Current status of the research: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to develop an efficient system of screening EFR mutants for their binding to elf18-CLas, and in vitro binding system was developed which could be used to screen a mutant phage display library. We demonstrated in vitro binding of both elf18-WT and elf18-CLas to fragments of EFR ectodomain, but not to the ones of related receptor FLS2. Binding of elf18-CLas was weaker than that of elf18-WT, but it was considered that improvements in binding achieved by phage display screening may not be evident considering the binding of WT EFR to elf18-CLas. In addition, recent structural information about FLS2 binding to flg22 indicates the involvement of BAK1 as a co-receptor directly binding the ligand. As the phage display system would not account for this interaction, it may yield mutants which would perturb the binding of BAK1. An alternative system based on split ubiquitin is currently in the process of being investigated, which will hopefully overcome these issues. Additional experiments have also been performed to determine which portion of elf18-CLas is non-functional, by making chimeric elf18 peptides with WT and CLas portions. Both WT-CLas and CLas-WT peptides fail to elicit ROS, indicating there are multiple issues with the function of the elf18-CLas peptide, and thus requiring further investigation. A collaboration is currently being set up with the laboratory of Prof. Chai at Tsinghua to obtain structural information surrounding elf18-WT and elf18-CLas binding to EFR, which would enable more straight-forward testable hypotheses. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Vectors are currently being constructed in the pCAMBIA backbone, under the expression of the 35S/FMV promoter. These constructs will contain: EFR; XA21; both EFR and XA21; and EFR and XA21:EFR chimeric. Cloning of these constructs should be completed in the next few weeks and will then be passed on to the Moore laboratory for transformation in citrus.
Within the last year and-a-half, researchers trying to find solution to HLB through production of transgenic plants have used every possible approach that offered some prospect for production of tolerant/resistant citrus plant. As a result, the Core Citrus Transformation Facility (CCTF) has indeed become the platform for testing the effect of different DNA sequences (‘genes’) on transgenic plants that could result in possible alteration of their ability to sustain pathogen attack. Since most of this work in its nature is theoretical, it is not well known how the introduction of certain gene into Citrus will affect production of transgenic plants. Some genes may be easily introduced into model organisms or may cause weak effect on their phenotype. However, very often situation is rather different in Citrus. From one client, CCTF has received a group of orders that all have common DNA sequence combined with other, different DNA sequences. Those orders consisted of six vectors that were supposed to be introduced into three different citrus cultivars in certain combinations. Since the time when these orders were placed and 9/20/2013, altogether 70 co-incubation experiments were performed with more than 38000 explants. Despite all this work, no transgenic plants were produced. From another research group came an order to produce plants with the gene that severely affected the phenotype of transgenic plants. Transgenic shoots were stunted and extremely bushy, making our efforts to graft them impractical. Work on this order stopped after about 30 PCR-positive shoots were produced at which time the agreement was reached with the client who will try to place new order where this gene will be controlled by an inducible promoter. The unintended consequence of the efforts of researchers to find the ‘gene’ that may render Citrus plants tolerant/resistant to HLB by using CCTF as a testing platform is low number of transgenic plants produced in the CCTF. In the last quarter, only 20 plants were produced. They belong to the following orders: pHGJ2 vector-one plant, pHGJ3 vector-seven plants, pHGJ4-three plants, pN5-five plants, pBI121-one plant, pN7-one plant, pW14 one plant, and pMED14 one plant. The second (out of ordinary) reason that contributed to low productivity within this quarter was the massive contamination in one of the common growth rooms where CCTF also keeps germinated plants. This contamination took place in June and July and wiped out 18% of our cultures from that period. Out of three orders for transgenic plants placed in the previous quarter, two were withdrawn. Within this quarter, six orders were placed. Work with two vectors is to proceed only to the early step in shoots production. For additional three vectors, the goal is to establish possible effect of introduced genes on genome activation/transformation success rate. Because of the presence of GFP as a reporter gene in these vectors, the work on these orders may also be completed at the phase of transformation when fully developed shoots are harvested from explants.
