Plant material -To get healthy and infected tissue around 60 virus-free plants of sweet were grafted into Rangpur lime, and kept in screen-house until use. Challenge with Candidatus Liberibacter spp -After reached 30 cm tall, a batch of 25 plants were grafted with budwoods infected with Ca. Liberibacter americanus (CLam) and another 25 were grafted with budwoods infected with Ca. Liberibacter asiaticus (CLas). All budwoods were checked by conventional PCR and RT-qPCR by the presence of the bacteria. Ten healthy plants were kept in the same conditions as control of no infected tissue. Every 30 days after grafting samples were collected and evaluated for bacteria. Since the plants were confirmed to be positive for the bacteria they were drastically pruned and transferred to a growth chamber at 22 to 24 oC and photoperiod of 16/8 hours. Total RNA-When the branches were around 15 cm (approximately 40 days after pruning) they were collected, and leaves and barks were separately grinding in liquid nitrogen. RNA was isolated using RNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions. Genomic DNA contamination was removed from the total RNA using the RNase-free DNase (Qiagen) following the manufacturer’s instructions. The concentration of total RNA was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). RNA integrity was verified using a Bioanalyzer 1000 (Agilent, Palo Alto, CA). Synthesis of cDNA and array hybridization: The RNA samples were sent to Roche NimbleGen Systems, where cDNA synthesis and Cy3 labeling was performed. Equal amounts of total RNA for each sample were converted to double stranded cDNA using the SuperScript II cDNA Conversion Kit (Invitrogen, Carlsbad, CA). Because this method uses an oligo dT primer, RNAs lacking polyA tails are likely to be under-represented. Hybridization, scanning, and image analysis of the arrays were performed by Roche NimbleGen Systems according to the manufacturer’s recommendations. The oligo-array includes 32,000 unigenes of sweet orange with six replicas of each unigenes with density of 340,000 spots. The preliminary results presented above were from the hybridization of RNA of plants infected with Ca. L. americanus. Other results with Ca. L. asiaticus were not evaluated yet. A set of primers for validation by RT-qPCR were designed. Ca. L. asiaticus and Ca. L. americanus were first detected 90 and 150 days, and the symptoms were observed 120 to 150 and 220 days after grafting, respectively. The rate of infection of Ca. L. asiaticus reached 95 % of the grafted plants whereas Ca. L. americanus was just detected in 30 % of the plants. Comparing two biological replicas of infected and healthy plants about 600 genes were up regulated in plants infected with Ca. L. americanus. Those genes were clustered in unclassified proteins (45%), metabolism (11%), protein fate (6%), classification not yet clear-cut (6%), cellular communication/signal transduction mechanisms (5%), transport facilitation (5%), cell rescue, defense and virulence (5%), transcription (4%), protein with bindind function or cofator requirement ‘ structural or catalytic (3%), subcellular localization (3%), control of cellular organization (2%), cellular transport and transport mechanisms (1%), energy (1%), systemic regulation of/interaction with environment (1%), storage protein ( 1%), and others categories presented inferior percentages to 1 % (cell cycle and DNA processing, development (systemic) and protein activity regulation). 200 genes were down regulated and they grouped into unclassified proteins (43%), metabolism (14%), subcellular localization (8%), transcription (6%), protein with binding function or cofator requirement [structural or catalytic] (7%), transport facilitation (5%), energy (5%), protein fate (4%), cellular communication/signal transduction mechanism (3%), %), control of cellular organization (1%), development (1%) and other categories.
Two phloem-specific promoter constructs comprising the -940 and -690 5′-deletions of the Arabidopsis SUC2 promoter sequences have been cloned into pCAMBIA vectors to drive expression of Arabidopsis resistance proteins (R proteins). The resistance protein coding sequences were cloned from cDNA derived from wild type and mutant lines of Arabidopsis plants. In the initial stages of the project we will evaluate phloem-specific expression of the R proteins in transformed Arabidopsis plants to see if any detrimental effects on plant growth or development occur. Our working hypothesis is that limiting expression of these proteins to the phloem may also limit the harmful effects of expression. At present we have wild type and constitutively active mutant forms of two R proteins, AtSSI4 and AtSNC1, cloned into the pCAMBIA vectors downstream from the two versions of the AtSUC2 promoter. In addition, each of the R proteins has been cloned in these vectors in versions where the leucine-rich repeats (LRR) have been deleted. The effect of removing this region of the R protein has not been well documented, but our rationale is this will uncouple the action of the activated R protein from signaling pathways related to pathogen invasion. Deletion of the LRR region may simplify the regulation of the R protein’s activity. Transformation of these constructs into Arabidopsis is scheduled for the fourth week of January, 2010. We expect to quickly move those constructs that are not lethal in Arabidopsis into citrus plants for evaluation regarding the survival and spread of Liberibacter from grafts with infected plants. It is anticipated that Objective 1 milestones will be met within year 1 from the actual start of funding. These include the evaluation of phloem-specific R protein constructs in Arabidopsis and citrus. The research of team of Dr. Eva Czarnecka and Lance Verner are now working on this project full time. The original proposal specified that Dr. Czarnecka (Co-P. I.) would be assigned 0.65 FTE.
