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.
Researchers at the USDA Ft. Pierce: Progress this past year in the various components of a mature citrus transformation system is as follows: Source of mature tissue) Four populations of adult phase trees were established in the greenhouse including Valencia sweet orange/Sun Chu Sha (73 trees), Ruby Red grapefruit/US812 (62 trees), US-942 citrange rootstock/Cleo (32 trees), Calamondin (31 trees), and Etrog Arizona 861-S1 citron (67 trees). Decontamination protocol) A decontamination protocol was developed that results in >90% clean explants, sufficient for tissue culture studies and practical applications. In vitro bud emergence and growth) A system was developed for the production of in vitro adult phase shoots from cultured nodes of greenhouse trees. Factors important in bud emergence and growth were identified and a system developed to initiate bud emergence and growth with reduced leaf drop. A manuscript has been prepared that documents this research. Shoot regeneration from mature tissue explants) A system was developed for the production of shoots from cultured internodes from greenhouse trees. Factors (e.g., pre-incubation tissue treatments, plant growth regulators, and incubation conditions) important in bud formation and shoot growth were identified and a system developed that is suitable for sweet orange, grapefruit, calamondin, and US-942 (Note: citron was just recently added so has not yet been tested). The system results in shoot and bud formation in 70-90% of the explants. A manuscript is in preparation that documents this research. Agrobacterium-mediated transformation of mature tissue explants) Transformation of mature internode explants from greenhouse trees has been demonstrated in grapefruit and US-942 using the GUS reporter gene. Though these are the first experiments, the results document that we have a functional mature tissue transformation system. Because explants were stained for GUS activity once shoot buds were observed, we can make no predictions on the efficiency of transformed shoot recovery. Current efforts are now directed toward identifying the factors important for a system of sufficient efficiency for routine transgenic plant production. At the CREC in the Gmitter lab, work is continuing on the use of Thin Cell Layers (TCLs) as explants for mature tissue transformation. Experiments have been done to induce regeneration in the TCLs by manipulating the amount of growth regulators, carbon source and also by pre-treating the TCLs with BA but regeneration is still problematic from these explants. In the Grosser lab, Hamlin mature budwood source trees were grown on a selected complex rootstock that seems to have superior nutrient uptake, and nutrition was provided by a new granular slow release product that has been showing excellent results with nursery and field trees. Six transgenic Hamlin lines stably expressing the GFP gene were regenerated from the 1st experiment using the first flush on the grafted trees as explants. In subsequent experiments, subsequent flushes on the same trees yielded no transgenics, indicating that the regeneration potential diminishes with sequential flushes. In the Machado laboratory in Brazil, regeneration ability of sweet oranges Pera, Valencia, Natal and Hamlin was evaluated by testing: different hormone combinations, the effect of the physiological age of the sprouting, and CTV infected and free tissue. Remarkable differences between genotypes regarding the capacity to transform juvenile tissues with Agrobacterium were observed. Optimum conditions, tissues and genotypes are being used in experiments with mature tissues. In the Moore laboratory in Gainesville, experiments have focused on using small peptides as vehicles to deliver cargos to plant tissues. If these techniques could be worked out they would have a number of applications for citrus transformation, perhaps even eventually allowing the transfer of genes or gene products to existing trees. Experiments this year demonstrated that enzyme (in this case GUS) could be delivered into plant tissues, including whole alfalfa seedlings, mung bean roots and citrus suspension cultures.
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. If it does not go forward on this site, we will make available existing HLB-infected trees for grafting of transgenics as a rigorous and rapid test for resistance/immunity/ 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 winter freeze, and trees are unscathed. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, and 1000 transgenic plants will be planted by July 1, 2010. An MTA is now in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Alphascents has provided an experimental 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. CLM damage also compromises ability to view HLB symptoms. It is requested that the second year of funding, at $84,405, be initiated on July 1, 2010. Thousands of additional transgenic trees will be planted and screened in the coming year. This budget includes $30,000 for land charges (standard USHRL fee is $3000/acre) plus $54,405 in funding for a GS-7 technician.
