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.
The goal of this project is to transform the citrus and Arabidopsis NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac. (3) Inoculation of the transgenic plants with the HLB pathogen and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. During the first year of studies, we have identified three transgenic lines overexpressing CtNPR1 and AtNPR1, respectively, by using Northern blot analysis. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants, suggesting that both CtNPR1 and AtNPR1 are functional in citrus resistance to canker disease. Establishment of resistance to Xac by CtNPR1 is particularly significant for engineering resistance in citrus in the future. Preparation of a manuscript describing these findings is in progress. We have also inoculated the transgenic plants expressing a truncated XIN31 and the preliminary data showed resistance to Xac306. We will further characterize these plants in year 2 of this project. To prepare for greening inoculation, we have grafted the six NPR1 (three each of CtNPR1 and AtNPR1) overexpression lines and the control onto more root stocks to propagate the transgenic population. A total of 7-15 individuals have been produced for each of the transgenic lines. All these plants are currently maintained in green-houses located at the Citrus Research and Education Center in Lake Alfred. A set of grafted NPR1 plants were recently inoculated with greening. We are currently monitoring disease development. Finally, we have finished the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. This construct may increase the expression of CtNPR1 in citrus phloem thereby maximizing the opportunity for resistance to greening. Citrus transformation of this construct is in progress. In summary, we have achieved most of the goals for year 1, which establishes a firm foundation for the research in next year. The delay for objective 3 in year 1 is largely due to the fact that greening inoculation requires the transgenic plants growing to relative bigger sizes. To continue our research, we request funds for the second year to achieve the following goals as proposed originally: (1) Inoculation of the characterized NPR1 transgenic plants with the HLB pathogen and monitoring of the bacterium in planta with quantitative PCR; (2) Characterization of transgenic plants expressing the truncated XIN31; (3) Transformation of SUC2::CtNPR1 into citrus; (4) Microarray analysis of the CtNPR1 plants in response to Xac or greening inoculations; (5) Examination of changes in hormone (abscisic acid, auxin, jasmonic acids and salicylic acids) levels in the CtNPR1 plants infected with Xac or HLB; (6) Plant maintenance. Accomplishment of these goals will very likely generate transgenic citrus plants with resistance to the HLB pathogen and advance our understanding of how citrus responds to these two diseases and could lead to new tools and strategies for the control of these two important diseases in Florida.
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. Thousands of putatively transformed shoots have been developed to produce citrus resistant to HLB and CBC or citrus psyllid. Many hundreds have been micrografted. 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. A wide series of promoters driving a reporter gene are being tested in transformed citrus. Tests of garlic-lectin transformed citrus are underway to determine effect on psyllid feeding and development. 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 the final steps 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 to permit transformation and resistance using citrus-only sequences. Border sequences and promoters from citrus are now in a construct driving GUS and will soon be inserted into citrus. 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, 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. 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 and planted in February 2010. the greenhouse complement to this study is showing earlier symptom development than field trees, especially from graft-inoculation. 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. Very sensitive methods have been derived and leaf extracts with and without spiked AMP have been tested. A material transfer agreement has been established with Texas A&M University and we have received their spinach defensin AMPs for in-vitro analysis. The first year of this project concludes May 1, 2010 and the second year of funding is requested at the current level.
Researchers at the USDA Ft. Pierce have established an in vitro system for growing mature tissue buds in vitro as a tissue source for transformation. One severe problem that they ran into was leaf drop. They have been able to reduce leaf drop substantially (though not eliminate it entirely) and are preparing a manuscript to document this work. The other area where they have made progress is shoot regeneration from mature tissue derived from greenhouse trees. We are preparing a manuscript on this as well. We have started transformation experiments but have run into some Agrobacterium overgrowth issues and are looking into that now. However, given the relatively high levels of shoot regeneration (Valencia, Ruby Red grapefruit, Calamondin, and US-942) that they are seeing they are anticipating that transformation should be pretty straightforward. Their best shoot regeneration is from greenhouse trees. Though they get shoot regeneration of in vitro derived shoots they do not believe that it is quite sufficient for routine transformation (Note: They are still working this part out, so it is not yet clear if in vitro tissue is a viable source of tissue for transformation). At the CREC in the Gmitter lab, work in continuing on the use of Thin Cell Layers (TCLs) as explants for mature tissue transformation. Experiments were done this quarter 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. Therefore they have recently additionally initiated experiments with internodal segments from greenhouse grown plants and are also planning to do preliminary experiments to establish axillary bud transformation. In the Grosser lab at the CREC, transformation experiments were tried this quarter with secondary citrus flushes. These were unsuccessful. Since they obtained positive results with first primary flush tissues, the results suggest that primary flush works better (which is in agreement with what the Spanish group has reported). In the Moore laboratory in Gainesville, experiments continued 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 quarter demonstrated that enzyme (in this case GUS) could be delivered into plant tissues, including whole alfalfa seedlings, mung bean roots and citrus suspension cultures. Work is now underway to determine whether DNA uptake can be achieved.
