Although Year 2 of this project officially began on July 1, 2010, funds did not reach any of the researchers until 9/9/10, and at this time, only the UF PI (Moore) and the UF subcontractors (Grosser, Gmitter) received funding. The USDA subcontractors did not get their funds until late in September due to procedures at UF and USDA. Therefore little new research was started in this quarter. Other funds were found to maintain materials and personnel.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene (with an enhanced, inducible defense response) have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. We recently started to propagate new lines from cuttings of 9 individually transformed plants: lines 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (We had to wait until the plants were large enough to withstand the taking of multiple cuttings). However, we have found that propagation by cuttings is difficult with certain lines and, even when it is possible, it may take several months for new growth on the cuttings. This process is still underway. Concurrently, we have standardized more probes and primers for the detection of SAR-associated citrus genes. Making these primers and probes requires knowledge of nucleotide sequence of the genes. Then the primers and probes must be tested and conditions optimized before experiments can be done. The list of genes we can test now includes: AZI1, BLI, CHI, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, PR1, R13032, R20540, RAR1 and SGT1, in addition to our controls 18S and COX. We chose these 15 genes because they are either important in the early induction and regulation of SAR (AZI1, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, R13032, R20540, RAR1 and SGT1) or are targets of the regulatory SAR pathway (BLI, CHI and PR1). In Objective 1 of this project, we propose to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA), by testing for expression of the above listed genes. This has been accomplished for the first set of lines. We intend to repeat this experiment with the increased number of transgenic lines once they are ready, with the increased number of genes we have identified, and using the commercial version of SA (Actigard, Syngenta Corporation).
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found two peptides that appear to effectively protect sweet orange trees from HLB. However, we and other labs continue screening for better genes that more effectively control HLB and can be approved for use in a food crop. In the California lab, we developed methods to rapidly screen anti-bacterial peptides against Ca. L. psyllaurous in tobacco plants. Tobacco plants were either inoculated with Ca. L. psyllaurous by using the tomato psyllid (Bactericerca cockerelli) and challenged one week later with recombinant Tobacco mosaic virus (TMV) expressing the specific peptides, or the plants first were inoculated with recombinant TMV, followed one week later by using B. cockerelli to inoculate Ca. L. psyllaurous. These assays are being analyzed presently. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
This project has three objectives: 1) gap closure of Ca. Liberibacter asiaticus (Las) found in Florida; 2) complete genomic sequencing to closure of Ca. L. americanus (Lam) strain S’o Paulo from Brazil, and 3) comparative genome analysis of Las and Lam to attempt to determine common factors enabling pathogenicity to citrus. Objective 1 Progress: The recently published Ca. Liberibacter asiaticus (Las) strain psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with Las to date. In the present study, shotgun sequencing and a fosmid DNA library of curated Las strain UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in Las and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. The SC-2 phage DNA appeared to stably replicate as an excision plasmid at a level 2-3X higher in planta than in psyllids. Objective 2 Progress: Similar phage DNA sequences (corresponding to both SC1 and SC2 and including putative lysogenic conversion genes) were found in Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected citrus in Brazil in collaboration with Dr. Nelson Wulff at Fundecitrus. We now have approximately 86% of the predicted Lam genome confirmed. Interestingly, both the SC1 and SC2 Las phage were found in Lam, and the gene order of the phage was also highly conserved. The 5 new and potentially pathogenicity related genes found in SC2 were also found on the equivalent Lam phage. This may be further evidence of the importance of these phage in lysogenic conversion of Liberibacter to become more virulent. This Lam genome is about one year from completion.