 150 days. All plants (10 with CLas, 10 with Clam, and 10 control) were pruned at the begin of Spring season (September) and transferred to well controlled environmental conditions (22 to 24 oC temperature and 16h/8h light/dark) in growth chambers (Conviron). The samples were collected in branches with three pair of leaves (about 30 days after pruning), and were pooled for each individual plant. 3 g of tissues (leaves and bark) have been sampled for total mRNA isolation (RNAeasy Plant Mini Ki, Qiagen). The integrity, concentration, and quality of RNA have been tested in denaturant electrophoreses 1 %, spectrophotometer and BioAnalyser (Agilent), respectively. All samples were confirmed to be bacteria positive by PCR. About 20 .g of total RNA of each sample were used in the hybridization experiments. Array set with unigenes of sweet orange Ð The database of 32,000 unigenes of sweet orange was submitted to Nimblegen for construction of the arrays. Each unigenes will be represented six times (density of 192,000 spots in two replicas per array) with different 60-mer oligo nucleotides. The complete experiments include: plants infected with CLas, plants infected with Clam (two conditions), two tissues (leaves and bark), and five biological replicas. Therefore, at least 2 (conditions) x 2 (tissues) x 5 (biological replicas) x 3 (technical replicas) = 60 (30 x 2) arrays will be produced. A first set of RNA was sent to Nimblegen (Madison) for hybridization. We are waiting for such results. A time course experiment is also underway to follow the infection process. Leaves above the grafting point were sampled 48 hours, one week and one month after grafting with infected budwoods. Total mRNA of such samples will be hybridized with the arrays by Nimblegen. The experimental design is three times of collect [48 h, 1 week and 1 month), two biological replicas, three not infected control, and two technical replicas (3 x2 x 3 x 2 = 36 (18 x 2) arrays].
The goal of this project is to transform the Arabidopsis and citrus 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. Overall the project is going very well. Among the PCR positive plants (see the progress report submitted on July 15, 2009), we have identified three transgenic lines overexpressing CtNPR1 and AtNPR1, respectively, by using Northern blot analysis. These NPR1 overexpression lines were inoculated with a strain of Xac 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. We are currently performing growth curve analysis to confirm our observations and also comparing the function of CtNPR1 and AtNPR1 by inoculating the plants with different concentrations of Xac inoculum. In addition, we have also grafted the six NPR1 (three each of CtNPR1 and AtNPR1) overexpression lines and the control onto more root stocks to propagate the transgenic population. Our goal is to produce more than ten individuals for each line. These plants are maintained in green-houses located at the Citrus Research and Education Center in Lake Alfred, and will be used for greening inoculations starting next month.
Objective II: Website creation and development. The Citrus Greening/HLB Genome Resources Website (http://www.citrusgreening.org/), brought online in the previous quarter, has been expanded to include a ÒNewsÓ column on the home page highlighting new genome analyses (discussed in more detail under Objective III), high interest genome-related publications with links to abstracts, and events relevant to genome researchers such as the upcoming HLB/Zebra Chip Meeting. In addition, a page listing a variety of citrus greening-related on-line resources has been created, linked to the home page under ÒAdditional InformationÓ. Objective III: Bioinformatic analysis of the Ca. Liberibacter genome. A. A post-doctoral associate with training in biological systems and computational genome analysis has initiated bioinformatic characterization of extragenic sequence features in the recently published, closed genome sequence for Ca. Liberibacter asiaticus. To gain insight into co-regulated sets of genes that the bacterium may be using to exploit changing environmental niches as it moves from the psyllid vector to plant phloem and establishment in the phloem, candidate promoter sequences are being mapped genome-wide using (i) models derived from characterized promoters in related bacterial species and (ii) novel sequence motifs found present in Ca. Liberibacter. Repetitive sequences and variation in sequence composition are also being characterized. B. To better assess its metabolic capabilities and limitations, unannotated regions in the published Ca. L. asiaticus genome sequence have been carefully analyzed, revealing the presence of numerous disrupted genes. Some disruptions are consistent with adaptation to a restricted environment while others are found in genes generally believed to be essential, suggesting possible errors in the published sequence. A listing of newly annotated pseudogenes can be found in in the “Access Data” section of the Citrus Greening/HLB Genome Resources Website in both table and Genbank format, the latter suitable for viewing with the Artemis genome viewer. Objective I: Assess community needs. The PI attended a special session on citrus greening at the 2009 Annual Meeting of the American Phytopathological Society (August 1-5) and engaged in fruitful discussions with interested researchers. The PI will be presenting a poster at the upcoming Joint Research Conference on HLB and Zebra Chip with the goal of promoting genome analysis tools available through the Citrus Greening/HLB Genome Resources Website, and establishing greater connections with researchers engaged in genome sequencing and genome scale analyses of additional strains and species of Ca. Liberibacter. Sequence comparison among isolates and species is predicted to yield important insights into the adaptation of this organism to different environmental niches.