In this quarter, seed from last spring’s new crosses to develop rootstocks and scions was planted in the greenhouse. New crosses were completed this spring with more than forty different genetic combinations. Fruit quality, yield, and tree size data were collected from four 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 rootstock and scion field trials. Two new rootstock field trials were planted into the field. Studies continue to assess citrus germplasm tolerance to HLB, CTV, and Phytophthora/Diaprepes in the greenhouse and under field conditions. More than fifty citrus genotypes and citrus relatives 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. Some hybrids with mandarin or trifoliate orange ancestry appear to be resistant or tolerant to HLB and/or the psyllid vector. All citrus germplasm and cultivars become infected with HLB when inoculated, but different germplasm responds to HLB infection at different rates and with different symptom severity. Some hybrid selections resembling mandarin, grapefruit, and sweet orange 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. Transformed trees containing 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. 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 highlighted. Summaries were prepared from ten different rootstock field trials and distributed to citrus growers. The release notice for US-942 rootstock was prepared.
Obj.1. The construction of six cDNA libraries have been completed from biologically relevant psyllid populations. Ca. Liberibacter was found (project #34) to be overwhelmingly present in guts but not in salivary glands, and liberibacter presence by qPCR in immatures and adults suggested that more information would be gained by focusing on whole adult- and immature instar liberibacter interactions, and on guts instead of salivary glands as originally proposed. The libraries were constructed from adult and immature psyllids, and dissected guts from psyllids, reared on HLB infected-infected and uninfected plants. Plants and psyllids from each treatment were tested by qPCR to confirm infection by, or absence of liberibacter (see report #21). Obj. 2. All six cDNA libraries have been constructed and two have been sequenced and annotated. The other four are presently being sequenced using Illumina short base read technology. The PG and PI libraries were assembled with PAVE and consensus sequences were annotated and display on a web-based summary and query-system. The unique transcripts (UniTrans) singletons and contigs were blasted against the UniProt Invertebrate subset (db Jan 4, 2010) and blast hits with e-values of >1e-20 were accepted for annotation. The PAVE query system offers the ability to query the UniTrans db by characteristic (e.g. number of ESTs, Invertebrate UniProt hit, EST library composition, R test statistic, etc.) and to query the UniProt Invertebrate proteins that matched the UniTrans. Each UniTrans is allowed multiple protein hits (1e-20 or better) from different organisms. Multiple proteins from the same organism were matched to a UniTrans and only the top match was accepted from that organism (‘non-redundant) match. Common proteins can be found in the UniTrans query system by asking for UniTrans with a high NRO count, indicating many organisms had a match to that particular UniTrans. The best annotation is defined as a protein matching a UniTrans with an e-value of 1e-40 and over 60% of the ESTs in the UniTrans matching the same protein with an e-value of 1e-10 or better. To assist with assignment of GO/GOSlim functions or pfam descriptions these annotations are added using the invertebrate matches. To search for protein annotation not yet known in UniProt’s invertebrate taxonomic set the assembly is blasted against full UniProt. Because the psyllid assembly contains bacteria, which will not match proteins in the UniProt databases, we blasted contigs against NCBI’s nucleotide database (Feb 10). Bast matches with e-values of 1e-20 or better were used from the full UniProt and from the nt databases. Obj. 3. We proposed a plan that would 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. To explore the number of guts and salivary glands that would be needed to produce sufficient RNA for RNA-seq quantitative sequencing (4-5 reps each time course AAP) we produced treatments of psyllids and dissected the guts and salivary glands from adult psyllids and isolated the RNA. We determined that 150 salivary glands and 75 guts would each yield about 1 ug of RNA, a sufficient quantity for quantitative analysis, per replicate per treatment. In the remainder of 2010 psyllids will be exposed to Liberibacter-infected citrus over a set of acquisition access periods and subjected to quantitative gene expression using RNA-Seq analysis.
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