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: We sequenced all of the constructs introduced into Arabidopsis which consisted of the AtSUC2 promoter (940 upstream from ATG) driving expression of the mutated (constitutive) and wild type forms of SSI4 and SNC1 (R proteins). For SSI4, which is derived from the Nossen cultivar of arabidopsis, two closely related genes (MUF8.3 and 8.2) are present in the Columbia cultivar. We cloned both, and the original from the Nossen cultivar, and cloned them behind the AtSUC2 promoter. These wild type versions of SSI4 will be used as controls for non-active forms of the R protein (pathogen activated). The four AtSUC2/R protein constructs (mutant and wild type of each of the two R proteins) were transferred from the pCAMBIA1305.1 vector, which confers hygromycin resistance, into pCAMBIA2301 with kanamycin resistance since the former is detrimental to transformation into citrus. The four constructs were submitted to the UF Citrus Research Facility at Lake Alfred for transformation into citrus. Transformation of these constructs into the Duncan variety of grapefruit is currently in progress at the Lake Alfred Citrus Research and Education Center (Dr. Vladimir Orbovic). Conclusions: Our hypothesis was that phloem-restricted expression of the R protein constitutive mutants would limit potential negative impacts on growth. The Arabidopsis transgenic plants expressing R protein mutants did not seem to be significantly affected in the majority of cases. Approximately 8% of the snc1 transgenics exhibited a stunted phenotype, very similar to the snc1 mutant expressed from its native promoter (not phloem specific). The first series of ssi4 transgenics (construct 5-2) had a point mutation (C>T) in the coding region that generated a premature stop codon and shortened the protein by 78 aa in the C-terminal region, past the leucine rich repeat (LLR). Five percent of these truncated ssi4 transgenics showed phenotypic differences mostly in the rosette appearance and lighter green, splotchy coloring. However, this effect will be investigated again in the new full-length ssi4 transgenic plants.
1- The physical construction/renovation of the growth room did not start yet. A draft of the final layout of the lamps and benches was presented. Several scenarios about air filtration and distribution were discussed as well as safety, security and the irrigation system. Regarding irrigation, it seems like there are concerns about the amount of run-off water leaving the growth room. A holding tank was proposed as a way to contain the water that is coming out of the facility, however the existence of a retention pond south of the building might be the best solution. No decision has been reached at this point regarding the disposal of the water. We make clear to everyone that we need hose bibs in the growth room and there will approximately 2000 gallons/week of run off water coming from the growth room, this is inclusive of watering the plants and maintaining cleanliness. 2 – The first material to establish mother plants from Hamlin 1-4-1 was released from Dr. Peggy Sieburth lab. The clean shoot tips are approximately 4 weeks old and they will need to be grafted in approximately 4 more weeks on rootstocks that are currently growing inside the lab. These rootstocks are being maintained inside the lab for 6 months under laboratory conditions and they needed to be transferred to bigger pots 2 months ago. Under these conditions the grafting will be delay until the growth room is available. Since these rootstocks are already suffering, a new batch was started as a backup. We hope that the shoot tips can hold a few months more until the grafting can be performed. We will need to transfer them several times which in normal conditions is not required. Maintaining shoot tips for a long time on in vitro conditions might also induce juvenility, which we will want to avoid. 3 – Construction of the growth room will start on October 25th according to information provided by the CREC maintenance supervisor. 4 – A grower was selected and he will be at CREC in a few weeks.
