The goal of this proposal has been to investigate whether Ca. Las is transmitted between infected and uninfected ACP adults in a sex-related manner to understand its role in disease spread in field. We found that Ca. Las was sexually transmitted from Ca. Las infected male psyllids to healthy females but not from infected females to healthy males or among psyllids of the same sex. The experiments were repeated this season to confirm the findings. Our findings were consistent with our previous findings and we were able to confirm that Ca. Las is sexually transmitted between the adult psyllids during routine mating. We were able to detect bacteria in ACP ovaries of recipient females with PCR. However, we were unable to detect the presence of bacteria in genital parts of male and female psyllids with scanning and transmission electron microscopy probably due to washing of bacteria during sample preparation procedures. However, we found some other unidentified bacteria like structures in ovaries of recipient females. In addition studies are in progress to determine if Ca. Las recipient females are able to new citrus plants. Our ongoing efforts focus on identification of unidentified structures and to exploit florescent in situ hybridization analysis to detect presence of bacteria in genital parts of recipient and of the donor psyllids. The procedures for this are being standardized in collaboration with microbiologists.
In the last few months, we have continued working on genetic transformation of mature material from the three sweet orange genotypes (Valencia, Hamlin and Pineapple) with the aim of improving transgenic regeneration efficiency and having a reliable mature transformation procedure for each type that could be reproduced in Florida. After some last refining, Valencia sweet orange is routinely transformed at IVIA now. The transgenic nature of the first plants acclimated to the greenhouse has been confirmed through Southern blot analysis. Hamlin is more difficult to transform but with appropriate modifications of the tissue culture media and the source material used we have been able to produce already many transgenic plants, as confirmed also by Southern blot. Pineapple is routinely transformed at IVIA since the 90’s and is being used as control. Transformation of mature Carrizo citrange was initiated later, simply because we had not enough space and personnel to work with all the genotypes at the same time. During the last quarter, we have been more focused on developing a reliable transformation system for this genotype. More than 100 mature transformants (PCR-positive shoots) have been produced so far. The key in this case is using proper source material. We have preparing new source material to attempt transformation of mature citrumelo and grapefruit in the coming months. Regarding our second objective, at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes are established in the greenhouse and we are now characterizing them in detail (genetic and phenotypically). Additionally, a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis has been synthesized and incorporated into Agrobacterium tumefaciens. It will be used to transform Carrizo citrange. In Florida, construction of the growth room has been finally initialed and according to the schedule it will be finalized before the end of February. The PI and his greenhouse manager are planning to travel to Florida next March to supervise and setting up plant growth conditions, to set up the healthy citrus mother materials, and to establish substrate, fertirrigation and phytosanitary treatments.
This is the first quarterly report from our project. Collaborators in Florida (M. Hilf, T. Gottwald) have been routinely sending a variety of citrus-greening infected citrus samples to the De La Fuente Lab, including leaves and fruits from grapefruit, pomelo, and orange. Methods have been developed for processing the samples to provide both DNA for molecular testing and bacterial suspensions for microscopic and culture-based investigations. To obtain DNA samples, a variety of tissues and tissue-disruption methods have been tested to determine which sample type and processing method produces the highest DNA yield of Liberibacter asiaticus (LAS). Leaves, pith, pulp, juice, seeds, and stems have been tested using chopping and bead-beating with various sizes and types of beads for tissue disruption. DNA was extracted from the disrupted tissue samples, and PCR was the molecular method used to determine which tissue type and disruption method yielded the highest concentrations of DNA from the bacterium. Two different PCR reactions were first compared using positive control samples and the PCR with the lower limit of detection was selected for subsequent analyses. Overall, seeds contained the highest concentrations of LAS, and the bacterium was most effectively released from the tissue using bead-beating with metal beads. Work has been conducted to implement a quantitative-PCR assay as well so the relative quantities of LAS in the samples can be measured. For microscopic and culture-based investigations of the citrus greening bacterium samples, tissue samples were initially processed in the same ways as for the DNA samples except under aseptic conditions. Samples of the bacterial suspensions produced by the tissue disruption were then plated on several kinds of low-nutrient media, some containing juice extracts from the fruits. Cultures of other bacteria (including Pseudomonas spp.) were also plated alongside some of the samples to potentially encourage growth of LAS. A collection of unknown bacterial isolates which grew from the plated bacterial suspensions from infected fruits were preserved in glycerol in the freezer for later identification and experimentation. The bacterial suspensions were also placed in sterile microfluidic chambers for observation under the microscope. Bacteria were observed in the samples in the microfluidic chambers, but it has not been confirmed if the visible bacteria were LAS. The dimensions of the bacterium observed in the chambers correspond with published data for LAS, but no growth was observed after 10 days of incubation.