We have collected and germinated seeds from several accessions from within the candidate categories listed in the proposal, including pummelos, intergeneric hybrids with Poncirus, wild citrus species, and various sweet orange lines for which there is emerging anecdotal evidence of differential sensitivity to HLB. Seedlings are being grown in DPI-certified greenhouses at the CREC to provide budwood for topworking and young trees to plant directly in the field; currently we have sufficient budwood to begin propagations for replicated field trials. In the 2009-10 fruiting season we collected seeds to increase the number and diversity of accessions that can be tested; these have been planted already, and are growing in our certified greenhouses to provide additional materials for tesing. We have a tentative agreement with one grower on the east coast of Florida to plant out the range of genetic diversity we hoped to test, both as seedlings and as top-worked trees, including some apparently tolerant types we have identified in an HLB-devastated grove in Florida. We are currently exploring other options within Florida, to be followed up with agreements to move ahead; these represent locations where growers have decided not to remove HLB-infected trees, so we expect there to be opportunities to challenge our replicated materials. We are in communications with our collaborators in China, to develop plans for importation of our plant materials there for testing in secure locations in Guangdong province.
Two trees have been found growing in HLB-ravaged orchards in Guangdong and one other in Guangxi province, that appeared to be free of HLB symptoms, while all other trees planted at the same time were either dead or declining, and replants likewise were afflicted. The trees from Guangdong were propagated at the Guangdong Institute of Fruit Tree Research facilities, and are being grown to conduct new tests of their reaction to HLB following deliberate inoculations. These original source trees have been tested twice after propagation using standard RT-PCR protocols, and they remain PCR negative for HLB; recent RT-PCR tests on the propagated trees have likewise proven to be HLB-negative. Two propagations of one of the selections have been replanted in an infected orchard location. The tree in Guangxi has been transplanted to a protected location in Guilin, at the Guangxi Citrus Research Institute; several propagations have been made from this tree and these were inoculated with HLB-infected budwood. Recently propagations of it have been shared with our colleagues in Guangdong and planted there in a field challenge to assess the tolerance/resistance of this selection under natural and high-disease pressure. To expand further our search for survivors, and to continue to learn about Chinese citrus industry adjustments in response to HLB, we have established contact and good communication with a citrus extension specialist in the Fujian Provincial Academy of Agricultural Sciences, Mr. Li, Jian. This contact will provide us access to Fujian, another very seriously HLB-affected region of China. Mr. Li is very familiar with the local industry, and production areas and practices. He is aware of the goals of our collaborative project with scientists in Guangdong and Guangxi, and he is enthusiastically interested to aid us in participation. We are currently planning another visit to China in mid-late spring 2010, to assess the progress of the work underway, to expand our explorations for HLB survivors through the new contacts in Fujian, and to continue to work with our collaborators in Guangdong and Guangxi in search of additional survivors. We will also plan new experiments together at that time, to begin to address the causes and underlying mechanisms of these apparently tolerant selections, using various molecular techniques including gene expression studies, confirmation of genetic identity of the materials, and repeat inoculations in the field. This return visit is central to encouraging the continuation of the collaboration, to participate in planning experiments for a more in-depth analysis of the nature and underlying mechanisms of this phenomenon, and most importantly to confirm that the resistance persists following further propagation and inoculation with HLB. A valuable side benefit of this project has been the opportunity in our search for “survivors” to survey regions where HLB devastation is severe and quite widespread, and in doing so we have also visited orchards that appear to be nearly completely unaffected by HLB though surrounded by severely declining orchards. These surprising locations have been visited both in Guangxi and Guangdong. We have been investigating the nature of their management programs that has enabled them to survive to eight years of age or more in apparently good health. We interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy orchards. We have reported on our experiences and the answers to our questions in recent editions of “Citrus Industry”. Although located in different provinces several hundred miles apart, the key elements outlined to us were the same. These include critically timed pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition. Our impressions have been presented likewise through talks given at various grower meetings in Florida.