The objective of this project is to screen citrus germplasm for resistance to the Asian citrus psyllid (ACP). Although citrus huanglongbing (HLB) is a century-old disease and the control of ACP is the key factor for HLB management, there is little information regarding citrus host resistance to ACP. Our preliminary results indicate there is ACP resistance in citrus germplasm. Historically, most (if not all) citrus resistance to HLB has been evaluated using graft inoculations, which sometimes resulted in plants becoming infected that appeared to have resistance in the field. What is needed is research to find varieties with resistance to the psyllid. Greenhouse and field evaluations of the USDA germplasm collection will be conducted. There already exists in China individual citrus plants that are thought to be HLB-resistant,but likely some of these are ACP resistant. The identification of a psyllid-resistant variety (or individual mutant) could revolutionize HLB management strategies. If we can identify ACP resistant germplasm, we can identify resistance genes for use in traditional and molecular breeding. Our intentions are to screen hundreds of sources of USDA and Chinese germplasm for resistance to ACP and to field test these to see if resistance to the psyllid negates HLB or greatly facilitates control. We expect to identify psyllid resistant citrus genotypes (or individual mutants from field-resistant collections) and/or citrus relatives within the Rutaceae that have psyllid resistance. We expect to determine traits that confer resistance and to identify traits that might be transferred to citrus varieties currently grown in order to make them resistant to the psyllid and thus less prone to contracting HLB. The ultimate return from this project would be an effective management strategy to control ACP and HLB that is less costly and friendlier to the environment and non-target organisms than the repetitive use of broad spectrum insecticides. A post doc was found and hired to conduct this research. A trip was made to China to firm up research plans with the Dr. Liu Bo and the Fujian Academy of Agricultural Sciences. Seeds representing the entire citrus/citrus relative collection at USDA-ARS-NCGR were obtained and have been planted in Ft Pierce. At the time this report was prepared, the Chinese government had not yet approved importation of germplasm from USA. A field planting of 87 citrus genotypes and relatives, primarily of the orange subfamily Aurantioideae, in Ft Pierce was screened for psyllid infestations during June, July, and August. There were significant differences in susceptibility of the genotypes as measured by the categorical rating of adults (F = 3.97, df = 86, P = 0.0001), nymphs (F = 7.56, df = 86, P = 0.0001) and eggs (F = 2.17, df = 86, P = 0.0001). Many of the genotypes were highly susceptible to infestations by the psyllid, but Glycosmis pentaphylla, Clausena harmandiana, and two genotypes of Poncirus trifoliata were completely avoided by the psyllid. The genera Glycosmis and Clausena are not members of the “true citrus fruit trees” and are not sexually compatible with Citrus however these groups could still serve as a source of psyllid resistance genes. However, Poncirus trifoliata, the trifoliata orange, readily forms hybrids with Citrus spp., is the dominant root stock in China and since 1892 has been used in Florida either alone or in hybrid form. This cold hardy genotype is highly resistant to citrus tristeza virus, Phytophthora-induced diseases, and citrus nematode. In recent psyllid no-choice oviposition studies using six different genotypes of Poncirus trifoliata, with Citrus aurantium and C. macrophylla as susceptible controls, a greatly reduced number of eggs were laid on the Poncirus trifoliata selections when compared with the controls.
1- The first objective of the second year was to build a plant growth room at the Citrus Research and Education Center in Florida (CREC). The physical construction/renovation of the growth room has not been started so far. The construction is expected to start at the end of September. The rootstocks that are currently growing in the lab are big and we will need to start growing a new batch until the growth room is ready. They cannot be transferred to another greenhouse because it will defeat the purpose of growing under controlled conditions. 2- Training of the manager Dr. Zapata has been completed at the IVIA under the supervision of Dr. Pena. 3- Initial material to establish the mother plants are being produced at the Department of Agriculture with Dr. Peggy Sieburth. She will start releasing the in vitro plants in September. Ideally these plants should be used immediately for grafting on the rootstocks; plants will be kept in vitro until the growth room is ready. The plants cannot be kept for more than 4-5 months on in vitro conditions. 4- A search for a full growth room technician started. The final hiring process will be completed once the growth room construction starts. We expect this technician to go for 2 weeks of training in Spain. At this moment, construction/renovation of the growth room is a major bottleneck for the progress of the project. Clean materials for the most important scion varieties of Florida have been obtained with the help of Dr. Peggy Siebuth through shoot-tip grafting. Clean rootstocks (stored at the lab at this moment-the only clean area we count with) are already 6-month-old and will be ready to be grafted with the clean scions for next October. However, we do not know yet when the growth room will be finalized. Only after this facility is fully operative, we will be able to perform the grafts with the clean materials. If the construction of the growth room is delayed further (more than 3 months), we have the risk of losing both the scions and the rootstocks, and consequently lose months of work (that could not be repeated until next year due to Dr. Sieburth’s agenda) and a lot of money. These delays (no growth room after 1.5 years of project) are making impossible to fulfil our objective for the end of this 3-year project. This situation is out of my understanding- Leandro Pe’a
