The overall goal of this project is to understand if a non-transgenic scion can be protected against HLB following budding to a transgenic SAR inducing rootstock. Progress has been made on all proposed objectives according to the time frame. This third quarter report of year 1 funding details progress made so far. All available transgenic lines expressing the AtNPR1 transgene have been evaluated in the laboratory for gene expression using qPCR. We have identified three distinct expression levels – low, medium and high. Selected lines that have a high level of NPR1 expression are also being evaluated by Western Blot using NPR1 specific antibody for confirmation. In some lines there is good corellation between NPR1 production in leaves vs roots while in others root NPR1 production is lower than leaves. Transgenic Kuharske and Swingle rootstock lines that highly express the AtNPR1 transgene have been clonally propagated and budded with non-transgenic Valencia scions. We detected enhanced levels of the PR1 gene following qPCR on the non-transgenic valencia scions. Additional lines are being clonally propagated for budding with non-transgenic Valencia in 2020. A subset of the existing trees is being side grafted with HLB infected budwood (Ct between 22.5 to 25.1) in the greenhouse while another set is being planted out in the field (Obj 1). A USDA-APHIS permit has been obtained for evaluation of these transgenic lines and the first set of trees will be planted in late 2019 (Obj 2). Three additional stacked gene constructs with NPR1 and the other listed genes in the project proposal have been produced and provided to the mature tissue laboratory for production of transgenic rootstocks expressing these transgenes (Obj 3). Trees are being produced with these genetic constructs. AtNPR1 overexpressing seed source trees budded onto US802 rootstock have been produced for field evaluation and will also be planted in late 2019 (Obj. 4). Additionally, transgenic NPR1 lines that have been observed to be tolerant to HLB under field conditions are also being propagated for use as interstocks in this study. In summary, several years of research has resulted in the develoment of this study and our progress thus far has been satisfactory.
We have proposed construction of several fusion genes and test them individually or in combination for their effects on improvement of transient expression or stable incorporation of T-DNA genes in citrus tissues. We have shown that the use of some genes we constructed can lead to 2- to 4-fold increases in transient expression activity of T-DNA genes or stable transformation efficiency using juvenile and mature citrus tissues as explants. However, our results are not always consistent in mature shoot tissues of Washington Navel and Valencia oranges. We believe that physiological conditions of explants may play a key role for the observed variations and inconsistence. We have also combined some of these genes and tested their effects. We did not see significant synergistic effects on transient expression or stable transformation efficiencies in mature citrus tissues of Washington Navel and Valencia oranges. We also used a Kn1 gene along with other genes in mature tissues of Washington Navel and Valencia oranges. Little synergistic effects in promoting transformation efficiencies have been observed. Some of the genes we identified may be of useful in improving transformation efficiencies of mature citrus tissues.
We have used root specific auxin biosynthetic gene, root specific-CKX fusion gene (cytokinin oxidase gene), a combination of both the root-specific iaaM and root-specific CKX genes, and other genes produce transgenic Carrizo citrange and HLB-tolerant rootstock US-897. We have produced a large number of independent transgenic citrus plant lines, respectively. Some of these plants have significant increases in root number and root length compared to the WT plants, consistent with our tobacco plant results. We have observed that adventitious root initiation of some of these transgenic citrus plants is faster and enhanced. Our results have also showed that the auxin overproducing plants have improved success rates of grafting. Enhanced root growth and biomass of rootstock may enhance citrus trees’ tolerance to abiotic and biotic stresses. Improved grafting success is desirable for commercial citrus planting because most citrus trees used for fruit production require grafting. We are currently using transgene free genome editing technologies to create the same traits in citrus rootstock.
We have tested a large number of chemical regulators on transient gene expression in mature citrus tissues. Among these chemicals and their combinations, we have identified that some can enhance Agrobacterium-mediated transient gene expression levels. Enhanced transient gene expression activity is important for producing non-transgenic gene-edited mutant citrus plants from mature explants. We have developed a method to produce non-transgenic genome edited mutants without going through sexual reproduction (Chen et al., 2018). We have also observed that that some chemical manipulations can promote shoot regeneration and stable transformation efficiencies of mature citrus tissues.
