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.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. A new 8 acre site has been bedded, supplied with irrigation, and a ground cover established. Several acres in the far NE corner have been prepared for Dr. Dawson’s proposed field test of modified CTV expression vectors designed to produce anti-microbial peptides in citrus host plants. APHIS specified that Dr. Dawson’s site be as far from existing commercial citrus groves as possible, and recommended the NE corner of the Picos Farm. There has been no recent word on the progress of APHIS approval for this project Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Trees were sprayed with microsprinklers throughout the recent freeze, and trees appear to be unscathed. USHRL has filed papers with APHIS to conduct field trials of their transgenic plants at this site. An MTA is now in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Discussions are underway with Alphascents to provide pheromone attract/kill product Malex to disrupt citrus leaf miner (CLM). Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids.
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 dozens 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 displayed Las development and show HLB symptoms. More active promoters have been identified and used in recent transformation 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. Peptide has been made corresponding to the transmembrane sequence and a phage display array system is being used to identify structures which are specific to this epitope. When identified, transgenics will be constructed and challenged with Las. 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 ‘M or less across all test bacteria. An additional 20 synthetic AMPs were assessed, revealing several AMPs that were highly active against all test species, with negligible hemolytic activity. Transformation constructs will be prepared to produce citrus with these AMP transgenes, having completed an agreement with entities who posses the rights to these AMPs. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. After 1 year in the field, the first trial shows similar levels of infection across all three methods of Liberibacter transfer. The complete experiment is being repeated with controlled graft and psyllid inoculations completed and plants in the nursery awaiting field planting in February 2010. 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 and we have received their spinach defensin AMPs for in-vitro analysis. Since this material is well down the regulatory pathway, it makes no sense to move forward with any transformed citrus which is not markedly superior to this benchmark material.
In this quarter, seed was collected from crosses completed in the spring to develop new rootstock and scion hybrids. Fruit quality, yield, and/or tree size data were collected from twelve rootstock and scion field trials. Greenhouse trees inoculated with CTV to evaluate supersour rootstocks for CTV tolerance were tested for virus titer in preparation for grafting. Cuttings were made from new supersour rootstock hybrids to propagate trees for field trials. Budded greenhouse trees for field trials were grown to planting size. Studies continue to assess rootstock and scion tolerance to Huanglongbing (HLB) in the greenhouse and under field conditions. Although all citrus cultivars tested become infected with HLB when inoculated, different rootstocks and scions respond to HLB infection at different rates and with different symptom severity. Some trifoliate hybrid rootstocks, including US-897 exhibit clear tolerance to HLB as seedling trees. Studies are underway to determine whether this seedling tolerance provides any benefit for trees with standard susceptible scions grafted on top. Experiments continued to assess the utility of different methods for testing germplasm for resistance or tolerance to Asian Citrus Psyllid (ACP) and HLB disease. A field experiment continued to identify rootstocks with resistance to the Phytophthora-Diaprepes Complex. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial and insect resistance genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker (CBC). Selected transgenic rootstocks were challenged with HLB and ACP to assess potential resistance and some selections were found to have possible improved resistance or tolerance. Research is continuing to use HLB responsive genes and promoters identified in the gene expression study published last year for engineering resistance in citrus. A study demonstrating no evidence for seed transmission of HLB was published in HortScience.
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 dozens 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 displayed Las development and show HLB symptoms. More active promoters have been identified and used in recent transformation 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. Peptide has been made corresponding to the transmembrane sequence and a phage display array system is being used to identify structures which are specific to this epitope. When identified, transgenics will be constructed and challenged with Las. 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 ‘M or less across all test bacteria. An additional 20 synthetic AMPs were assessed, revealing several AMPs that were highly active against all test species, with negligible hemolytic activity. Transformation constructs will be prepared to produce citrus with these AMP transgenes, having completed an agreement with entities who posses the rights to these AMPs. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. After 1 year in the field, the first trial shows similar levels of infection across all three methods of Liberibacter transfer. The complete experiment is being repeated with controlled graft and psyllid inoculations completed and plants in the nursery awaiting field planting in February 2010. 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 and we have received their spinach defensin AMPs for in-vitro analysis. Since this material is well down the regulatory pathway, it makes no sense to move forward with any transformed citrus which is not markedly superior to this benchmark material.