Obj. 1,2. Construct cDNA libraries from (a) adult/immature psyllids, dissected gut, salivary glands and accessory salivary glands; Sequence random cDNA clones, assemble ESTs, and select unigene sets; Obj.3. Produce RNA-seq libraries and compare transcript levels in psyllids and digestive organs at key acquisition access periods (AAP) to determine the relative abundance of insect mRNAs affected by bacterial infection. NCGR assembled the Illumina reads and made available files of the UniTran sequences and expression levels for each library. We assembled the 454 UniTrans (previously assembled with PAVE) with the Illumina UniTrans. The 454 assembly has 59002 singletons and 18998 contigs, and the Illumina had 121444 contigs; these assembled into 153482 Unitrans (singletons and contigs) and 12708 has at least one 454 sequence and one Illumina sequence. The assembly has been annotated as described in the last report (i.e. with UniProt, GO and GOSlim); 18339 UniTrans have a protein hit. As the expression levels for the new super-Unitrans must come from the original assemblies, the PAVE schema and software have been modified accordingly, and we are currently modifying the interface to for correct display and query of the expression levels of this super-assembly. The Hunter ESTs have been downloaded from GenBank and are currently being split into libraries and assembled. B. cockerelli libraries analogous to the ACP cDNAs were prepared as paired end libraries during the last quarter. Libraries are expected to be submitted to the sequencing lab in January 2011. Data-mining, qPCR analysis of ACP transcripts, FISH probe design and optimization using our epifluorescence microscope equipped with a filter for Cy5-labeled probes commenced. Ten psyllid genes have been selected for initial validation by PCR, RT-PCR and FISH localization as potential targets for RNAi constructions. Additionally ten Ca. Liberibacter genes potentially involved in the infection cycle were selected from the published genome sequences. Primers have been designed and are under validation by PCR and RT-PCR. Transmission studies continue in order to determine % efficiency transmission by single B. cockerelli psyllids to tomato plants using liberi-infected and -uninfected colonies over a range of AAPs and IAPs. Key time points will be identified and results validated by qPCR for extrapolation to time point feeding for ACP and RNAseq analysis. Asian citrus psyllid colonies are successfully being reared on citrus plants, now showing 80% infection with selected plants are confirmed positive by qPCR. Time course feeding will commence when flush growth appears during Spring 2011 and psyllids will be collected for RNAseq library construction (7-8 reps each).
Obj1-DNA bar coding to establish the identity and diversity in south Florida (Stansly, Brown).PCR primers were used to obtain a ~1200 bp product; analysis underway for 50 seqs plus sequences obtained for potato psyllid (outgroup). Obj2-qPCR detection of Ca. Liberibacter in potato psyllids is optimized; in progress for the asian citrus psyllids (from etoh). Obj3-Define the gross association of Ca. Liberibacter in thick sections’develop a ‘gross anatomical road map’ of Ca. Liberibacter accumulation in key organs, tissues, and cells; Obj4-Using the resultant ‘thick section road map’, elucidate at the TEM level the specific organs, tissues and cells where Ca. Liberibacter accumulates in the vector. Adoption of the potato psyllid for study here in Arizona has proven to yield windfall savings in time, expense and manpower toward understanding the anatomical particulars of Liberibacter transmission. The most important observations came from dissection and staging of guts, from all instars, for the scanning electron microscope (SEM). As this exercise was initially very labor intensive, specimens were processed in small lots. As observations and correlations came into view, we realized how powerful they were, revised our approach to the Objectives, and proceeded to increase our sample number (‘n’) many fold in association with the skill sets gained from the experience. We are now able to process larger lots rapidly and simultaneously, and so far, have examined a total of 195 uninfected and 138 infected guts. Results from these stagings have allowed us to develop several hypotheses and conclusions on the relationship between insect and pathogen, how the pathogen is transmitted, and the cost and benefit to the vector. We are now ready to draw up a manuscript on these findings for publication. We are continuing this approach, and have redefined our ‘unit specimen’ to include salivary glands and guts of each individual in separate clusters on the SEM stub. We can now directly correlate the degree of infection in the gut with the condition of the glands of each same individual. We have attached a fluorescent label to the bacterium inside extirpated guts, and want to move to the next logical step- development of techniques to label the bacterium inside the nonextirpated gut, the latter residing in its native configuration inside the deshelled abdomen, surrounded by all the other organs and blood. We’ve developed the skills to deshell the exoskeleton from the fixed tissues inside, and are experimenting with methods to eliminate non-specific labeling. We will then commence time-course studies in association with the acquisition access studies summarized below. Lastly, two directives for transmission electron microscope studies are in continuance. The first focuses on developing the probes needed to label the bacteria with colloidal gold. The second uses the techniques of Elliott (2007 Microscopy Today 15: 30-33) to stack Z-sections of the oral box into looped animations using PowerPoint and Macromedia Flash so that access by the bacteria can be studied in 3 dimensions. Time course IAP and AAP studies are underway. Subsets of adult psyllids are allowed defined feeding times, and assayed by qPCR to confirm bacterial presence, or prepared for SEM/TEM-hybridization analyses.