Funding is now in place among all the partners of the International Citrus Genome Consortium (US, Brazil, Spain, France, and Italy) to move forward with the project to sequence a haploid citrus genome. This genome sequence, when completed, will be THE reference genome for citrus, as it will be of the highest quality technically possible. DNA samples for sequencing have been prepared, and the strict quality control standards required by the sequencing centers (JGI in the US, Genoscope in France, and IGA in Italy) have been met. DNA samples have been shipped to the three centers, and sequencing has begun at Genoscope. Unfortunately, library preparations at JGI failed to achieve the needed insert size, so new DNA preps will be sent from Spain to JGI in January 2010.The Brazilian group remains in negotiations with JGI over contract language, but resolution is anticipated no later than February 2010. A meeting was held at Genoscope (Paris) with representatives from the sequencing partner nations, to revisit plans and progress. Progress was reported on the genetic linkage map, on haploid transcriptome sequencing, and genome sequencing (GS has completed 2.1x already); most importantly, a new time-line was produced that could lead to availability of gene sets by October 2010. Two sets of plants of sweet orange, rough lemon, and Volkamer lemon, representing the more susceptible and more tolerant types respectively, were inoculated with HLB in an environmentally controlled greenhouse. Plants were monitored for symptom development and for Las by qPCR. RNA samples were prepared from all plants at regular intervals, from inoculation through symptom development to be used in microarray experiments. Hybridization of RNA with Affymetrix and Agilent citrus chips (the latter developed by us at UF) has been completed and data sets have been generated; these are currently being analyzed to compare gene expression profiles over the time course.Though funding to the collaborators at UCR was delayed, they proceeded with their objective to update the HarvEST-Citrus EST database, including EST sequences from colleagues in Brazil and Japan, to provide an improved database for gene expression studies; currently there are more than 465,000 publicly available ESTs. Likewise, the collaborator in Spain was delayed in receipt of funds, but he has worked with us in preliminary experiments to test the feasibility of tissue-specific gene expression analysis that may be conducted. The sweet orange genome sequencing project is a collaboration between UF, JGI, and Roche/454 using next-gen sequencing platforms. Sequencing runs have been completed, and we now have >30x sweet orange genome coverage. In addition, 1.2 million ESTs were produced by one Titanium run of an RNA library from leaf tissue, to aid in subsequent assembly, gene prediction, and annotation. Six preliminary assemblies of the genome sequence have been attempted using various versions of 454’s assembly program, Newbler. To this point, the work has yielded fragmented assemblies upon which gene prediction models had a difficult time to work. Roche/454 will continue their efforts at assembly. Both genome sequences, when assembled and annotated, will be housed in a new database, Tree Fruit GDR, which was funded by an SCRI grant to include citrus. Further, the sequences will be available also through the JGI plant genome portal, and will be deposited with NCBI, as well. The plans to exploit genome sequence information for a better understanding of the interactions of citrus plants with the pathogen causing HLB are ultimately most dependent on having the genome assembled and annotated; for this reason, our main focus will be on accomplishing that goal, while continuing to conduct experiments using microarray analyses and deep transcriptome sequencing.
Transgenic citrus trees (900 containing 15 constructs with potential for resistance to HLB/canker) were planted at 2 field locations with severe pressure. Gene constructs have been prepared for new transformation experiments, including lima b, a ‘consumer-friendly’ sister to lima a which has shown promise against HLB. At least 1500 new transformations with various new constructs and promoters, mostly using sweet orange selections, have been produced for greenhouse tests against HLB/canker; many of these should be field planted in fall 2010. We have verified 4 tissue-specific promoters that target gene expression only to phloem against Las. Testing of previously produced transgenics continues, with several showing either delayed, reduced or no symptoms following HLB-inoculation. Transgenic Carrizo plants were produced with insecticidal genes; preliminary feeding tests showed the snowdrop lectin gene killed aphids and psyllids. Several transgenic and hybrid plants have been identified with tolerance to canker and citrus scab. New disease resistance genes were cloned from grape and tested in tobacco for efficacy; preliminary results identified two potential candidates for citrus. A newly developed plant-derived anthocyanin marker allows visual selection of transformants, and will be tested in citrus, an alternative to non-host plant markers used in most genetic transformations. Our Agilent microarray was tested with salicylic acid-treated grapefruit and 2 labeling protocols; results confirmed the array’s value for disease resistance research. Comparisons of sugar and starch metabolism in HLB-infected and healthy plants revealed that