Objective 1: Transform citrus with constitutively active resistance 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 have transformed arabidopsis plants with a total of 12 constructs comprised of two versions of the AtSUC2 phloem-specific promoter driving expression of three variations of two resistance proteins, AtSSI4 and AtSNC1. The R genes were introduced as wild type, as constitutive expression mutants and as deletion mutants lacking the LRR region thought to be involved in signal perception. Overexpression of constitutive mutants of these two R proteins has been reported by others to exhibit enhanced SA accumulation and constitutive pathogen resistance; however, the transformed plants show dwarfism. Overexpression of wild type AtSSI4 showed no stunting, while the evidence in not as clear with overexpression of AtSNC1. In our experiments, restricting expression of the R proteins to the phloem cells caused no signs of stunted growth with any of the R protein constructs. While this was true for the majority of transformants, some plants exhibited stunted growth for some of the constructs. For example for the Atssi4 constitutive mutant, 2 plants out of 41 showed stunting. For Atsnc1, 5 out of 60 transformants showed a stunted phenotype. We are currently determining the level of expression of the transgenes and of their predicted target genes (PR1 and PR2). Conclusions: Our hypothesis was that phloem-restricted expression of the R protein mutants would limit potential negative impacts on growth. If results confirm that the wild type and constitutive mutant forms of the two R proteins are expressed in the transgenic arabidopsis plants, then this important requirement in our overall approach has been met. Our next step is to transfer these R protein constructs to citrus and test for expression and disease resistance.
The main objective of our project during this first year was hiring a Florida-based faculty scientist that could be trained under our supervision in Spain, for the purpose of learning the mature tissue transformation technology and transferring it to Florida. Moreover, we had the commitment to establish genetic transformation systems for mature materials from the most important sweet orange varieties grown in Florida and the Carrizo citrange rootstock. The Florida-based faculty scientist was hired (Dr. Cecilia Zapata) on October 2009 and a few weeks later started the training at the Instituto Valenciano de Investigaciones Agrarias (IVIA). She has been trained in all tissue culture techniques associated with mature citrus transformation, starting with preparation of the source of material, and ending with the acclimation of transformants in the greenhouse. She will finalize her second 3-month-stay in our lab next July 2010. In this final stage, we are focusing on improving transformation methods for more recalcitrant types, making molecular analysis of the putative transformants and on starting plant material preparation at the greenhouse. Transformation experiments were performed with three (3) sweet orange varieties: Hamlin, Valencia and Pineapple (used as readily transformable control). Mature Valencia was very responsive to transformation and organogenic regeneration and transgenic plants have been already acclimated in the greenhouse. Hamlin was more difficult to transform due to quality problems with the starting material and tissue culture media, and procedures specific to this genotype were needed. To date, transformants have been also obtained from this orange type and verified as positive using PCR. The plants are still growing in vitro and will be transferred to the greenhouse within a few weeks. Carrizo citrange transformation experiments were initiated later but putative transformants have been already generated and micrografted in vitro. The second objective of this project is related with the necessity of implementing new cultural practices to be able to survive with the HLB disease in Florida until a definitive solution is found. We have proposed the use of strategies to control tree size and productivity by genetic modification of either the rootstock or the scion through over-expression of flowering-time or gibberellin biosynthesis genes. This could permit to establish reduced but highly-productive trees at higher planting density which would facilitate flush management and mechanical fruit harvesting. For generating more compact and productive varieties, we are ectopically expressing the flowering time genes FT or AP1 from sweet orange in juvenile sweet orange. Additionally, we are overexpressing these same genes in Carrizo citrange in an attempt to modulate its architecture and reduce its size. More than 10 independent transgenic lines have been generated for each construct and genotype. In both genotypes, overexpression of the FT transgene led to early flowering in vitro and poor regeneration. Once transferred to the greenhouse, transgenic plants continued flowering and consequently their vegetative development was generally very poor, indicating that this transgene could be of interest for other biotechnological application but not to modify the architecture of either a scion or a rootstock. In the case of AP1, some of the transformants showed a compact and branched phenotype. This phenotype remained once plants were established in the greenhouse. We are waiting them to flower and fruit possibly next spring. Moreover, for generating a dwarf-dwarfing rootstock, we are making a construct aimed to induce RNA interference to downregulate the expression of a crucial gene in gibberellin biosynthesis, CcGA20ox1, in Carrizo citrange. We will focus in this sub-objective during the second year of the project.