The Mature Citrus Biotechnology Facility (MCBF) continues to increase our capacity for genetic transformation of mature scion budded onto immature rootstock. To this end, we have double budded immature rootstocks with mature scion. The number of explants will be significantly increased if this double budding scheme works well. Thus far, the results are promising for grapefruit but we are still waiting for results in sweet orange. Sweet orange is not as vigorous as grapefruit. We are also increasing the number of transgenic events produced by transforming immature rootstock. Different transgenic/wild-type combinations of rootstock/scion will be tested in field studies. A number of clients have provided genetic constructs important to imparting tolerance to citrus canker. Genetic transformations of Valencia using two constructs obtained from Dr. Wang’s lab have been performed and putative transgenic shoots are regenerating which will be micro-grafted in the future. Transformations using a construct from Dr. Mou’s lab (originally provided by Dr. Dong at Duke, NC) have been used with Hamlin, two batches of Pineapple and Ray Ruby grapefruit. Mature shoot explants of Valencia will be transformed in the near future as explants become available. This construct has also been used with explants of Carrizo. Swingle and Macrophylla rootstocks will be transformed in the near future. Additional genetic constructs will be obtained from citrus researchers as the capacity of our lab increases. Molecular analyses of putative transgenics, transformed with marker genes, are underway, and these plants are expected to flower in the near future. The number of transgenes for each putative transgenic is being determined by qReal-time PCR or Southern blot. Additional laboratory equipment and consumables are being purchased and another employee hired to accomplish this objective. The temperature, photoperiod, and light source have been changed in Growth Room A to induce flowering in these transgenics. Dr. Pena indicated that thorns in mature citrus transgenics regenerated from tissue culture is normal, transitory, and a sign of vigor. During the last quarter, additional experiments were conducted with marker genes (Table 1). The number of positive shoots recovered in some experiments is relatively low, but still acceptable for mature citrus transformation. Table 1. Mature citrus transformation experiments showing transformation efficiencies. Cultivar Date Batch Plasmid Explants Positive Transformation Shoots Efficiencies (%) Ham 19 6/11/13 36 p2301 240 1 0.8 Ham 20 7/2/13 31 p2301 300 2 2.2 Pine 8 6/11/13 35B p2301 240 1 0.8 Pine 9 7/9/13 40 pE121 260 3 4.5 Val 20 5/21/13 30 pE121 810 28 2.1 Ray 2 7/30/13 37X pE121 640 7 6.7 Book chapter from previous lab manager: Orbovic, Shankar, Peeples and Hubbard (in press) Citrus Transformation using Mature Tissue. Edited by Kan Wang, IN Agrobacterium Protocols Vol 2
This quarter work has been divided among between all of the proposal objectives. For the primary objective of transformation via CPPs, a new approach in the transient expression assays has been evaluated in order to enhance the transformation protocol. The new approach has been adapted from Chugh and Eudes, 2008. Instead of 3 days of transfection of citrus seedlings, testing has been done following one hour, two hours, or three hours following incubation of the seedlings with peptide and constructs. Early data indicate that one hour is insufficient for transient expression. As another option to improve upon this method, new and different peptides are being tested, including the classic ‘Tat’ peptide penetratin, and a chimeral peptide ‘R9-Tat’. These experiments did not have as much tissue as normal, since the ‘Pineapple’ sweet orange (40 of 80, 50%) and the ‘Duncan’ grapefruit (24 of 240, 10%) both had low germination rates. The next quarter should produce more plant tissue and results from these first experiments. In addition to these new transformation events, a second reporter assay has been run on the 36 ‘Carrizo’ plants. These plants did not report the GUS gene and are presumed negative. DNA from these plants have been purified and a PCR will confirm, but the plants are probably escapes. Finally, work has been done to create plasmid constructs for a silencing experiment. The citrus analog of NPR1 has been sequenced. Using this sequence, we hypothesize that two different constructs can lead to the same desired, silenced effect. The first construct is a full length gene inserted in reverse. The second is a dual vector, where the genes will hairpin due to their complementary nature. Currently, the second vector is half finished, as only one side has been confirmed, and the full-length plasmid needs to be tried again. Finishing these constructs and more transformation on the fresh seedlings will be the focus of next quarter.