Juvenile sweet oranges grafted to several rootstocks with potential to induce scion precosity were grown for entry into our RES (rapid evaluation system) to encourage precocious flowering and fruit set. Seedlings of selected sweet oranges, including high juice quality precocious selections, were grown for entry into the RES, as needed to determine differences in minimum time to flowering/fruiting. ‘ Several key components of the Agro-transformation system have been investigated in order to improve transformation and regeneration efficiency. We have optimized the following components for efficient transformation and data has been published. o media formulations o hormonal combinations o pre transformation incubation conditions o bacterial growth conditions o co-cultivation conditions and o plantlet regeneration conditions ‘ Produced the first transgenic plants (containing the LIMA lytic peptide construct) of 5 new high quality sweet orange selections, including 3 precocious somaclones and two Rhode Red Valencia somaclones. ‘ New transformation methods are being developed in order to improve the transformation efficiency for sweet oranges and to allow for transformation of important citrus cultivars that are recalcitrant to standard Agro-transformation. A system for embryogenic callus transformation has been developed and numerous transgenic plants of OLL8 sweet orange, and specialty fruits W Murcott tangor and Ponkan tangerine have been produced. Overexpression of a FLOWERING LOCUS T (FT) gene in transgenic plants leads to accelerated flowering: Experiments with the citrus FT genes (there are three of them) continue. Many putatively transformed citrus (Carrizo) plants have been produced and are being analyzed. Shoots from one construct continue to flower in culture. The three construct. Publications: 1. Dutt, M. and J.W. Grosser. Evaluation of parameters affecting Agrobacterium-mediated transformation of Citrus. 2009. Plant Cell, Tissue and Organ Culture 98: 331-340. 2. Dutt, M., V. Orbovic, and J.W. Grosser. Cultivar dependent gene transfer into citrus using Agrobacterium. Proceedings of the Florida State Horticultural Society (accepted).
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. 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 initiated plans to establish ~1 acre of transgenic plants expressing genes expected to provide HLB/canker resistance. USHRL has filed papers with APHIS to conduct field trials of their transgenic plants at this site.
At the USDA, mature tissue transformation technology using in vitro derived shoots is being developed. Established greenhouse-grown source plants Ð of four populations including Valencia sweet orange, Ruby Red grapefruit, and US-942 on two different rootstocks are in place. A procedure to disinfest plant material derived from greenhouse grown plants has been developed that results in little phytotoxicity and little contamination (< 10%). Conditions suitable for in vitro shoot growth have been identified; a variety of conditions were screened including those relating to substrate, growth regulators, basal medium composition, ethylene, and ventilation. Experiments have been initiated using in vitro grown shoots. At the CREC, budwood was collected from 'Midsweet' sweet orange (Citrus sinensis L. Osb.) trees growing in the field and grafted onto Volkameriana rootstock in the greenhouse. The first flush from the grafted buds was disinfested, cut transversely into thin cell layers (1-2mm) (TCLs) and placed horizontally on MS supplemented with BA and NAA with or without the addition of Plant Preservative Mixture (PPM). PPM prevented some level of contamination, but also may have inhibited regeneration. In other experiments, Hamlin mature budwood source trees were grown on a selected complex rootstock that seems to have superior nutrient uptake, and nutrition was provided by a new granular slow release product that has been showing excellent results with nursery and field trees. Six transgenic Hamlin lines stably expressing the GFP gene were regenerated from the 1st experiment using the first flush on the grafted trees as explants. The 2nd experiment, using the 2nd flush on the same trees yielded no transgenics, indicating that the regeneration potential diminishes with sequential flushes. Similar experiments are being initiated in Brazil. In Gainesville cell penetrating peptides are being evaluated as an aid to transducing compounds into mature citrus tissue.