In the past quarter, we have succeeded in developing a transgene construct for citrus which is transcriptionally activated by TAL effector proteins delivered by Xanthomonas citri. TAL-induced promoter activation triggers a localized hypersensitive response (HR), a typical plant disease resistance response which generally results in a reduction of bacterial growth. The transgene was developed based on the two key features of the pepper Bs3 gene; a tightly regulated pathogen-inducible promoter and an encoded protein, Bs3, that triggers an HR and mediates resistance to Xanthomonas. Because it was not know if the activity of Bs3 as an HR-inducing protein would function in citrus, we tested another gene in parallel for the protein known as AvrGf1 from the X. citri Aw strain. Unlike the predominant A strains, Aw strains elicit an HR on all known commercially grown citrus species, specifically due the expression of AvrGf1 (Rybak et al., 2009, Mol Plant Pathol 10:249-262). Using transient transformation assays, we looked for the production of an HR caused by expression of Bs3 or AvrGf1 in Duncan grapefruit leaves. Indeed, constitutive expression of AvrGf1 produced a robust HR, demonstrating that ectopic expression of AvrGf1 in plants is sufficient to trigger localized cell death. Analysis of constructs in which the AvrGf1 gene is under transcriptional control of the Bs3 promoter demonstrated that in the absence of TAL effectors no reaction was evident on leaves. However upon co-inoculation with X. citri strains containing the TAL effector AvrBs3, the transgene construct produced a robust HR. The tight regulation of the Bs3 promoter and its transcriptional activation by AvrBs3 through its specific recognition sequence is well characterized in pepper and tobacco (Romer et al, 2007, Science 318:645-648), and now confirmed in citrus. We further tested this reaction with X. citri strains that are deficient in their system for delivering effectors into plant cells, and we observed a loss of the HR, confirming that the reaction specifically requires the presence of AvrBs3 in the plant cell. Thus far, we have not observed an HR in response to Bs3 expression in transient assays and continue to test Bs3 in stable transformation assays and particle bombardment experiments. AvrGf1, however, makes an effective alternative to Bs3. Additionally we are in the process of testing a complex Bs3 promoter that has 14 recognition sequences for all currently known X. citri TAL effectors. We will test whether stable citrus transformants containing the complex promoter driving AvrGf1 or Bs3 expression confer an HR in response to a range of X. citri strains. We have initiated experiments to examine the effect of TAL effector-induced AvrGf1 expression on bacterial growth in grapefruit leaves. In these assays, X. citri is inoculated onto leaves transiently transformed with control or test Bs3 promoter constructs and the growth of bacteria is assessed over time. In our first experiment, we observed that the presence of the Bs3 promoter:AvrGf1construct lowered the amount of an X. citri strain carrying AvrBs3 1000-fold compared to controls lacking the construct. This result is very promising and suggests that the constructs we are developing are capable of conferring disease resistance to citrus canker by restricting X. citri growth in transgenic citrus plants.
This is a 3-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, the AtMKK7 gene has been subcloned into the CTV-based expression vector and transition expression of MKK7 in citrus leaves is ongoing. The AtMKK7 gene has also been subcloned into the plant binary vector pBI1.4T (a pBI121 derivative) and transformed into citrus using the Agrobacterium-mediated approach. The AtMKK7 transgenic plants are growing. Conformation of the presence of the AtMKK7 gene in the transgenic plants by PCR and analysis of the expression levels of AtMKK7 in each transgenic line are underway. Resistance of the transgenic lines to citrus canker and greening (HLB) will be characterized when the transgenic plants are ready. For objective 2, Hamlin suspension cells have been used 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 have tested the resistance of hypocotyls of citrus seedlings to the selective compound sodium iodoacetate and found that citrus hypocotyls are very sensitive to this inhibitor. A concentration of 0.2 mM could completely inhibit the growth of the calli generated from hypocotyls. We will use 0.2 mM of sodium iodoacetate in selection of the hypocotyl-derived calli. We have done irradiation for the first batch of Duncan grapefruit cuttings on 11/2/09. The irradiation dosage was 40G. We found that the irradiated cuttings generated significantly fewer shoots than the control and calli were formed on both irradiated cuttings and the control. The shoots and calli generated on both the irradiated cuttings and the control will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. We are preparing another batch of explants for irradiation.