The person hired to work on the project has not arrived and therefore no significant results can be reported.
The person hired to work on the project has not arrived. The genes to be constructed were designed and the DNA sequences were verified. Also, the sources of citrus plant materials have been identified.
The first task in this proposal is to find citrus version of the two proteins that make up the functional design of a chimeric antimicrobial protein previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have completed the discovery of the replacement of the first component the human neutrophil elastase (HNE) the surface binding component. Since HNE is a serine protease with elastasin as a substrate and whose crystal structure has been determined, we used 3D shape criteria and electrostatic properties to search the PDB data base for a plant protein with the identical active site structure. Focusing on a set of 288 non-redundant plant derived proteins from the entire PDB database, we used CLASP to search for a match using the electrostatic and structural features of amino acids that make up the active site. This was successful and the tomato PR14a was identified as an exact match. Using the tomato amino acid sequences we then searched for a similar citrus protein by searching citrus genome information in Phytosome (http://www.phytozome.net). This was successful and we have identified a single protein that is identical in the Citrus sinensis (Cs) and clementina (Cc) genomes. We are focusing on the 165 amino acid protein from Cs which we call CsP14a. We have analyzed this sequence and have determined that it is a secreted protein and contains a 25 amino acid signal sequence. We are focusing on the 137 aa mature protein and begun the construction of two genes that encode this protein. The first is a CaMV35S expression cassette that will be used to express just the mature form of the CsP14a protein, however, we have included a signal peptide (22aa) that we have used before and know works really well at secreting proteins to the plant apoplast and xylem. This signal peptide has been added at the N-terminal of this protein and we have added a Flag Tag also at the N-terminal so that the protein can be easily detected and purified. The Flag tag is part of the secreted protein after cleavage of the signal peptide. The second version is like our CAP protein and has Cecropin B (CecB) at the C-terminal linked via a flexible linker as we have described earlier. This protein is designated CsP14a-CecB. These genes will be used for in planta expression of the proteins CsP14a and CsP14a-CecB that will be isolated and used to test their efficacy against the bacteria as we described in our last report.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. A total of 3,805 transgenic plants have passed through inoculation process. A total of 76,160 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we have performed the designed microarray experiment to identify genes that are induced by NAD+ in citrus. The microarray data is currently under analysis. For objective 2, in last quarter we cloned two non-host resistance genes against citrus canker into the T-DNA vector pBI1.4T and mobilized the plasmids into Agrobacterium. In this quarter we have started genetic transformation of citrus ‘Duncan’ grapefruit with the Agrobacteria. Several putative transformants have been identified.
Certain citrus cultivars, such as Cleopatra mandarin, have been reported to be incapable of supporting the full developmental life cycles of psyllids. Preliminary experiments with hybrids in a Cleopatra-derived family, using caged psyllid nymphs on pesticide-free, field grown trees, indicated possible genetic transmission to some of the progeny. We will evaluate this effect, in replicated experiments conducted under controlled conditions. Identifying genetic control of the suppression of psyllid feeding and reproduction in host citrus plants potentially points to future strategies aimed at capitalizing on this phenomenon as another tool to mitigate HLB disease effects in integration with other genetic, cultural, and chemical control strategies. A total of 91 trees in three families produced by crossing Cleopatra mandarin with three male parents were selected from field plantings for the project and for future evaluations. Rootstock seedlings were produced previously, and these have been used to propagate replicates from each individual for future assessments of psyllid reproduction and feeding behavior in controlled greenhouse and laboratory conditions; these trees are being grown now using management approaches to have trees available this autumn for greenhouse and lab studies. Plans have been made for new caged psyllid experiments in the field, to confirm previous observations, and to provide baseline information on responses of psyllids to specific individuals from within the families.