Continued efforts to improve transformation efficiency: ‘ The effect of antioxidants lipoic acid, glycine betaine and glutathione are being evaluated for increased transformation efficiency. Some treatments are showing a significant increase in transformation efficiency across a range of citrus genotypes.’ We have successfully developed an efficient transformation system for embryogenic citrus callus, (publication in Plant Cell Reports in press). This system works well for polyembryonic mandarin types (i.e. W. Murcott, Ponkan) that are seedless or more recalcitrant using the common Agrobacterium-mediated citrus method. The method should also work well for lemons. Horticultural manipulations to reduce juvenility in commercial citrus: The RES (Rapid Evaluation System). ‘ Commercial sweet orange, grapefruit, and specialty mandarin cultivars were propagated and planted in the RES. Several juvenile hybrids from our breeding program flowered and set fruit after only one year – our goal is to force flowering and fruit set in juvenile sweet oranges and grapefruits in 1-2 years. If successful, the same approach could be applied to transgenics. A two-year old field trial using a juvenile Valencia budline on more than 70 rootstocks is showing significant rootstock affects on precocious bearing. Transformation of precocious but commercially important sweet orange clones: transgenic plants of precocious ‘Vernia’ sweet orange somaclones were regenerated and micrografted for further study of early flowering. Transgenic approaches to reduce juvenility: ‘ Whole plants generated from ciFT and empty vector control transformation experiments of Carrizo are being evaluated by both PCR amplification assays and a repeat of the screening histochemical GUS assay. No obvious phenotypes have yet been observed among the whole plants, however, flowers have occasionally been observed to occur on in vitro shoots. ‘ Putative transformed Duncan grapefruit whole plants in soil and shoots being rooted in vitro have been generated. We will be doing additional transformation experiments as soon as fresh seed becomes available. We will shortly have T1 seed from additional plants and will soon be able to proceed with assays to phenotype this generation and compare the effects that each of the ciFT genes has on expression and morphology. ‘Through a project being conducted by an HHMI-sponsored undergraduate student, Melanie Pajon, we will also be cloning the tomato FT ortholog and using it to obtain citrus transformed by a heterologous FT gene.Transformation of Samsun tobacco with the ciFT genes has resulted in a number of T0 plants of each of the 3 ciFT constructs, some of which have produced T1 seeds. We will shortly have T1 seed from additional plants and will soon be able to proceed with assays to phenotype this generation and compare the effects that each of the ciFT genes has on expression and morphology. Phenotypes of the T0 plants have ranged from early flowering, multi-branching, dwarfs to ones very similar in architectures in the wild type parent.
During the first quarter of funding, Core Citrus Transformation Facility (CCTF) continued to process the orders for transgenic Citrus material. Demand for genetically transformed citrus plants stayed high resulting in influx of new orders listed here by names of genes of interest or plasmids: p6; p33; p7; p10; pMOG8000; pAS7; pAS13*; pNAC1; pMKK7; pMOD1; and pSucNPR1. The work also continued on the old orders that were previously partially completed. Considering that transgenic Duncan plants carrying NPR1 gene exhibited significant resistance to Citrus canker, CCTF received order for production of commercially important Flame grapefruit cultivar transformed with the same NPR1 gene (order completed-NPR1 gene: 10 plants). A gene thought to be superior to NPR1 (so-called superNPR1) was introduced into Duncan grapefruit (superNPR1 gene: 12 plants). Introduction of NPR1 into Hamlin orange cultivar and superNPR1 into Flame is half completed. Work on the order pAS7 that is associated with HLB tolerance/resistance is half done (5 Duncan plants). Order that included use of pLC plasmid is also completed (8 Hamlin orange plants). Ten Mexican limes transformed with gene in pHK plasmid were produced, but satisfaction of this order will include production of more plants per client’s request. Five plants of Mexican lime transformed with p33 gene were also produced. CCTF produced more plants for the old orders: N1* gene: 3 Duncan; C5*: 3 Duncan; CL1 gene: 2 Valencia, 3 Duncan; CL2 gene: 1 Duncan; PiTA gene: 1 Valencia; CIT108p: 1 Flame. About thirty more soil-adapted plants will be submitted to the PCR testing as a secondary proof of their transgenicity before delivery to clients. Some of the funds from the grant were used to hire additional help through the summer resulting in seasonal increase of CCTF capabilities. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
Since the end of April and the completion of the first year of our grant, we have pursued the following: – We have continued with population and grapefruit leaf disease/resistance studies to examine the effect of transiently expressed Bs3 promoter constructs on the growth of an expanded range of X. citri strains. These studies concur with the preliminary results showing that the constructs limit X. citri growth and produce HR against a number of strains. – We have continued to grow out stably transformed Duncan grapefruit lines and identified positive transgenics by PCR. – We have had some discussions with parties in Argentina about their interest in this genetic approach to controlling citrus canker. – We are selecting tobacco plants with the Bs3 promoter constructs driving reporter genes to aid in our investigation of the interaction of various X. citri TAL effectors with the promoter elements.