starch, sucrose, and glucose accumulated in infected leaves, maltose decreased and fructose levels were unchanged; studies are underway comparing activity of cell-wall bound invertase, a critical enzyme involved in sucrose metabolism and plant defenses. Using the iTRAQ technique, proteomic analysis was used to compare healthy and HLB-diseased mature leaves of sweet orange; 19 proteins were differentially expressed, out of which 9 proteins involved in stress and defense responses were highly up-regulated. Previously, microarray experiments highlighted canker defensive genes in kumquat; real-time PCR confirmed roles of one kumquat R-gene, two kinases, and one transcription factor, new targets for transformation. Cybrid grapefruit plants were produced with kumquat, and preliminary results show that some behave like kumquat in challenges. New DNA samples from a genetic population are being genotyped to contribute to the ICGC sequencing project. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses made 2009 are growing off. Previous work to develop rootstocks tolerant of or resistant to other maladies (such as CTV, blight, Phytophthora, Diaprepes, calcareous soils, etc.) continues, as we collected data from replicated trials and plantings. 15,000 trees were propagated from > 100 advanced UF-CREC rootstock selections, mostly for a 40-acre planting density/open hydroponic system trial located in Indiantown. The remaining trees will go to other PD/OHS trials to evaluate their potential value in closely-spaced groves under intensive cultural management; planting to be complete in Spring 2010. > 200 candidate rootstock hybrid seedlings were selected following a screen for Phytophthora tolerance /hi-pH soil adaptation. New pummelo-grapefruit seedless hybrids were created, and others available were found to be more tolerant of canker than grapefruit. New grapefruit-type candidates have been found this season, with selection based on fruit quality attributes. Sugar Belle, UF’s 1st fresh release, was licensed to NVDMC, and first crops of Sugar Belle fruit were successfully marketed. Six new seedling orange selections were approved for release by IFAS, as unpatented and unlicensed varieties, 5 of which are early maturing with attributes superior to Earlygold, and another with reported canker tolerance. Finally, our first UF-bred orange cultivars, Valquarius and N7-3, are being licensed currently; these will provide 6-8 week early and later maturing Valencia quality fruit.
Various factors were tested in efforts to develop a genetic transformation protocol for Murraya paniculata, such as varying OD values of the Agrobacterium cultures, the duration of explant incubation time, duration of co-cultivation, and the amount of antibiotic used for selection of transgenic shoots and for Agrobacterium removal using various plasmids and Agrobacterium tumefaciens strains; these include pCAMBIA2301 and pGreen0029 in AGL1, pTLAB21 in EHA101 and pCAMBIA2301 in EHA105. At this time, no transgenic shoots have successfully been regenerated. To overcome this regeneration barrier, consequently, we have pursued in parallel the regeneration of Murraya from axillary buds obtained from in vitro grown seedlings. Conditions suitable for micropropagation of axillary buds as well as induction of multiple shoot regeneration from axillary buds have been standardized; a variety of conditions were screened including those relating to the amount of growth regulators, basal medium and carbon source. The axillary buds are being used as a possible alternative regeneration system for the transformation experiments. Experiments to confirm the uptake of Agrobacterium by the axillary buds are currently underway by co-cultivating intact, nicked and/or pricked axillary buds. Depending on the response of these axillary buds, various treatments such as OD600 of Agrobacterium cultures, duration of incubation time, duration of co-cultivation and amount of antibiotic used will be carried out.
Various factors were tested in efforts to develop a genetic transformation protocol for Murraya paniculata, such as varying OD values of the Agrobacterium cultures, the duration of explant incubation time, duration of co-cultivation, and the amount of antibiotic used for selection of transgenic shoots and for Agrobacterium removal using various plasmids and Agrobacterium tumefaciens strains; these include pCAMBIA2301 and pGreen0029 in AGL1, pTLAB21 in EHA101 and pCAMBIA2301 in EHA105. At this time, no transgenic shoots have successfully been regenerated. To overcome this regeneration barrier, consequently, we have pursued in parallel the regeneration of Murraya from axillary buds obtained from in vitro grown seedlings. Conditions suitable for micropropagation of axillary buds as well as induction of multiple shoot regeneration from axillary buds have been standardized; a variety of conditions were screened including those relating to the amount of growth regulators, basal medium and carbon source. The axillary buds are being used as a possible alternative regeneration system for the transformation experiments. Experiments to confirm the uptake of Agrobacterium by the axillary buds are currently underway by co-cultivating intact, nicked and/or pricked axillary buds. Depending on the response of these axillary buds, various treatments such as OD600 of Agrobacterium cultures, duration of incubation time, duration of co-cultivation and amount of antibiotic used will be carried out.