In Florida, first year’s milestones were: 1) Build a greenhouse for growing citrus for mature transformation. The University of Florida IFAS facilities has the specifications for the greenhouse and still are in the planning stages. The original specifications did not include a headhouse for maintaining clean entry and supply for the greenhouse. Facilities are modifying in an attempt to stay within budget. 2) Hire a manager of the mature transformation facility and allow this scientist to begin training in the Spanish laboratory. Dr. Cecilia Zapata Carrero has been hired as the mature transformation facility manager. She is now making plans to travel to Spain to begin learning the mature transformation technology. In Spain, we have to develop genetic transformation systems for mature tissues of the most important sweet orange varieties of Florida, and then transfer the methodology to Florida, once the greenhouse is constructed and implemented in Lake Alfred for this purpose. Valencia sweet orange has been highly responsive to regeneration and transformation. We have been able to produce a good number of transformants from the first experiments. The first Valencia transformants are already acclimated to the greenhouse. More important than that, the procedure has been shown to be reliable and consistent, meaning that we have been able to generate independent transformants with 1-2% transformation efficiency from each of the four experiments performed so far. This frequency is lower than that obtained with the readily transformable Pineapple sweet orange type that we use as responsive control, but we think transformation rates can be still increased for Valencia sweet orange. In any case, this result means that we are able to generate 1-2 independent transformant/s per 100 explants (about 20 shoots) agroinoculated, which we consider a very good rate considering that mature material is being used. Hamlin sweet orange is easy to transform from seedlings. However, we have been unable to prepare the type of starting plant material needed for mature transformation in the two first experiments performed, and this resulted in poor callus formation and regeneration. A third experiment is underway with important adjustments of the basic protocol. Plant material from mature Carrizo citrange has been prepared and it is ready to use in coming days for the first time. In addition, we are generating shoots from Pineapple sweet orange seedling explants transformed to ectopically over-express CsAP1 (AP1 from sweet orange) and CsFT (FT from sweet orange) flowering-time genes with the aim of modifying tree architecture. Interestingly, many shoots transformed with the CsFT construct are flowering in vitro soon after regeneration.
The diseases Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of genes conferring resistance to these diseases or the HLB insect vector is a promising way to solve these problems. Transformation vectors, suitable for incorporating genes into citrus trees, have been prepared for five antimicrobial peptides (AMPs) with many 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 and some further propagated for replicated evaluation. 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 developed HLB symptoms. More active promoters are in the pipeline to achieve better results. Tests of garlic-lectin transformed citrus are underway to determine effect on psyllid feeding and development. Efforts are underway to use Liberibacter sequence data to develop a transgenic solution for HLB-resistance, targeting a transmembrane transporter. Collaboration is underway with a USDA team in Albany, CA to provide constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are also being identified from citrus genomic data to permit transformation and resistance using citrus-only sequences. 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, with minimum inhibitory concentrations at 1 µM 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. 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. The tomato cultivar ÔM82Õ was transformed with the AMP D4E1 and Garlic Lectin as a model system for more quickly assessing resistance than is possible using citrus. D4E1-transformed tomatoes were challenged by inoculations with Agrobacterium tumefaciens: no immune plants were identified, but some produced only very small galls and overall gall mass was 30% lower in D4E1-transformed vs. control ÔM82Õ. Transformed plants have been propagated and D4E1- transformed vs. control plants will be challenged with Xanthomonas campestris pv. vesicatoria to assess resistance. Garlic-lectin-transformed tomatoes vs. control ÔM-82Õ will be tested for effects on the phloem-feeding whitefly. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ÒnaturalÓ psyllid inoculation in the field are being compared. 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. A material transfer agreement has been established with Texas A&M University to permit lab and greenhouse comparisons with the spinach defensin expressing grapefruit and ‘Hamlin’. They also are providing the 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.