Six cDNA libraries were constructed from (a) adult/immature psyllids, dissected gut, salivary glands (PSGs) and accessory salivary glands (ASGs). The cDNA synthesis was based on the total amount of RNA: (a) The yield of total RNA for uninfected 1000 guts (PG) was 10.22 ug at a concentration 511 ng/ul in 20 ul. ESTs were trimmed and 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 at a concentration 572 ng/ul in a total volume of 20 ul) from tube ‘P-INF’ 8/12/2009: infected) for the library construction. Cataloging genes/proteins is underway for six libraries. We have made good progress optimizing the FISH assay on whole psyllids (adults, immatures) and dissected organs to minimize auto fluorescence and maximize signal using a probe for the primary endosymbiont 16S rRNA. Having sequenced random cDNA clones, assembled ESTs, and initiating data mining toward gene validation using FISH, we will next make libraries for short base read sequencing (RNAseq) to quantify expression in adults, immature instars, and eventually organs. We will carry out extensive sequencing for HLB+/- stages, instars, and organs (instead of microarray analysis) 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 adult and immature psyllids, guts and salivary glands. Based on preliminary mining the pyro-sequencing and Illumina sequencing were highly successful; hits predict psyllid and primary endosymbiont, as well as other prokaryotic genes, including Ca. Liberibacter in infected psyllid colonies. In addition the putative ‘uninfected’ psyllids gave no Ca. Liberibacter hits, confirming colonies are HLB free, as has been indicated by qPCR results. Further, to date no phytoplasma sequences have been detected. Because studies indicated that 5th instar nymphs might better support Liberibacter accumulation over the adults, we constructed the following EST libraries: LB+/- adult psyllids; HLB+/- adult guts, and whole HLB+/- 4-5th instar psyllids. The rationale is that PSG/ASG transcripts will be present in the whole adult and 4-5th immature instar HLB+/- libraries. The salivary gland libraries will be constructed in Yr 2 for the potato psyllid because it is a more tractable system; once parameters are established to identify the point at which the bacterial titer is highest in these same organs for the Asian citrus psyllid PSG/ASG libraries will be made. Quantification can be achieved based on the downstream random cDNAs sequenced from HLB+/- adults, given a range of AAPs (0-40 days), 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, and presently we are considering HLB+/- whole immature instars reared on infected plants. This 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.