Objective I: Assess community needs The PI presented a poster entitled ‘Analysis of Ca. Liberibacter asiaticus Psy62 (Las) genome sequence data and creation of the CG-HLB genome resources web site’ at the Joint Research Conference on HLB and Zebra Chip, November 2009 for the purpose of publicizing the available resources, and engaging with other researchers involved in genome analysis of Liberibacter. Objective II: Website creation and development. The Citrus Greening/HLB Genome Resources Website (http://www.citrusgreening.org/) has continued to expand. To assist CG/HLB researchers in navigating the growing number of Liberibacter-related datasets generated by 3rd party sites, links to these resources have been added to the Citrus Greening/HLB Genome Resources Website. Examples include pathway maps of Liberibacter metabolism generated by the Kyoto Encyclopedia of Genes and Genomes, and lists of genome structural features at the Genome Atlas Database. Links to other CG/HLB relevant sites including the Citrus Greening and Citrus canker publication list and web site for the International Psyllid Genome Consortium are also provided. Objective III: Bioinformatic analyses of Ca. L. asiaticus sequence data. Understanding Las biology and pathogenicity depends not only on knowing its raw genetic capability but also how and when individual genes are expressed. A list of predicted regulatory proteins encoded by the Las genome has been compiled. To initiate characterization of sites in the genome where these proteins bind (with implications for expression of downstream genes), sequences of experimentally characterized binding sites in related bacteria (e.g. Sinorhizobium, Rhizobium, and Agrobacterium) were assembled and used to create binding site models for computational analysis. These models are being applied to the Las genome sequence in order to identify candidate binding sites for three regulatory proteins: (RpoH (heat shock response), RpoD (constitutive expression), and RirA (response to iron availability). The models continue to be refined, but preliminary analyses suggest that in contrast to free-living bacteria which have distinct sets of co-regulated genes, Las has a much simpler regulatory profile. First, Las has many fewer regulatory proteins than do related free-living bacteria. Secondly, locations of predicted binding sites suggest that many genes that in free-living bacteria are tightly regulated in response to specific environmental conditions may be constitutively expressed in Las. Repetitive AT-rich sequences are also found in the promoter regions of several candidate virulence genes. It is hypothesized that their presence may enhance gene expression by allowing for easier separation of the DNA strands. The models described here can be readily applied to other Liberibacter strains and species as their genome sequences become available, with the potential to reveal sources of heat tolerance and other differences in environmental adaptation observed among isolates.
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, the Arabidopsis MKK7 (AtMKK7) gene has been transformed into citrus using the Agrobacterium-mediated approach. The MKK7 transgenic plants are growing. Conformation of the presence of the AtMKK7 gene in the transgenic plants by PCR and analysis of the expression levels of AtMKK7 in each transgenic line are underway. Resistance of the transgenic lines to canker and HLB will be tested. For objective 2, we have tested the resistance of hypocotyls of citrus seedlings to the selective compound sodium iodoacetate and found that citrus hypocotyls are very sensitive to this inhibitor. A concentration of 0.2 mM could completely inhibit the growth of the calli generated from hypocotyls. We will use 0.2 mM of sodium iodoacetate in selection of the hypocotyl-derived calli. We have done irradiation for the first batch of Duncan grapefruit cuttings on 11/2/09. The irradiation dosage was 40G. We found that the irradiated cuttings generated significantly fewer shoots than the control and calli were formed on both irradiated cuttings and the control. The shoots and calli generated on both the irradiated cuttings and the control will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. We are preparing another batch of explants for irradiation.
In efforts to standardize the genetic transformation protocol for Murraya paniculata, new experiments have been designed and implemented using pTLAB21 harbored in Agrobacterium tumefaciens strain EHA101, as previous efforts with standard citrus transformation strains and vectors were not successful. We have also tested pCAMBIA2301 and pGreen0029 in AGL1 for comparison. Epicotyl segments obtained from in vitro grown seedlings of Murraya were used as explants and a tissue culture medium designated as M10 (Murashige and Skoog’s (MS) standard medium supplemented with predetermined levels of BA and NAA) was used as the regeneration medium for all the transformation experiments. Various factors are being tested in efforts to develop a standard protocol for transformation, such as varying OD values of the Agrobacterium cultures, the duration of explant incubation time, duration of co-cultivation, and the amount of antibiotic used for selection of transgenic shoots and for Agrobacterium removal. At this time, no shoots have successfully been regenerated. Consequently, we are pursuing in parallel the regeneration of Murraya from axillary buds obtained from in vitro grown seedlings; this technique is being standardized with the aim of using it as a possible alternative regeneration system for future transformation experiments. Different concentrations of BA alone and in combination with NAA are being tested to induce multiple shoot regeneration from axillary buds.