We have published two articles and two more are under preparation.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. Detached leaf assays, with CLas+ ACP feeding, have been conducted and lines with the most promising results have begun greenhouse and field studies. Greenhouse studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic.Data collection from the first round of field plantings with Mthionin transgenic Carrizo rootstock (45 plants) with non-transgenic rough lemon continues. Initial results show transgenics maintaining higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. A large second planting of Mthionin transgenics went into the ground in April, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) and WT/WT controls (16 plants) with data collection scheduled in October. Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) on transgenic rootstock are being made for follow up plantings. Mthionin construct transformations have also been completed on 450 Valencia and 300 Ray Ruby explants to provide additional transgenic material of these critical varieties. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted on lines representing chimera constructs TPK, PKT, CT-CII, scFv-InvA, scFv-TolC, TBL, BLT, LBP/’74’, `73′, and `188′. Detached leaf feeding assay protocols were adjusted to improve sensitivity and increase transmission rates (See section 4). Multiple lines from several constructs have been moved forward into greenhouse studies based on these results, as noted below.Initial ACP inoculations conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’) showed a statistically significant reduction (13x) in CLas titer for `74′ transgenics vs WT in the CLas+ plants. However, many plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All ACP inoculations for greenhouse experiments are now using an improved protocol. Through a combination of selecting smaller plants, more aggressively trimming larger plants and close observation, we have been able to extend the caged ACP infestation time from 7 days to 21 without severe mold or cage damage to the plants. We expect this longer infestation period to greatly improve inoculation rates. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using this improved protocol with the first samplings and data collection scheduled for 9.26.19. We are also emphasizing parallel field trials for all phenotyping efforts. Preparations are now being made for a field planting of ~400 `74′ and `188′ transgenics is scheduled for spring 2020. Completed so far are 196 grafts of WT scions (Hamlin, Valencia, and Ray Ruby) onto transgenic Carrizo root stocks. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT rootstocks are underway. All plants will be ready for the post Hurricane planting season.Seven new transformations, totaling over 3000 explants, have been completed to generate Valencia, Ray Ruby, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and, with all appropriate permitting now completed, will be moved to the field after hurricane season. An additional 129 rooted cuttings are propagated for follow up plantings.The 3 month data from the 150 plants from the 2nd group of scFv lines (12 lines) that were initially no-choice ACP inoculated showed an insufficient infection rate. These plants have now been graft inoculated with HLB+ RL and qPCR sampling is scheduled for November 2019. The 370 scFv rooted cuttings already propagated for a 3rd round of ACP-inoculations will use the higher pressure 21 day infestation protocol described above. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. The use of a phloem specific endogene would allow for samples to be normalized to phloem cells instead of total citrus cells, more accurately evaluating bacterial titer and potential therapeutic effects with the phloem limited CLas.The best performing lines of Mthionin, chimeras `74′ and `188′ and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. 3 lines of Hamlin Mthionin transgenics and 2 lines of Carrizo/Mthionin have been returned certified clean. Objective 5, Transgene Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and both His6 and Flag tagged variants of scFv constructs have been generated and confirmed for transgene expression by RT-qPCR. Experiments are underway using these plants to track the movement and distribution of transgene products in parallel to direct antibody based approaches.
Objective 1. Create hybrid rootstocks which combine germplasm from parental material with good rootstock traits and HLB tolerance, propagate the most promising of these hybrids, and establish replicated field trials with commercial scions.
Hybrid seed from 2018 crosses were grown, including SuperSour type parentage of unique HLB tolerant types, and 51 hybrids were selected for continuing work. Seed source trees were propagated for several new hybrids being prepared for release, and additional trees propagated as a source for cuttings. A study was completed evaluating seed propagation of some of the newest US rootstocks and presented at the FSHS meeting. Information was prepared and distributed to nurseries about accurately identifying many of the new citrus rootstocks.
Multiple budded trees of about 130 selected new rootstock hybrids were grown in the nursery, in preparation for field planting other new rootstock field trials later in 2019. These nursery trees included many of the most promising SuperSour hybrids identified in ongoing trials established in previous years, and several commercial standard rootstocks. These nursery trees also include other new and different hybrids chosen because of newly available information about parentage and characteristics best associated with outstanding traits. Three new field trials with sweet orange scion will be planted from these trees later in 2019, including one trial in the East coast region, one in the Central ridge region, and one in the Southwest region.
Objective 2. Collect field performance data from early-stage replicated rootstock field trials and release new rootstock cultivars as justified by superior performance in multiyear field trials.