This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment activities. The project’s funding came available in November 2012, but as this is based on the foundation of the long-term breeding program, activities have proceeded on a continuing basis. Individual assessments of HLB field tolerance have been carried out in the vast collection of raw germplasm that exists on UF and collaborating growers’ property, from fall 2012 through June 2013. Twelve individual rootstock trials planted in SW Florida, the Indian River region, and along the Ridge have been carefully observed for performance against HLB. In several cases these observations were made in a quantifiable fashion, measuring tree growth and estimating severity of symptom expression. In rootstock trials with early, midseason, and late season scions, we have collected data to document yields, fruit and juice quality, and fruit drop, and to correlate the disease rankings with yield performance; there are striking examples of very healthy, albeit infected trees, showing high yields of normal fruit, depending upon the rootstock. Rootstock seedlings of 100 accessions were previously grafted with HLB-positive Valencia budwood, and those growing out normal flush were selected and exposed to hot ACP populations in greenhouse conditions for one month. Under DPI permit, these trees were planted in a high-pressure, unsprayed grove on the east coast; we have continued to assess these trees. Newly produced rootstock hybrids have been entered into the same screen recently; removed seedling tops were rooted to provide seed source trees, after the infected scion budwood was grafted for testing. As the citrus industry has become aware of the unusual performance of assorted rootstocks from our breeding program that are planted in field trials affected by HLB, there has come a huge interest in propagation and planting of these trees on a large scale in the various growing areas in the state. We had already provided substantial quantities of seed from some of these outstanding HLB-tolerant candidates to a major grower for propagation of trees to be planted out in large blocks (up to one acre in size per rootstock). CRDF has approached us to produce a list of our top candidates for such plantings with several other growers through a process that is being developed in conjunction with the CRDF and their Commercial Product Delivery Committee. This list has been created and includes several tetraploid rootstocks of complex parentage, diploid new citranges, and sour orange types, all of which have shown excellent production with few symptoms even though affected by HLB. Seed trees exist for some, but not all of these selections; for those which saw their source trees burned a few years ago during canker eradication efforts, we have lifted roots to stimulate shoot development do we can recover these potentially valuable rootstocks for increase and production.
This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment activities. The project’s funding came available in November 2012, but as this is based on the foundation of the long-term breeding program, activities have proceeded on a continuing basis. Individual assessments of HLB field tolerance have been carried out in the vast collection of raw germplasm that exists on UF and collaborating growers’ property, from fall 2012 through early spring 2013, and this will continue through the coming months. Twelve individual rootstock trials planted in SW Florida, the Indian River region, and along the Ridge have been carefully observed for performance against HLB. In several cases these observations were made in a quantifiable fashion, measuring tree growth and estimating severity of symptom expression. In rootstock trials with earlier maturing scions (Hamlin orange, Sugar Belle mandarin hybrid, and grapefruit), we have collected data to document yields, fruit and juice quality, and fruit drop, and to correlate the disease rankings with yield performance; there are striking examples of very healthy, albeit infected trees, showing high yields of normal fruit, depending upon the rootstock. We have now completed yield and juice quality analysis in some rootstock trials that include Vernia and Valquarius sweet oranges, and plans are in place for performing the same data collection tasks in trials with Valencia scions. Rootstock seedlings of 100 accessions were previously grafted with HLB-positive Valencia budwood, and those growing out normal flush were selected and exposed to hot ACP populations in greenhouse conditions for one month. Under DPI permit, these trees were planted in a high-pressure, unsprayed grove on the east coast; nearly one year after planting, these trees were assessed and nearly 80% of them were found to be free of HLB symptoms. Newly produced rootstock hybrids have been prepared for the same screen in the coming season; removed seedling tops were rooted to provide seed source trees, after the infected scion budwood was grafted for testing. Seven rootstocks were entered into the DPI Parent Tree Program for cleanup via shoot-tip grafting, followed by indexing, to provide certified budwood to nurseries upon release. A new filed trial using sour orange type hybrids was planted near Lake Wales, and another rootstock trial with grapefruit scion was planted at the IRREC. More than 100 rootstock seed source trees were planted on UF properties. Four new sweet oranges were approved for release by the UF-IFAS Cultivar Release Committee, including one that appears to be less sensitive to HLB than other oranges.