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. The Brazilian group remains in negotiations with JGI over contract language, but UF and JGI came to terms in late September 2009. Because of the various contractual delays, both here and elsewhere, and the current decreased capacity for Sanger sequencing at JGI, the ICGC goal to have the genome sequence completed and available to the citrus research community in mid-2010 will not be achieved. Meetings will be held this autumn to revisit the plan and coordination among the sequencing centers, to move forward at the quickest possible pace. Meanwhile, work has proceeded at the UF-CREC to produce sample materials needed for the microarray experiments planned, using Affymetrix GeneChips, a new array platform developed by the co-PIs at UF using Agilent technology, and for the cDNA platform available through our co-PI in Spain. To this end, two sets of plants of sweet orange, rough lemon, and Volkamer lemon, representing the more susceptible and more tolerant types respectively, have been inoculated with budwood from HLB-infected Carrizo citrange (Carrizo is resistant to CTV, so viral interaction complications will be avoided) in an environmentally controlled greenhouse. Plants have been observed for symptoms, and qPCR has indicated that we have successfully infected the plants. Samples of RNA have been prepared from all of the plants at regular intervals, to be used in microarray experiments. We are nearly finished the plan time course of RNA sample collection. Though funding to the collaborators at UCR has been delayed, they have proceeded with their objectives. The HarvEST Citrus EST database is being updated, to provide an improved database for gene expressions studies. The EST sequences from our colleagues in Brazil and Japan have all been downloaded and reassembled, increasing the number of publicly available citrus ESTs to more than 465,000. Likewise, the collaborator in Spain was delayed in receipt of the funds allocated, but he has been engaged with us in establishing experimental designs for array experiments using the cDNA platform, as well as some tissue-specific gene expression analysis that will be conducted. 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 establish the experiments and collecting the samples that will be used for subsequent microarray analyses and deep transcriptome sequencing.
This project was an extension in time and financial resources to the previously funded project titled “The International Citrus Genome Consortium (ICGC) sequencing project; Part 1, Sequencing” which was intended first to initiate the ICGC haploid genome sequencing project using Sanger sequencing. Those funds were allocated as a first investment intended to demonstrate good faith commitment on behalf of the Florida citrus industry. That goal was achieved, as evidenced by current funding in place for 4x coverage by our French collaborators through Genoscope, 2x coverage by our Italian colleagues through the Istituto di Genomica Applicata (IGA), 2x coverage through EMBRAPA funding of colleagues in Brazil plus 2x coverage through FCPRAC from the US together with the USDOE Joint Genome Institute (JGI). Because the timing to use the allocation first made by FCPRAC through this grant did not allow for our investment in Sanger sequencing through JGI (they require the project to be conducted at one time, not in pieces), I requested and was granted a revision to the objectives, to begin a sweet orange genome “re-sequencing project”, using next-gen sequencing (454 of Roche Diagnostics). This is a collaborative effort between my lab, Dr. W Farmerie (UF-ICBR), Dr. D. Rokhsar (JGI), and Dr. T. Harkins (Roche Diagnostics/454). This project is in concert with the ICGC sequencing initiative plans to use next-generation sequencing technologies on several diploid genomes, as an added resource to the Sanger haploid sequencing project currently underway. Currently, all sequencing runs of the sweet orange genome have been completed, including 8x coverage of 454/FLX WGS, 6x coverage 454/Titanium WGS, 9x coverage of 3 kb insert libraries paired-end sequences (PE), and 6.7x coverage of 8 kb insert libraries (PE). Combined with a 1.2x Sanger sequencing effort by JGI several years ago, we now have >30x sweet orange genome coverage. In addition, 1.2 million ESTs have been produced by one Titanium run on 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 by 454 scientists, using various versions of their in-house assembly program, Newbler, and two more assembly efforts will be made using modified parameters. To this point, this work has yielded fragmented assemblies upon which gene prediction models had a difficult time to work. Roche/454 is continuing their efforts at assembly. When the challenges of assembly can be overcome, it is likely that the sweet orange genome sequence will be made available to the research community sometime in the first half of 2010, following gene prediction and annotation procedures, to make it a useful resource for subsequent research. The sequence will be housed in a new database, Tree Fruit GDR, which was funded by a recent SCRI grant to include citrus. Further, the sequence will be available also through the JGI plant genome portal, and will also be deposited with NCBI. Though funding for this project was terminated in September 2009, the work will continue to completion. This will be a valuable tool for all research projects aimed at understanding HLB infection and disease, as well as empowering for the full scope of citrus genetic improvement objectives addressed by breeding and genetics research programs.
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; recently propagations of it have been shared with our colleagues in Guangdong and planted there in a field challenge. 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. Plans are being discussed for a possible visit to Fujian sometime within the next 6 months, and follow-up visits to Guangdong and/or Guangxi. This trip seems central to encouraging the continuation of the collaboration, and to participate in planning experiments for a more in-depth analysis of the nature and underlying mechanisms of these apparent resistant phenotypes, and most importantly to confirm that the resistance persists following further propagation and inoculation with HLB.
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