We completed the construction of two quarantine houses, one for Citrus saplings infected by psyllids collected from the field, and another for Citrus grafted to infected root stock, and ramped up rearing to high numbers of insects with Ct values indicative of infection (Stansly). Two infected colonies were initiated in April 2009: (1) ÔCarrizo’ citrange and ‘Pineapple’ sweet orange plants (total 33) were inoculated with Candidatus Liberibacter asiaticus through grafting bark tissue from a PCR positive HLB infected plant, and (2) 300 field collected psyllids that tested 10% HLB positive by PCR (N=52) were released in two cages, each containing 6 ‘Carrizo’ plants. All plants tested negative for HLB before they were inoculated and became infected. After about one month, dark green mature leaves were PCR tested and 85% (orange) and 95% (‘Carrizo’) grafted plants, respectively, were HLB positive, whereas, plants exposed to infected psyllids were negative. Then, grafted plants were pruned to induce new shoots that tested 31% and 35% HLB positive at 2wk and 90% and 100% positive at 4wk when 97% of mature leaves also tested positive. Two months after exposure, 83% psyllid-infested plants tested HLB positive based on qPCR using dark green mature leaves. We optimized qPCR by developing a standardized curve to determine threshold levels of detection in adult and nymphs (Roberts). Adult psyllids collected on 8/12/09 from both infected colonies and a control colony reared on Murraya paniculata were qPCR tested, and reported to be 61% and 67% HLB positive, grafted and psyllid-infected plants, respectively. Our efforts to maintain quarantined colonies with a high HLB titer have been tracked by lengthy and systematic qPCR. The latest report consisted of 148 reps from 3 HLB infected colonies and 1 uninfected colony, as well as 35 corroborative replicates tested in Riverside, CA. We performed exploratory qPCR analysis of psyllid organs (guts) for the presence of HLB. Detailed methodology is per Li et al. (2006) for plant material and Manjaunth et al. (2008) for psyllid samples (Roberts; Stansly). Isolation and purification of mRNA from 1000 non-infected adult D. citri gut extirpations (#1UNIFadult-gut), previously reported, yielded ~10 ug of total RNA (511 ng/ul in a total volume of 20 ul), from 1ml of Trizol sample #4INFwhole-adult (50-100 mg adult psyllids), at 572 ng/ul in 20 ul (or enough for cDNA synthesis) (Gang, Brown). Two cDNA libraries have been successfully constructed from samples #1 and #4. These libraries were sent for DNA sequencing using 454 technology ~2 weeks ago. The first DNA sequence data will soon be assembled and processed. We are developing a new protocol for total RNA extraction from psyllid larval samples #5INFlarv and #6UNINFlarv. Preparations yielded ~10 ug of total RNA (~500 ng/ul in a total volume of 20 ul) from 1ml of Trizol sample (50-100 mg psyllids). Sequencing of libraries under construction will commence once we are sure of the quality of the current data. To complement the 1000 uninfected guts extirpated during the last quarter, we have commenced extirpation of 1000 guts from adults reared from eggs on grafted Citrus. We have stockpiled 200 guts to date. Only adults raised from egg on graft-infected plants were used. This follows the finding of Inoue et al (2009) that Liberibacter multiplies to high titers in 5th instars but not in adults transferred from uninfected plants (Cicero, Brown).
CCTF continued its expanded operation with the new organization of the work reported in the previous quarterly report. The facility continued to service orders mostly for FCPRAC funded researchers wanting transgenic plants with various disease resistance constructs. The list of transgenic plants that were produced during the quarter and confirmed by the presence of the reporter gene and appropriate validating PCR reaction: LIMA gene: 1 Flame grapefruit; LIMA gene: 9 Mexican lime; CIT108 gene: 1 Valencia sweet orange; CIT108p gene: 3 Flame; CIT108p3 gene: 5 Flame; CIT108p17 gene: 16 Flame; PITA gene: 3 Duncan grapefruit; F3* gene: 2 Duncan; AF1 gene: 1 Hamlin sweet orange, 1 Duncan; NPR1 gene: 1 Duncan; p6 gene; 3 Sour orange; pTLAB21 vector: 3 Duncan; pTLAB32 vector: 3 Duncan. CCTF also produced approximately 30 soil-adapted plants that were not tested by PCR for the presence of the transgene of interest. These fall into two groups: 1) plants that were selected on the basis of the presence of reporter gene, 2) plants obtained after co-incubation of explants with the Agrobacterium strain that carried binary vector with the reporter gene.