Seed from new crosses to develop rootstocks and scions were planted in the greenhouse. New crosses were completed with more than forty different genetic combinations. Fruit quality, yield, and tree size data were collected from 16 rootstock field trials. Propagations from supersour rootstock hybrids were prepared for budding to produce trees for disease testing and field trials. Rootstock liners were budded with scions to prepare trees for trials. Budded greenhouse trees for field trials were grown to planting size. Two new rootstock field trials were planted into the field. Three new field trials were planted at the Whitmore Farm in Lake County to study inheritance of fruit quality factors in sweet orange-type material from populations of hybrids between high quality pummelo and mandarin parents. One of these was planted on trellis to also examine the effect of tree manipulations on the length of time for transition from juvenility to maturity. Studies continue to assess citrus germplasm tolerance to Huanglongbing (HLB) and Phytophthora/Diaprepes in the greenhouse and under field conditions. Greenhouse trees inoculated with Citrus tristeza virus (CTV) were tested for virus titer in preparation for CTV-induced decline evaluation of supersour rootstocks. More than fifty citrus genotypes and citrus relatives, as well as thousands of progeny from crosses, have been challenged by natural inoculation with Liberibacter in the field, and data are being collected on HLB symptoms and Liberibacter titer by PCR. Detailed information is being collected on HLB tolerance and tree performance in four rootstock field trials. All citrus germplasm and cultivars become infected with Liberibacter when inoculated, but different germplasm responds to HLB infection at different rates and with different symptom severity. Some trifoliate hybrid rootstocks, including US-897, exhibit tolerance to HLB as seedling trees. Some hybrid selections resembling mandarin, grapefruit, and sweet orange also appear to exhibit some tolerance to HLB. Greenhouse and field studies are continuing to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial, insect resistance, and other genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker. Transformed trees containing seven different promoters and three new anti-bacterial genes were prepared for greenhouse testing with HLB. Genetic transformation was used to introduce the citrus FT gene for induction of early flowering into citrus scion and rootstock germplasm. Manipulation of this gene with inducible promoters will drastically accelerate the pace of cultivar development (shortening the generation time from 6-15 years to 1 year) and can also be used to increase early cropping of commercial trees. To date, the early flowering gene has been introduced into Hamlin, Ray Ruby, US-812, and US-942. Four new hybrid rootstocks, US-1235, US-1239, US-1225, and US-1241, were identified as especially promising for expanded field trials in the coming year. Promising new scion cultivars were released, including the seedless mandarin cultivar ‘Early Pride’. The new hybrid rootstock US-942 is being released for commercial use because of outstanding performance in many trials. Research is continuing to use HLB responsive genes and promoters identified in the gene expression study published last year for inducing or engineering resistance in citrus. New studies were initiated to examine gene expression and metabolic changes associated with HLB disease development and apparent resistance to Liberibacter in particular selections. This will provide additional insights about how to engineer HLB resistant cultivars. A study demonstrating no evidence for seed transmission of HLB was published in HortScience. A field day was held at the Ft. Pierce USDA farm to highlight progress in development of new cultivars, and performance information from several rootstock trials was presented.