For Dec 2010 Methodology The plants were put in a growth chamber (photoperiod of 16/8 hours, temperature 24/22 oC, at 60 % relative humidity) day/night, 22/6, 24/16), as described in the previous report, and monitored periodically for presence of bacteria and symptoms of HLB. After confirm the presence of both Ca. Liberibacter asiaticus (CLas) and Ca. L. americanus (CLam) the tissues of five plants were collected for RNA extraction. Since the infection of Ca. L. asiaticus progresses faster than the infection of Ca. L. americanus, sample were collected at different infection time. The tissues of symptomatic plants infected were processed as described in the previous report, and was later performed the extraction of total RNA (RNeasy Mini Kit, Qiagen). The RNA was then analyzed for integrity, quality and concentration. The next step was to send the samples to Roche NimbleGen for microarray hybridization as previously described. Time course experiments – Since the proposal experiments were with Ca. Liberibacter americanus (there was publication with Ca. L. asiaticus), and considering the importance of time course experiments for a better understanding the gene network involved in the response to infection, we decide to set up a time course experiments with both bacteria. Results The first round of hybridizations were performed by Roche Nimblegen with a customized 385K chip containing 32,000 unigenes of C. sinensis. The raw data were pre-processed using the Robust Multiarray Average (RMA) by the NimbleScan software. Normalized data were imported to R package (Bioconductor) where statistical analyses were performed. A Bayesian moderated t-test statistic was calculated and p-values were adjusted for multiple comparisons by the false discovery rate correction. A total of 634 genes with P- values ‘0.05, fold change |logFC| ‘ 2.0 and odds probability ‘ 0.95 were considered differentially expressed at a statistically significant level. From those, 419 were up- and 215 down-regulated in symptomatic plants. In order to identify the most representative metabolic pathways regulated in infected plants we applied a gene set enrichment analysis approach by using the information of Gene Ontology and KEGG annotation. We found 45 different biological processes, including those involved in response to jasmonic acid stimulus, signaling and metabolism. There is partial agreement between our results with Ca. L. americanus and Albrecht & Bowman (2008) results. They have found 279 to 515 differentially expressed genes according with the infection time. Although the statistical approaches are similar (R package, Bioconductor, RMA, fold change, and False Discovery Rate), our results are based in wide DNA chips with only sequences of sweet orange. But the overall tendency is pointed out that the interaction of sweet orange with Ca. Liberibacter americanus is quite similar to the interactions with Ca. L. asiaticus.
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
The modifications to the citrus economic investment model and the citrus reset model to enable comparison of the enhanced foliar nutrient program to the traditional HLB management program were completed. An economic analysis using these models was done that estimates the annual HLB infection rate that switching from the traditional HLB management program to an enhanced foliar nutrient program maximizes profits. The results of this analysis will be presented at the 2nd International Research Conference on HLB in Orlando, Florida in January, 2011. Prior to the discovery of HLB, or citrus greening, in Florida in 2005, resetting diseased/unproductive trees was usually the most economically viable strategy to maximizing the economic life of citrus groves. In some situations, replanting the grove was required but the replanting decision was obvious since the grove had probably been destroyed from a freeze, hurricane or been removed as part of the canker eradication program. However, the introduction of endemic greening into Florida citrus groves has made the grove replacement decision critical to maximizing the profitability and economic life of citrus groves. It is assumed that the grower’s objective is to maximize the net revenues generated by the operation of a citrus grove and its replacements over their lives. For grove replanting decisions, this objective is best accomplished through marginal analysis, where the net revenues from operating the grove for another season are compared with the opportunity to earn higher future net revenues that would be initiated by replanting the grove during that season. The model has been developed and is now in preliminary form. It is designed to estimate the net revenues at which citrus groves infected with HLB should be replanted and what type of technologically improved grove to replant. The data used in this analysis were from a commercial grove planted to a density of 270 trees per acre in the late 1980’s. Expected fruit prices, yields, tree attrition rates, and operating costs for both the existing grove and the replacement grove are all key variables that affect optimum replacement times. Also, capital required for replanting and any other variables that will affect the performance of the replacement grove must be included in the analysis. Thus no industry-wide grove replanting policy can be developed from this model. This analysis showed that replanting HLB-infected groves to advanced production systems (APS) groves gives the grove perpetual life if tree resetting is practiced and an economic life greater than 25 years, if resetting is not practiced. Thus, APS technology offers the potential for growers to economically survive until a cure is found for HLB. We plan to publish preliminary results of this grove replacement model in Citrus Industry Magazine in the spring of 2011. Final results will be developed and published when data on an advanced production system grove are available.