HLB’s impacts have led to grower-directed interest in advanced production and harvesting systems with the potential for early and sustainable yield, ease of harvest and other management efficiencies. In the absence of a long-term HLB solution, grove life may be only 12-15 years. A different production approach is required, and higher density plantings with smaller trees managed with intensive cultural systems may be a solution. This project will identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Though funding became available 1 November 2012, the project was built on many years of previous efforts in rootstock development and field trial testing. Existing field trials were monitored for tree growth and disease incidence, including a portion of the St. Helena project planted with dwarfing selections, and a 40-acre Hamlin/Valencia cooperative [GFC] rootstock trial with trees planted between 300-500/acre. The latter planting is 3 years old, and yield data were collected on the Hamlin portion of the planting. Seed trees for selected dwarfing rootstocks, already showing good performance, are being propagated, to support expanded trials in the future. Four rootstocks selected for their potential in high density plantings through good tree size control were entered into the DPI Parent Tree Program for cleanup by shoot tip grafting followed by indexing, to provide certified budwood of these rootstocks for commercial nurseries upon their release. A new rootstock trial with Ray Ruby grapefruit and containing several tree size control candidates coming from crosses made using Flying Dragon as a seed parent (to capture the dwarfing trait, crosses must be made in this direction) was planted this spring, and the remaining selections for the trial have been grown off in our greenhouses to be planted in late summer 2013; more than 2000 trees will have been planted when this is finished. Seed trees from 5 selected tree size controlling rootstocks have been propagated and planted to provide a seed source for use upon their release, which will likely occur in the next three years.
HLB’s impacts have led to grower-directed interest in advanced production and harvesting systems with the potential for early and sustainable yield, ease of harvest and other management efficiencies. In the absence of a long-term HLB solution, grove life may be only 12-15 years. A different production approach is required, and higher density plantings with smaller trees managed with intensive cultural systems may be a solution. This project will identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Though funding became available 1 November 2012, the project was built on many years of previous efforts in rootstock development and field trial testing. Existing field trials were monitored for tree growth and disease incidence, including a portion of the St. Helena project planted with dwarfing selections, and a 40-acre Hamlin/Valencia cooperative [GFC] rootstock trial with trees planted between 300-500/acre. The latter planting is 3 years old, and yield data were collected on the Hamlin portion of the planting. The best yield was only ca. 0.5 boxes per tree across a whole bed planted to one rootstock, in this case C-35 citrange. Observations of severity and frequency of HLB symptoms were recorded on the dwarfing rootstocks at the St. Helena planting, prior to recording a third year of yield data in the coming months. Seed trees for selected dwarfing rootstocks, already showing good performance, are being propagated, to support expanded trials in the future. Four rootstocks selected for their potential in high density plantings through good tree size control were entered into the DPI Parent Tree Program for cleanup by shoot tip grafting followed by indexing, to provide certified budwood of these rootstocks for commercial nurseries upon their release. Two dwarfing rootstock hybrids have been budded with vigorous lemon scions to test their potential for tree size control using a very vigorous citrus scion type. A new rootstock trial with Ray Ruby grapefruit and containing several tree size control candidates coming from crosses made using Flying Dragon as a seed parent (to capture the dwarfing trait, crosses must be made in this direction) has very recently been planted.
A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified over 1,150 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a highly valuable pool of potential candidate genes from which to identify these true HLB tolerance genes can be identified. This project aims to screen these potential candidate genes using high throughput target capture, massively parallel sequencing of targeted gene regions, and genetic association and linkage analysis to find the most likely candidate gene(s) for HLB tolerance in Poncirus and rough lemon. Toward this goal, we will sequence the genome of rough lemon, and together with the genome sequence of Poncirus currently being produced, we will design a target capture system based on the Agilent SureSelect system. The custom-designed gene capture system will be used with Illumina HiSeq 2000 to rapidly identify sequence variations in the candidate genes in Poncirus and Citrus genotypes and to identify genes most likely associated with HLB tolerance. We had recruited a post-doctoral research associate, but this person decided to decline our offer, and currently we are trying to identify another qualified candidate. In the meantime, sequencing of Poncirus has been initiated and plans have been made to sequence the rough lemon genome, as well. Some of the plant materials to support the project are being collected and grown, to be prepared for downstream applications.
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