Seventeen rootstock trials planted prior to summer 2018 (as described in the Proposal Appendix ii) were monitored and used for data collection on field performance, as appropriate during this quarter for the scion involved. During this quarter, data collection in established trials was focused on tree size and tree health. Plans were made to begin posting results from USDA rootstock field trials to the site www.citrusgreening.org, a webpage sponsored by USDA-NIFA, and that information will be updated at least once per year for each active trial. The first trial summary data for posting to this site has been assembled from a cooperative trial in the Ft. Meade area (Planted 2014, Valencia scion, 24 rootstocks, 7 replicates), and has been modified and provided in suitable format to the host Boyce Thompson Institute.
The three new USDA rootstocks released in November 2018, identified as SS1, SS2, and SS3, are being propagated in nurseries for numerous planned commercial plantings. Additional information is being collected from established trials and other experiments on these newest rootstocks. Made significant contributions to completion of 4th edition of the Florida Citrus Rootstock Selection Guide, the newest revision of the IFAS extension publication.
The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data In this quarter, we have focused on the following three activities: (1) Fourty-nine transgenic plants (30 ‘Hamlin’ sweet orange plants expressing transgene 1, 6 ‘Duncan’ grapefruit plants expressing transgene 1, 6 ‘Duncan’ grapefruit plants expressing transgene 2, 6 ‘Duncan’ grapefruit plants expressing transgene 3, and 1 ‘Duncan’ grapefruit expressing transgene 4) were planted on May 9th, 2019 in the field in Ft Pierce USDA ARS with the help from Dr. Ed Stover. Twenty health plants (10 ‘Hamlin’ plants, 10 grapefruit plants) were purchsed from Briteleaf Nursery and were randomly planted together with the trangenic plants. All plants currently grow vey well and will be analyzed. (2) Continue propagating CTV and CLas free transgenic plants. More budwoods that are negative for CTV and CLas have been grafed onto rootstocks. We will keep propagating plants whenever budwoods are available. The propageted progeny plants will be analyzed for transgene expression by Western blotting. (3) A major citrus defense gene was cloned. This gene is expected to provide resistance or tolerance to HLB when overexpressed in citrus. We plan to generate cisgenic citrus plants using this gene. The full-length cDNA was sequenced and cloned into a bacterial expression vector. We are expressing protein for antibody development. The antibody would be very useful for identifying cisgenic citrus plants that accumulate high levels of the defense gene product, which would help shroten the time-consuming screening process.
Citrus transformation Facility continued its operation during the second quarter of 2019. Within this period, we accepted seven orders. Six of the orders required production of transgenic Duncan grapefruit plants and one was for production of transgenic Mexican lime plants.Altogether, CTF produced 67 plants in this quarter. They belong to following orders: eight Duncan plants BB3, seven Duncan plants BB4, seven Duncan plants HGJ74, six Duncan plants ZM3, five Duncan plants ZM6, two Duncan plants ZM12, 11 Duncan plants JJ8, three Mexican lime plants LM2, five Mexican lime plants M2SF, three Pineapple orange plants AL2, nine Pomelo plants HGJ68, one Valencia plant BB4. A new employee was hired in April to work for the next six months on the CRDF-funded project lead by Jeff Jones. With the departure of one of the employees at the end of June, there are presently six members of staff in CTF.The fruit of Duncan grapefruit and Valencia oranges that we stored in the cold room at CREC, provide steady source of seeds. The other seeds were acquired from the Lyn Citrus nursery in California.