The goal of this project is to transform the citrus and Arabidopsis NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac. (3) Inoculation of the transgenic plants with the HLB pathogen and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. During the first nine months of studies, we have identified three transgenic lines overexpressing CtNPR1 and AtNPR1, respectively, by using Northern blot analysis. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants, suggesting that both CtNPR1 and AtNPR1 are functional in citrus resistance to canker disease. Establishment of resistance to Xac by CtNPR1 is particularly significant for engineering resistance in citrus in the future. Preparation of a manuscript describing these findings is in progress. We have also inoculated the transgenic plants expressing a truncated XIN31 and the preliminary data showed resistance to Xac306. We will further characterize these plants in year 2 of this project. To prepare for greening inoculation, we have grafted the six NPR1 (three each of CtNPR1 and AtNPR1) overexpression lines and the control onto more root stocks to propagate the transgenic population. A total of 7-15 individuals have been produced for each of the transgenic lines. All these plants are currently maintained in green-houses located at the Citrus Research and Education Center in Lake Alfred, and will be used for greening inoculations in year 2 of this project. Finally, we have finished the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. This construct may increase the expression of CtNPR1 in citrus phloem thereby maximizing the opportunity for resistance to greening. Citrus transformation of this construct will be started soon. In summary, we have achieved most of the goals for year 1, which establishes a firm foundation for the research in next year. The delay for objective 3 in year 1 is largely due to the fact that greening inoculation requires the transgenic plants growing to relative bigger sizes. To continue our research, we request funds for the second year to achieve the following goals as proposed originally: (1) Inoculation of the characterized NPR1 transgenic plants with the HLB pathogen and monitoring of the bacterium in planta with quantitative PCR; (2) Characterization of transgenic plants expressing the truncated XIN31; (3) Transformation of SUC2::CtNPR1 into citrus; (4) Microarray analysis of the CtNPR1 plants in response to Xac or greening inoculations; (5) Examination of changes in hormone (abscisic acid, auxin, jasmonic acids and salicylic acids) levels in the CtNPR1 plants infected with Xac or HLB; (6) Plant maintenance. Accomplishment of these goals will very likely generate transgenic citrus plants with resistance to the HLB pathogen and advance our understanding of how citrus responds to these two diseases and could lead to new tools and strategies for the control of these two important diseases in Florida.
Each group of collaborators on this project are exploring a different approach to achieve the goal of the project: genetic transformation of mature citrus tissue obtained from trees grown under the challenging conditions present in climates such as that in Florida, thus shortening the time from transformation to evaluation in the field of citrus trees containing promising genes. USDA Ft. Pierce: Development of an in vitro shoot induction and multiplication process to serve as a source of in vitro adult phase tissue explants for transformation. An in vitro tissue culture system was developed using adult phase citrus nodes from calamondin, grapefruit, and sweet orange. Various types and amounts of plant growth regulators, salts and agar concentrations were evaluated for shoot growth. However, grapefruit in particular showed a large amount of leaf abscission in two weeks. Therefore ethylene action was inhibited using silver nitrate and vented lids to prevent abscission. Using a combination of these techniques, we have established a medium and procedure for the in vitro system. The appropriate preincubation tissue treatments, plant growth regulator types and concentrations, and incubation conditions for shoot organogenesis from intermodal explants from greenhouse grown citrus plants were evaluated. As a result, we have identified a protocol that provides shoot regeneration from mature tissue for a majority of explants. Shoots are being micrografted to month old rootstock seedlings. Following this success, we have initiated transformation with Agrobacterium carrying the GUS reporter gene. CREC Grosser: Transgenic lines of ‘Hamlin’ sweet orange stably expressing the GFP gene have been produced. PCR analysis of 4 of these lines has validated the presence of the C. sinensis codon optimized CEMA antibacterial gene. In all cases, explants for transformation were grown on the vigorous experimental rootstock tetrazyg ‘Orange 19’. Three of these lines are large enough and have been propagated by the ex vitro micrografting technique onto vigorous rootstocks. Optimization of protocol for Florida specific conditions is currently underway. Seed from several experimental tetraploid rootstocks selected for vigor and enhanced nutrient uptake were planted to provide liners for a 2010 study to investigate rootstock effects on mature tissue transformation efficiency. CREC Gmitter: Thin cell layers (TCLs) are being investigated as explants. In order to overcome the inhibitive effect of the contamination reduction medium additive PPM on the TCLs, axillary buds from the first flush were cultured onto medium with the addition of PPM and the shoots obtained from these axillary buds were used as the explant source for TCLs cultured onto medium without the addition of PPM. However, these explants have not yet shown any regeneration even though no contamination or browning has been observed. Experiments are being carried out to induce regeneration in the TCLs by manipulating the amount of growth regulators and carbon source. Brazil Machado: The Brazilian collaborator has been delayed in starting the project due to difficulties in getting funding from UF to his institution in Brazil. This has been rectified