The following transgenic plantlets have been produced in Dr. Grosser’s lab: Beta glucanase with 35-S constitutive promoter: Vernia 15 plants; Valencia 8; OLL orange 5; Carrizo 10; Duncan 2, and Beta glucanase with Suc2 phloem-limited promoter: Vernia 6, Valencia 5; OLL 7; OLL#8 9. All of these plants and some earlier ones are being maintained and/or increased in a greenhouse. Some earlier produced plants have been challenged with the HLB bacteria. Additional plants transformed with Liberibacter virulence genes are being maintained by Dr. Wang’s group. As these plants mature, they will be challenged with Liberibacter bacteria and symptom expression will be monitored. Samples were taken from 131 Poncirus plants at two sites to see if any of these plants are PCR positive for HLB. Carrizo and Swingle plants at one of these sites were positive last year, unlike their symptomless expression when grown in a greenhouse or growth chamber.
During the second period, we accomplished manual verification of structural and functional predictions for Candidatus Liberibacter asiaticus proteins incorporated in KEGG pathways. And as a pilot study for a more in-depth analysis of a crucial subsystem, we carried out a project to classify all the ABC-transporters in Liberibacter. More specifically, in order to understand the function of a protein in the context of the whole organism, we manually analyzed proteins in subsystems or pathways from KEGG. For all proteins included in KEGG (about 500 proteins, half of the genome), we selected the best homologous template for structure modeling, analyzed the domain architecture and predicted the function by combining the evidence from homology, structure prediction, surrounding genes in the genome, and the integrity of each pathway. For 95% of the proteins in these major pathways, we were able to provide a confident structure prediction and most of their functions can be predicted. By comparing with the pathways in close bacteria, e.g. Rhizobium etli, we noticed several inconsistencies within the Liberibacter genome. Some key enzymes appeared missing, and others did not fit in the context of a pathway. The former cases may reveal non-homologous iso-functional enzymes which can be used as possible targets to control the bacterium. On the other hand, we carried out an in-depth study of all ABC-transporters in Liberibacter. Starting with ABC-transporter ATPase, which is a conserved family of proteins with diagnostic sequence and structure motifs, we assembled all the component proteins of ABC-transport system. We attempted to clarify their functions by comparative analysis, classified their structures and studied their evolution. The manuscript describing the results is currently in preparation. Briefly, we found 17 ABC-transporter ATPases in the genome and identified 15 ABC-transport complexes consisting of 39 proteins in total. The two remaining ABC-transporter ATPase homologs apparently do not function as ABC-transporters. One of them was homologous to SufC, which is part of the iron-sulfur cluster assembly complex. The other one (gi|254780233) is homologous to ChvD, which is involved in regulation of virulence gene expression in Agrobacterium tumefaciens. Given its important function in virulence gene expression of a close bacterium, we proposed that this gene should be of interest. Currently, we are trying to expand the manual structure-functional analysis to proteins that are not mapped in KEGG. These proteins usually have fewer homologs available to base our predictions on, so for many cases, ab initio methods to fold proteins in silico will be applied. In addition to ABC transporters, we also will carry out in-depth analyses of other interesting systems in the bacteria, such as other transporters and sugar metabolic pathways. We believe that such an analysis will result in better understanding of the physiology of this bacteria and the mechanisms of its virulence.
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.
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.
After stabilization in the hydroponics system, greenhouse plants have been subjected to the three levels of boron fertilization (high, normal, and deficiency). As the nutrient levels stabilize to the new fertilization treatments we will inoculate the seedlings with HLB and monitor for Las titer and symptom development. Determining the mechanism of this possible HLB symptom escape will provide important information about how Las causes disease and provide a possible strategy for HLB management by citrus growers.
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.
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