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate transformants of rootstock and scion genotypes. More active promoters, derived from ubiquitin genes, have been identified and used in recent transformations. A wide series of promoters driving a reporter gene are being tested in transformed citrus and show very different levels of expression. Liberibacter sequence data are being used to develop a transgenic solution for HLB-resistance, targeting a transmembrane transporter. Peptide has been made corresponding to the extra-membrane sequence and a phage display array system is being used to identify structures which are specific to this epitope, with tests against an E. coli containing the Liberibacter protein underway. When identified, transgenics will be constructed and challenged with Las. Collaboration with a USDA team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are being identified from citrus genomic data, from Carrizo citrange generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Antimicrobial peptides (AMPs) continue to be 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, along with the Tachyplesin (which is among the most effective AMPs in Dr. Dawson’s CTV expression vector study), 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, and some of these were constructed using key functional elements from the horseshoe crab-derived Tachyplesin. Four new and very potent variants have been tested in the last few months. Transformation constructs will be prepared to produce citrus with these AMP transgenes. Transgenes are being developed to suppress a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. 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 and planted in February 2010. the greenhouse complement to this study is showing earlier symptom development than field trees, especially from graft-inoculation.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene (with an enhanced, inducible defense response) have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. Some transgenic ‘Carrizo’ citrange lines (lines 854, 857, 859 and 884) transformed with the AtNPR1 were produced in Year 1 of this project. In this quarter we were able to start to propagate new transgenic lines from cuttings: 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (the plants were now large enough to propagate). We have also identified sequences for several additional citrus genes that are associated with SAR, including AZI1, BLI, CHI, R13032, R20540, RAR1 and SGT1. These genes were preciously undescribed for citrus, however our microarray studies indicated that these sequences were differentially regulated by chemical and pathogen treatment. R13032 and R20540 belong to the NPR1/NPR3 family of genes in citrus and our experiments show they are all differentially expressed during SAR. Objective 1 of this project proposed to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA). This has been done with the first set of transgenic lines but we wish to repeat the experiment when the new plants have been propagated so we have more replications.
Note that this report corresponds to the first of the two additional quarterly updates mandated in amendment #1 of the no-cost extension for the first year of grant #123. Objective III: Bioinformatic analysis of Ca. L. asiaticus sequence data Bacterial proteins mediating interactions with the environment and with host organisms are commonly found in the periplasm and bacterial outer membrane. To identify the likely set of Las-encoded proteins targeted to regions of the cell outside the cytoplasm, all predicted Las proteins were evaluated for the presence of predicted signal peptides and lipoprotein signals using the SignalP and LipoP programs. Resulting predictions are posted on the CG-HLB Genome Resources website together with the repetitive sequences, transcription factor binding sites, and horizontally transferred regions predicted previously. Objective II: Website creation and development To better display the accumulated data on bioinformatic characterization of Ca. L. asiaticus, a new genome viewing utility is being added to the CG-HLB Genome Resources website for presentation at the upcoming Annual Meeting of the American Phytopathological Society (APS) Meeting (August 7-11). Guidelines and materials for use of the Artemis Genome Viewer will remain on the site; however, the GBrowse based viewer (see http://gmod.org/wiki/Ggb/ for typical display) is more easily accessed by a wider audience and can be readily expanded to include a broad range of bioinformatic data and comparative analyses. Data presented in the GBrowse viewer is organized as a linear display of genes as they are found in the Las genome, and through which the user can scroll or zoom. Tracks corresponding to different aspects of genome characterization are graphically displayed below the main genome entry with hyperlinks to other databases included as appropriate. Tracks have been installed for all predicted proteins with links to records at NCBI, all predicted proteins having links to the COG database (as a source of functional information), subcellular localization as predicted by Psortb, and operon predictions generated using DOOR. Transcription factor binding sites, repetitive regions (of particular interest for diagnostic purposes), and signal peptide predictions will also be incorporated. In addition to functioning as a central data clearinghouse for analyses performed by my group as well as by other genome-related databases, this type of display can also accommodate analyses such as the 3D protein structural predictions currently being generated by the Grishin group and orthology between the published Las genome sequence and those of other strains and species of Liberibacter. The new genome viewer is expected to go live during the next couple weeks. At that time, all registered users of the CG-HLB Genome Resources Website will be invited to try out this new feature, and feedback and additional data will be solicited. Different features of the viewer as well as the component analyses will be discussed at the ‘Candidatus Liberbacter/ Epidemiology & Ecology’ technical session at the upcoming APS meeting.
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