and he has now initiated experiments. Gainesville Moore: Since all of the other collaborators are making progress on developing in vitro systems, we have focused our efforts on the evaluation of cell penetrating peptides (CPPs) and the possibility of using them as vehicles to transport compounds into mature citrus tissue without the necessity of a tissue culture step. CPPs have been best characterized in mammalian and bacterial systems but there are new and surprising reports that the compounds can also work in plants. CPPs are short peptides that, when present either attached or not attached to another compound, allow the compound to be taken up by cells. Compounds that have been taken up with certain CPPs and under certain circumstances include DNA molecules, RNA molecules, proteins, and antibodies. We are presently evaluating various CPPs in model plant systems to determine how to visualize uptake and identify the most promising CPPs to evaluate in citrus.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene and 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. Transgenic ‘Carrizo’ citrange plants (lines: 854, 857, 859 and 884) transformed with the AtNPR1 gene were propagated by cuttings. Previous tests showed that lines 854 and 857 overexpressed the endogenous marker gene PR1 (considered a marker of SAR). On the other hand lines 859 and 884 did not express the AtNPR1 transgenic gene and did not show overexpression of the endogenous PR1 gene, hence were considered as negative controls. Subsequently we grafted a number of these plants with wild type (WT) ‘Duncan’ grapefruit. We also grafted WT ‘Carrizo’ plants with ‘Duncan’ grapefruit as controls. We also treated these plants with either salicylic acid (SA) or water (as negative control) and compared their response using TaqMan Real Time PCR. In preparation for the real time experiments we sequenced a number of genes of interest (NPR1, NPR3 and PR1) from both ‘Carrizo’ and ‘Duncan’ to guarantee that the target probe/primer sequences within the genes were identical and that any observed differences in expression were not due to differential efficiency in annealing of the probes and/or amplification. We also standardized the real time reaction for the marker PR1 gene and AtNPR1 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. Using Real time PCR we confirmed the expression of AtNPR1 in lines 854 and 857. The expression was about twice as high in the SA-treated plants compared to the water treated plants. Plants from these two lines also exhibited levels of PR1 expression up to 200 times higher than those of transgenic controls or wild types, confirming our previous results. We will repeat the SA treatment experiment to confirm the results and analyze the expression of more genes as stated in our objectives. In addition the same group of plants will subsequently be analyzed as proposed in objectives 2 and 3 for their response to HLB infection. For this purpose we propagated HLB-infected material and standardized the real time PCR detection of the pathogen so we are confident we can conduct the proposed experiments.
The Core Citrus Transformation Facility (CCTF) had the best year since it opened. Addition of one more technician funded by this grant helped to re-organize the work in the laboratory and bring it to levels that are about 50% higher than they were. Facility continued to service multiple orders and deliver transgenic Citrus plants with success. Transformed plants of seven different Citrus cultivars were produced within last year, further stressing facility’s ability to satisfy varying demands of researchers. Transgenic plants of Duncan grapefruit and Hamlin sweet orange that CCTF produced for one of the recent orders may represent a breakthrough in the fight against Citrus canker. In a challenge experiments with canker-inducing bacteria, these plants exhibited significant increase in resistance to this disease. In the short period of last nine weeks, facility received a large number of new orders. When grouped, these orders came from five different IFAS faculty (F.G. Gmitter, W.O. Dawson, J.H. Graham, N. Wang, and Z. Mou). All of these faculty are presently involved in research projects associated with NAS/FCPRAC funded efforts to produce Citrus plants resistant/tolerant to huanglongbing (HLB). Therefore, CCTF continues to be an irreplaceable element in the fight against Citrus diseases and especially HLB. Activities of CCTF on a few orders that have to do with improvement of Citrus not associated with disease resistance are continuing as well. The list of transgenic plants that were produced and confirmed by the presence of reporter gene and appropriate PCR reaction for validation: pSUC-LIMA1: Hamlin sweet orange-23 plants; pCIT108P(17): Flame grapefruit-5 plants; pCIT108P: Flame grapefruit-1plant; pCIT108P(3): Flame grapefruit-6plants; pCIT1070: Mexican lime-9 plants, Hamlin sweet orange-1 plant; pCL2: Duncan grapefruit-18 plants; pLIMA: Mexican lime-5 plants; p6Cass: Mexican lime-2 plants; pSuperNPR1: Duncan grapefruit-3 plants; pC5*: Duncan grapefruit-16 plants. There are about 50 plants that have been soil-adapted under laboratory conditions, but not yet tested for the presence of the gene of interest. Most recently, the new method for detection of foreign DNA in plants that requires minute amounts of tissue as a source of template was adopted by the facility. This will allow for faster detection of transgenic plants because screening can be done on a small shoots before they get micro-grafted on the rootstock plants. This is very important for those orders where binary vectors have no reporter gene. As a consequence, the delivery time for production of PCR-confirmed transgenic plants is expected to be shorter. CCTF’s role as a reliable partner to researchers within the University of Florida community and Citrus industry continues to grow through the increase of the CCTF activity. Boosted activity of CCTF represents a guarantee that the process of production of plants resistant/tolerant to HLB or canker will not be slowed-down because of generation of transgenic material.