This is a 3-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (MKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, we have subcloned the Arabidopsis MKK7 gene into the CTV-based expression vector and transition expression of MKK7 in citrus leaves is underway. We have also subcloned the Arabidopsis MKK7 gene into the plant binary vector pBI1.4T (a pBI121 derivative) and transformed the MKK7 gene into citrus using the Agrobacterium-mediated approach. The MKK7 transgenic plants are growing and characterization of the MKK7 transgenic citrus plants is underway. For objective 2, we have decided to use the Hamlin suspension cells as starting materials for the selection. The Hamlin cell suspension culture has been scaled up in Murashige and Tucker (MT) liquid medium. Several flasks of the culture are maintained for subculture. To determine the concentrations that will be used in the selection, the Hamlin cells from the suspension culture were grown on MT medium plates supplemented with different concentrations of sodium iodoacetate ranged from 0 to 0.2 mM. Hamlin suspension cells were found to be highly sensitive to the inhibitor. A concentration of 0.1 mM of sodium iodoacetate could completely arrest their growth. Therefore, 0.1 mM of sodium iodoacetate has been used in the selection. We are also testing hypocotyls of citrus seedlings because plants can be easily regenerated from hypocotyl-derived callus cells. Hypocotyls were grown on MT medium plates supplemented with different concentrations of sodium iodoacetate ranged from 0 to 0.4 mM. Hypocotyls were also very sensitive to the inhibitor. A concentration of 0.2 mM could completely inhibit the growth of calli generated from hypocotyls. We will use 0.2 mM of sodium iodoacetate in selection of hypocotyl-derived calli. We are currently generate mutations in the hypocotyls using fast neutron-mediated mutagenesis. Calli will be generated from fast neutron-treated hypocotyls. The hypocotyl-derived callus cells will be selected on MT medium plates supplemented with 0.2 mM sodium iodoacetate.
We have propagated by cuttings the following ÔCarrizoÕ citrange AtNPR1 transgenic lines: 854, 857, 859 and 884. Lines 854 and 857 show high overexpression of the endogenous marker gene PR1 (considered a marker of SAR). Lines 859 and 884 do not express the AtNPR1 transgenic gene and do not show overexpression of the endogenous PR1 gene, hence are considered as negative controls. Subsequently we have grafted a number of these plants with wild type (WT) ÔDuncanÕ grapefruit. We also grafted WT ÔCarrizoÕ plants with ÔDuncanÕ grapefruit as controls. We have also treated the plants with either salicylic acid (SA) or water (as negative control) and are in the process of comparing their response using TaqMan Real Time PCR. In preparation for the real time experiments we have also sequenced a number of genes of interests (NPR1, NPR3 and PR1) from both ÔCarrizoÕ and ÔDuncanÕ to guaranteed that the target probe/primer sequences within the genes are identical and that any observed differences in expression are not due to differential efficiency in annealing of the probes and/or amplification. We have also standardized the real time reaction for the marker PR1 gene and continue to do so for the rest of the genes. This will allow us to analyze the response of the plants as proposed in objective one. The same group of plants will subsequently be analyzed as proposed in objectives 2 and 3. For this purpose we have also been propagating HLB-infected material and have standardized the real time PCR detection of the pathogen so we are confident we can conduct the proposed experiments.
Assessment of Bs3 activity in citrus remains our primary initial objective. We have three approaches under development: 1) Transient transformation by Agrobacterium infiltration. We have been working to establish a reproducible Agrobacterium-based citrus transformation system. Our first efforts have focused on leaf infiltrations with fluorescent (GFP) reporter gene constructs and examination by confocal microscopy. The method has not performed adequately thus far, with the appearance of fluorescent materials in the GFP channel which may be phenolic compounds. Although we can subtract out autofluorescence, these compounds are still visible and complicate the interpretation of the experiments. Consequently we resynthesized gene constructs with the GUS reporter gene. We continue to test the GFP constructs with appropriate controls, but we have now also begun the use of the GUS constructs. We have also tested transient transformation by dip inoculation of various citrus plant parts, as well as infiltration of intact citrus fruits. Most of the testing has been with 35S:GUS marker gene constructs, and we have observed GUS staining of albedo in sweet orange and other citrus fruits. We will continue to examine the Bs3 GFP and GUS constructs in the fruit assay to assess the function of both marker genes and Bs3 by these approaches. 