This project has three objectives: 1) gap closure of Ca. Liberibacter asiaticus (Las) found in Florida; 2) complete genomic sequencing to closure of Ca. L. americanus (Lam) strain S’o Paulo from Brazil, and 3) comparative genome analysis of Las and Lam to attempt to determine common factors enabling pathogenicity to citrus. Objective 1 Progress: Within the recently published Las strain psy62 chromosomal genome (Duan et al. 2009), many unique genes of unknown function are found, and several phage related genes are found integrated into the chromosome, but no replicating phage DNA was found. We reported last quarter the finding of two complete circular lytic phage genomes, SC1 and SC2, with the copy number of SC1 replicating an average 10X higher in citrus and 20X higher in periwinkle, than when the phage is integrated into the chromosome in infected psyllids. [Gabriel & Zhang, 2009. Phytopathology 99 (6): S38]. Neither phage were previously reported. A fosmid DNA library from curated Las strain UF506, isolated from an infected Florida citrus tree, was surprisingly biased towards phage-related DNA inserts. Two highly related circular phage genomes (SC1 and SC2) were assembled from the UF506 library, revealing 5 new genes not previously identified in psy62. Annotation revealed multiple genes on SC2 which were pathogenicity related; since SC2 also appeared to lack lytic cycle (ie., lysis) genes, SC2 may be involved in lysogenic conversion of Las to become more virulent. Phage particles associated with Las were found in the phloem of infected periwinkles by transmission EM. Southern blot and PCR analyses were used to: 1) confirm the presence of replicating SC1 and SC2 circles in Las infected citrus and periwinkle and replicating SC2 circles in Las infected psyllids; 2) map the cos sites and confirm gene order on both SC1 and SC2, and 3) determine the genomic DNA integration sites of both SC1 and SC2 as prophage. Semi-quantitative RT-PCR revealed that the copy number of (lytic cycle) SC-1 in infected citrus and periwinkle averaged 10X and 20X higher, respectively, than in (lysogenic cycle) infected psyllids. 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: In collaboration with Fundecitrus in Brazil, Lam strain ‘S’o Paulo’ DNA samples extracted from citrus and purified on pulsed-field gels by Dr. Nelson Wulff has resulted in 1,095,921 bp of partially confirmed Lam genomic DNA sequence obtained by 454 sequencing in 318 contigs, which corresponds to 86% of the predicted Lam genome. A high degree of syntenic gene order was observed between Las and Lam. 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. We are in the process of making another full 454 sequencing run, and are on track to meet target deadlines of the original proposal.
Our group made outstanding progress in the first year of FCATP funding, accomplishing all of our original first year goals and making significant progress on our second year goals already. In brief, we determined the binding sites for all fourteen known Xanthomonas citri TAL effectors and combined these into new Bs3 promoter constructs. Thirty-two constructs and controls were prepared in the Lahaye lab with different Bs3 promoter and Bs3, AvrGf1, and reporter gene combinations and sent to Gainesville. Constructs were introduced into grapefruit in both transient and stable transformation experiments: – A transient Agrobacterium transformation and assay system was successfully developed for grapefruit leaves. In this system, promoter constructs were introduced into Agrobacterium strains and infiltrated into leaves. Infiltrated areas were assessed over several days for the appearance of a hypersensitive response (HR) produced by the Agrobacterium strain or following co-inoculation with strains of X. citri carrying various citrus TAL effector/Pth A genes. We demonstrated that only the combination of specific X. citri strains and Bs3 constructs containing binding sites recognized by TAL effectors in those strains produced a strong HR. – Stable transformations were carried out with nine different promoter constructs, producing more than 300 plantlets on tissue culture, with more than 50 rooted and transferred to soil. There are many more transformed epicotyls in progress. Positive transformants will be identified and tested by X. citri inoculation. In addition, we tested our constructs in growth assays to assess their effect on X. citri growth in grapefruit leaves. Whereas the original Agrobacterium strains used in our study had little to no effect on X. citri growth and Agrobacterium strains carrying the Bs3 promoter constructs had a small effect on X. citri growth, the population of X. citri on leaves co-inoculated both with Agrobacterium containing Bs3 constructs and X. citri strains with corresponding TAL effectors was dramatically reduced (see below). Population studies will be carried out on stable transformants to identify lines for product development. Growth of X. citri at 6 days post inoculation on grapefruit leaves, following transient transformation treatments (average from two experiments): Pre-inoculation treatment: X. citri growth (Log 10 CFU/ml): Agro strain BS3 promoter construct TAL effector – – – 8.50 + – – 8.41 + + – 7.43 + + + 4.80 Lastly, we have begun testing the responses of the diverse accessions of X. citri available to the Jones lab.