Obj. 1. Construct cDNA libraries from (a) adult/immature psyllids, dissected gut, salivary glands (PSGs) and accessory salivary glands (ASGs). Results: Our cDNA synthesis is based on the total amount of RNA: (a) The yield of total RNA for the uninfected 1000 guts (PG) is 10.22 ug with the concentration 511 ng/ul in 20 ul. Total nt=92,850,628; filtered: 399,147/220 bases; bimodal distribution 50-520 and 520-760 of filtered data. ESTs were trimmed and then assembled, organized, and annotated using PAVE. NCBI nr db reveals short read matches to psyllid primary endosymbiont, while short and long EST reads were annotated using Uniprot. Prelim conclusion: endosymbiont nr db matches primarily, the primary sym Carsonella ruddii; ESTs encode psyllid proteins; (b) For psyllid Ca. Liberi-infected adults (PI), total RNA was obtained @11.44 ug with the concentration 572 ng/ul in a total volume of 20 ul) from tube ‘P-INF’ 8/12/2009: infected) for the library construction. Total nt = 480,465; trimmed: 480,465/294 bases; bimodal distribution, preliminarily, represents prokaryotic nr nt hits and eukaryotic ‘insect’ Uniprot hits. This is consistent with the type of data (nucleic acid or EST translated/proteins) that are available in the NCBI database for bacteria and insects sequenced to date. The availability of psyllid sequences for these libraries will aid future annotation for this and other insect transcriptomes. Cataloging of genes/proteins is underway for each library, to be followed by a comparison of PG/PI transcript composition using the PAVE web-based software system. Conclusions: The first mRNA and cDNA/pyrosequencing is highly successful; hits predict psyllid and primary endosymbiont, as well as other prokaryotic genes, including Ca. Liberibacter in infected psyllid colonies. In addition it is highly promising that the putative ‘uninfected’ psyllids gave no Ca. Liberibacter hits, indicating that colonies are HLB free, as has been indicated by qPCR results. This is very important because several reports indicate reversion from HLB-infected to uninfected (or vice versa) psyllid colonies, whose basis is entirely unclear. Because new information indicated that 5th instar nymphs might better support Liberibacter accumulation over the adults, we modified our plan for constructing the remainder of the EST libraries. Instead of making PSG/ASG libraries at this stage, libraries will consist of: HLB+/- adult psyllids; HLB+/- adult guts, and whole HLB+/- 4-5th instar psyllids. The rationale is that all possible PSG/ASG transcripts will be present in the whole adult and 4-5th immature instar HLB+/- libraries. Thus quantification can be achieved based on the downstream random cDNAs sequenced from HLB+/- adults, given a range of AAPs (0-40 days), as compared to ESTs from adult or immature instars born and reared on HLB+/- plants. In this way we will learn how 4-5th immature instars compare to adults as reproductive hosts. Since immature instars are not highly mobile, we will consider only HLB+/- whole immature instars reared on plants at this time. [Even so, downstream analysis will reveal gene expression patterns in immature psyllid gut ESTs, as they can be identified based on adult gut ESTs. This plan will allow us to quantify gene expression in the various treatments, stages, and organs, while requiring fewer insects and organs (for mRNA) from time-course studies; see below] Obj. 2,3. Sequence random cDNA clones, assemble ESTs, and select unigene sets for quantitative analysis and construct a microarray chip to quantify expression in instars and organs. We propose a revised plan in place of the unigene sets that will better and more cost effectively allow us to carry out quantitative analysis of whole immature instars, versus whole adults, guts, and PSG/ASGs from time course exposure (AAPs) to HLB plants within a defined time frame (steady-state qPCR-based titer). This involves extensive direct sequencing of random cDNAs from HLB+/- stages, instars, and organs, and is proposed because the relative cost of sequencing has declined, as the extent of coverage vs. cost has increased. In this way we can more effectively compare expression levels between whole adults and immatures, and adult guts and SGs.