2) Transient transformation using particle bombardment. We have initiated this additional transient approach to test a standard assay for resistance gene function – co-bombardment of reporter genes with or without a resistance gene that cause cell death. In these assays, a GUS reporter gene produces individual transformed cells that turn blue, whereas in the presence of the R gene, the blue signal is lost due to a highly localized hypersensitive response. These experiments require a different set of gene constructs, which have now been made and will be testing with a particle gun at UF. 3) Stable transformation using Agrobacterium. Our stable transformation protocol requires citrus seedlings, which must be germinated from seasonally-available seeds. Seeds are now available, and the first experiments have been set up. These experiments included the following constructs: (i) 35S:Bs3-GFP (constitutively expressed Bs3 gene) (ii) 35S:Bs3m-GFP (constitutively expressed inactive Bs3 gene) and (iii) Bs3:Bs3-GFP (the Bs3 gene under control of its own promoter). We will assess GFP expression, cell death, and growth of X. citri. First results will be available later in October. In addition to the transformation work, we have completed assembly of a multi X. citri strain-inducible Bs3 gene construct. The original Bs3 promoter, which has an activation site for AvrBs3, was modified to contain fourteen additional activation sites for TAL effectors from all four X. citri A strains, as well as A*, Aw, C and B strains (Table 2 of our Project Narrative). This promoter is currently used to drive the GUS reporter gene, will be used in planta to test which X. citri strains activate the complex promoter. The design is facilitated by the recent discovery of a simple code between amino acid residues in TAL effectors and specific DNA bases defining protein-DNA interaction, in the manner of zinc finger proteins (in press, Science). The results will inform commercial constructs with Bs3 conferring disease resistance. 2Blades has gained the capability to do whole genome sequencing in our new laboratory, and we will begin sequencing of several X. citri strains to examine TAL effector sequence diversity. Coordination of the project is facilitated by participation of all the parties in regular conference calls to discuss results and next steps.
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: Within the recently published Las strain psy62 chromosomal genome (Duan et al. 2009), many unique genes of unknown function are found, and several phage related genes are found integrated into the chromosome, but no replicating phage DNA was found. We reported last quarter the finding of at least one complete circular phage genome and a large contig that appeared to represent another phage. Neither were previously reported. We have now quantified the copy number per cell of the two phage DNAs relative to the main chromosome, and have confirmed that the SC-1 phage DNA appears to replicate in Las when infecting citrus to a level averaging 10X higher than the Las chromosome when Las is extracted from infected psyllids, and that SC-1 replicates to a level averaging 20X higher than the Las chromosome when Las is extracted from infected periwinkle. The SC-2 phage DNA appears to replicate in Las to a level 2-3X higher than the Las chromosome in both citrus and periwinkle. These data strongly indicate that we have discovered two lytic phage and was reported at the last APS meeting [Gabriel & Zhang, 2009. Phytopathology 99 (6): S38]. Since some of the SC-1 and SC-2 phage genes were found by Duan et al (2009) to be integrated into the Las genome, both phage likely have a lysogenic cycle. Lysogenic phage are known to mobilize bacterial genes when they repackage into infectious particles and then horizontally transfer these genes between different bacteria that they infect. Any genes carried on a phage that replicates to a level 10X higher than the host chromosome also replicates the copy number of all genes it carries to a level 10X higher than the rest, dramatically increasing gene expression levels, possibly affecting virulence. Following that lead, several new Las genes were found on SC1 and SC2, encoding: 1) a hemagglutinin; 2) an ABC dipeptide secretion component of a Type I secretion system; 3) a porin type major outer membrane protein; 4) a lysozyme and 5) a fatty acid desaturase, any of which may contribute to pathogenicity. None were reported in the psy62 genome. The hemagglutinin was found on both SC-1 and SC-2. Hemagglutinins are secreted by Type I secretion systems and can be effectors of pathogenicity. Complete Type I systems are found in the Las genome. Since this new hemagglutinin is encoded by two different phage, and since the phage can replicate to ca. 10X-20X copy number in plants per Las cell, this hemagglutinin will be investigated experimentally for a potential role in HLB disease. Objective 2 Progress: In collaboration with Fundecitrus in Brazil, Lam strain ‘S’o Paulo’ DNA samples extracted from citrus and purified on pulsed-field gels by Dr. Nelson Wulff has resulted in 78 kb of partially confirmed Lam genomic sequence obtained by 454 sequencing assembled into contigs greater than 1 kb. Additional sequencing is currently being performed, and will be analyzed together with data from our previously reported Lam fosmid libraries in comparison with the Las genome and the yet to be published sequence from the Liberibacter causing “zebra chip” disease of potato (Objective 3). We are on track to meet target deadlines of the original proposal.
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