The seeds of Murraya paniculata were procured from the USDA-ARS National Clonal Germplasm Repository for Citrus and Dates, as well as from the local sources in Florida. Seeds have been available only periodically for our experiments. These seeds were germinated in vitro and used to establish cultures for experiments designed first to improve the efficiency of plantlet regeneration from epicotyl explants through adventitious organogenesis. 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. In efforts to standardize the genetic transformation protocol, experiments were carried out with plasmids pCAMBIA2301 and pGreen0029 harbored in Agrobacterium tumefaciens strain AGL-1 and pTLAB21 harbored in Agrobacterium tumefaciens strain EHA101. Various factors were 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. Unfortunately and despite substantial efforts, we were not successful in recovering transgenic plantlets from any of these experimental treatments. To overcome this regeneration barrier, consequently, we have pursued in parallel the regeneration of Murraya directly from axillary buds obtained from in vitro grown seedlings. A variety of conditions were screened including those relating to the amount of growth regulators, basal medium and carbon source; as a consequence, conditions suitable for micropropagation of axillary buds, as well as induction of multiple shoot regeneration from axillary buds, have been standardized. Prior to genetic transformation experiments, the amount of antibiotics required to inhibit micropropagation culture growth has been determined using non-transformed axillary bud cultures. To study the response of the explants to wounding, wounded (nicked and/or pricked) non-transformed axillary buds have also been cultured. The intact, nicked and/or pricked axillary buds have been co-cultivated with plasmids pCAMBIA2301 and pGreen0029 harbored in Agrobacterium tumefaciens strain AGL-1 and pTLAB21 harbored in Agrobacterium tumefaciens strain EHA101. An experiment was performed using two different sizes of explants while the OD600 of Agrobacterium cultures, duration of co-cultivation and amount of antibiotic were kept constant. The size of the intact as well as the wounded explants influenced their survival. Putative regenerants will be tested for the presence of marker genes. 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 to define the requirements for successful genetic transformation of Murraya.
Funding is now in place from the International Citrus Genome Consortium partners (US, Brazil, Spain, France, and Italy), and the project to sequence a citrus genome is well underway. This will be THE reference genome sequence for citrus; it will be of the highest quality technically possible, by virtue of its haploid condition and the use of Sanger sequencing technology. DNA samples 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, and sequencing is ongoing at Genoscope and IGA. Unfortunately, library preparations at JGI failed, so new DNA preps have been sent to JGI recently; a backup plan has been established for timely delivery of new samples, should current samples prove inadequate. The HudsonAlpha Institute in Alabama, a JGI affiliate, will sequence the Brazilian part of the collaboration; this brings Drs. J Schmutz and J. Grimwood, two world authorities in sequencing, to the project. A meeting was held last December at Genoscope (Paris) with representatives from the partner nations, to revisit plans and progress on the genetic linkage map, and haploid transcriptome 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. The sweet orange genome sequencing project is collaboration between UF, JGI, and Roche/454 using their next-gen sequencing platform. Sequencing runs have been completed, yielding 30x sweet orange coverage. In addition, 1.2 million ESTs were produced from an RNA library from leaf tissue, and several additional libraries for sequencing are being constructed with RNAs from 16 different biotic and abiotic conditions, to broaden gene coverage to aid subsequent assembly, gene prediction, and annotation. Six preliminary genome sequence assemblies were attempted using various versions of 454’s assembly program, Newbler, yielding fragmented assemblies upon which gene prediction models could not work. Roche/454 continued their efforts, and a 7th and useful assembly has been produced; currently gene prediction and annotation have begun. 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. 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. Two sets of plants of sweet orange and rough lemon, representing 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 RT-PCR. RNA samples were prepared from all plants at regular intervals, from inoculation through symptom development, to be used in microarray experiments. Hybridization with Affymetrix and Agilent citrus chips (the latter developed by us at UF) has been completed and data sets have been generated; these are under analysis to compare expression profiles over time; some genes clearly up-or-down regulated differentially have already been confirmed by RT-PCR. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from colleagues in Brazil and Japan, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs. We are working with our collaborator in Spain on experiments to examine tissue-specific gene expression. New experiments have been designed and are being implemented.
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. These greenhouse grown trees have now been inoculated with HLB-infected budwood, but no symptoms have yet been observed. Several 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; these are being closely monitored for symptoms and by RT-PCR. Recently propagations from Guangxi were shared with our colleagues in Guangdong and planted in a field challenge to assess the tolerance/resistance 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 join us in the project. We are currently planning another visit to China in October-November 2010, to assess the progress of the work underway, to visit the original trees, 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 observations have been presented likewise through talks given at various grower meetings in Florida and California.
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