Protocol Improvement: The Agrobacterium-mediated juvenile citrus transformation protocol was improved to increase speed and efficiency by improvement in: A. manipulation of hormonal combinations to increase shoot regeneration; B. pre-transformation incubation conditions; C. bacterial growth conditions;D. co-cultivation conditions; and E. shoot regeneration conditions. Following these combined improved procedures, is possible to produce transgenic plants that can be ready for mass propagation within 6 months of transformation. This has allowed us to cut the time by half (compared to previous protocols) from transformation to propagation of transgenic materials. Finally, a preliminary experiment testing a new antioxidant in the selection medium doubled the transformation efficiency for 3 cultivars tested. Experiments to validate this result are underway. Improvement of Rapid Micrografting Technique: (Modification of the Skaria technique) to stabilize the micro-graft union, a thin strip of Nescofilm’ was used to wrap around the wedge-micrograft. This increased micro-grafting success rate to approximately 95%. Transformation of Selected Precocious or Potentially HLB-Avoiding Sweet Oranges: Using the improved protocol, transgenic plants of high quality precocious Vernia sweet orange somaclones C2-1-1, C2-1-2 and C2-2-1, and Rhode Red Valencia clones avoiding HLB infection (B4-79 and B10-68) in a heavily HLB-infected Martin County grove, containing the LIMA anti-bacterial gene were produced. Embryogenic Callus Transformation: A protocol was developed for the direct transformation of embryogenic callus, and numerous transgenic plants were recovered from OLL-8 sweet orange, W. Murcott tangor,(Afourer/Nadercott), and Ponkan tangerine. This technique clearly extends transformation methodology to other important polyembryonic commercial citrus cultivars, particularly those that are recalcitrant to Agro-bacterium mediated transformation (ie. fresh market mandarin types). Rootstock Effect on Length of Juvenility: Juvenile sweet orange scion (OLL-8, a high quality Valencia-type with high solids and enhanced juice color) grafted to 6 selected precocious rootstocks and Carrizo as a control were single-stemmed in preparation for planting in the RES (Rapid Evaluation Structure) in March. The goal is to apply horticultural procedures to induce flowering in these plants as early as spring of 2011. Rootstock effect on speed of flowering will be determined. Effect of Scion Genetics on Length of Juvenility: Seedlings of several high quality processing sweet orange clones selected for precocious bearing were planted, and seedlings are now growing well, for subsequent planting in the RES. Transfer of Induced Precocious Flowering: We have cloned each of the genomic sequences of ciFT1, ciFT2, and ciFT3 (Arabidopsis Flowering Locus T genes) into a plasmid vector in which their expression is constitutively driven by the 34FMV promoter. Agrobacterium tumefaciens mediated transformation experiments have been performed with each of the ciFT constructs and the empty vector using juvenile tissue of the citrus hybrid Carrizo citrange. We currently have planted in soil a collection of GUS+ regenerated plants from each of the four transformation constructs as well as from non-transformed controls. Confirmation of transformation by PCR analysis is underway. We have observed novel in vitro flowering to frequently occur in transformation experiments using the ciFT3 clone. The occurrence of in vitro flowering also suggested that replacement of the constitutive 34FMV promoter with an inducible promoter may provide a better system for controlling precocious flowering, particularly when ciFT3 serves as the transgene. We have obtained vectors for an estradiol inducible system and the experiments to test inducibility in citrus have begun.
Progress on first year’s objectives: 1) Build a greenhouse in Florida for growing citrus for mature transformation. The preliminary planning work to build the greenhouse for growing citrus for mature transformation was completed. It included a detailed analysis of all project components and necessities. Conceptual designs were fully developed to allow for a more accurate estimate of project costs. A first estimate showed that the initial project is over budget. Modification of the initial project is underway to adjust the budget by redesigning the greenhouse and/or looking for additional funds. 2) Training of the Florida manager (Dr. Zapata) at IVIA in Spain. Dr. Cecilia Zapata arrived to the IVIA in November 11th to learn the technology to transform mature citrus. Several transformation experiments were set up with Valencia, Hamlin and Pineapple sweet oranges and Carrizo citrange, and screening of putative transformed plants regenerating from the in vitro cultures is underway. She has been trained in all tissue culture techniques associated with citrus transformation, with preparation of the source plant material at the greenhouse, and with acclimation of transformants to the greenhouse. Parallel to this, she is learning how to start and maintain a greenhouse to support a mature transformation facility. 3) Establishment of genetic transformation systems for mature materials from the most important sweet orange varieties grown in Florida and Carrizo citrange rootstock. Mature Valencia sweet orange is being routinely transformed, as well as Pineapple, at the IVIA. Randomly chosen transgenic lines coming from different experiments are being transferred to the greenhouse to follow their growth and flowering-fruiting response. Hamlin is being more difficult to transform, but we are learning to prepare the starting materials properly and adapting the tissue culture media and procedures to this genotype. Although we did not get any transformant yet, many experiments are running and the aspect of the cultures and regenerating shoots is very promising. Regarding Carrizo citrange, we are just getting the first putative transformants. 4) Strategies to improve tree management. We have decide to focus so far in the overexpression of flowering time genes for generating new orange types putatively more compact and productive. 25 sweet orange seedling transformants with CsFT and 7 with CsAP1 have been obtained. In less tan one year, 68% of CsFT plants and 14% of the CsAP plants have produced flowers. They are being characterized at the phenotypical and molecular level. In order to compare this with the effect of overexpression of the same flowering genes in other citrus types, we have genetically transformed juvenile Carrizo citrange explants with the CsFT and CsAP1 genes. All these experiments are under way in greenhouse and tissue culture phases, respectively.
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