During the second quarter of 2019, we continued our horticultural assessments according to the objectives outlined in the proposal. In April 2019, we collected fruit samples from the Valencia trees at both the Lake Wales and the Basinger location. Because of the high cost, only fruit on all rootstocks that were fully replicated were analyzed for the following quality parameters: fruit weight, % juice, % solids, % acid, brix/acid ratio, and juice color. Fruit quality analyses were conducted at the CREC Processing Pilot Plant. Fruits were harvested in both locations shortly thereafter and yield data were collected from trees on all rootstocks, even if not fully replicated, as they may provide important information to the breeders.At the Basinger location, the average yield across all trees and rootstocks was 33 lbs. fruit/tree for Valencia and 13.6 lbs. fruit/tree for Hamlin. Hamlin yields were low because of citrus canker, which spread through this grove after hurricane Irma and caused many fruits to drop. Although adjacent to the Hamlins, Valencia fruit were not as affected. The average tree size at the Basinger location was 5.4 ft for Valencia and 5.6 ft for Hamlin. At the Lake Wales location, the average yield across all trees and rootstocks was 26 lbs. fruit/tree for both Valencia and Hamlin. The average tree size was 5.3 ft for Valencia and 5.6 ft for Hamlin. The largest trees across all trials were those on X-639 and C-54 with an average height of 6.7 ft and 6.3 ft, respectively. The highest yielding trees across all trials were those on C-22 and FA-517 with an average of 32-33 lbs. fruit/tree, followed by C-146, US-897, and UFR-5 with an average of 29-30 lbs. fruit/tree. Significant differences among trees on different rootstocks were also found for most fruit quality parameters (Valencia). The largest fruit were produced on trees with the Spanish rootstocks. The highest percentage of solids was imparted by FA-517 and several of the UF rootstocks including UFR-5 and UFR-6. The brix/acid ratio was highest in fruit on FA-517 and another Spanish rootstock.To conduct proper statistical analyses, we only included fully replicated rootstocks, which resulted in approximately 30 rootstocks depending on the scion and the trial location. Our analyses showed some statistically significant trends among rootstocks although results varied with scion and location. Overall, the most prominent differences were found for tree size and yield efficiency imparted by different rootstocks. For example, trees on some rootstocks (e.g. X-639) were large and exceptionally healthy looking but yielded few and lower-quality fruit (low yield efficiency). In contrast, trees on other rootstocks that were not remarkable in appearance yielded significantly more fruit than trees on X-639.
This new project is a continuation of work that was ongoing under CRDF 15-010; the official start date for the project was 1 February 2019. We report below on the activities in the first two months of the project by objectives. 1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Trees that were selected from the Gauntlet screen from 2018 crosses were stick grafted with CLas-infected Valencia budwood for further selection of tolerant types and are now under observation. Several crosses were made in spring 2019 for rootstock improvement at the diploid and tetraploid levels, using parents that have previously demonstrated good genetic combining ability, to generate new families for selection that combine tolerance of high pH calcareous soil and Phytophthora, with appreciable tolerance of HLB in grafted scions. Previous good parental combinations have included A+HBP x Sour Orange + Rangpur, among others at the tetraploid level. Recently we have used LB8-9 Sugar Belle as a seed parent with various pollen parents and have selected several strong survivors in the Gauntlet. We have repeated some of these crosses to increase the likelihood of finding superior performers. We are using DNA fingerprinting techniques to verify the origins of the Super-Root Mutants that have been selected from in vitro propagations in a commercial nursery. 2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. A large number of crosses were made in the spring 2019, to produce families for selection of individuals that may meet this objective and be valuable for commercialization following advanced testing, as well as to continue the process of parent development to improve future outcomes from breeding. The former will meet the needs of the industry in the near-term while the latter will lay the basis for a future of quantitatively enhanced tolerance of HLB. Over 25 crosses were made for grapefruit development, using at least one parent that has been shown to be highly tolerant of HLB and citrus canker; 10 crosses were made to create families of sweet orange like hybrids, including 2 using Parson Brown as pollen parent at the recommendation of some citrus growers; and 40 crosses for mandarin hybrid development and parent building. Some of the crosses for mandarin improvement may yield also sweet orange-like hybrids. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We evaluated the fruit quality attributes of several very tolerant types previously identified, but we have not carried out whole collection assessments of tree health vis a vis HLB in these two months.4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. We have completed anatomical studies of HLB-tolerant LB8-9 and Bearss lemon, and demonstrated that phloem regeneration is an obvious physical mechanism of their apparent tolerance.
Two students were successfully recruited to work on the project during the present reporting period, March 15 – June 15, 2019. Mr. Chad Vosburg has accepted an offer of admission as a M.S. degree student in the Penn State Department of Plant Pathology graduate program. He has experience working with HLB and is currently involved in HLB research as a technician. Chad will begin his studies in August, 2019 and will be working on the citrus infection and HLB disease measurement parts of the project. Mr. Jeremy Held, who was rotating in the PI’s lab starting in Februrary, 2019, has elected to stay in the PI’s lab as a Ph.D. student in the Intercollege Graduate Program in Plant Biology at Penn State. Jeremy will be working on the tree molecular characterization objectives of the project. Jeremy has already successfully detected the FT-scFv protein by protein gel immunoblotting and verified the pollen viability of trees producing the FT-scFv protein. Personnel to complete this project are now in place. The PI visited the lab of collaborator Dr. Tim Gottwald at the United States Horticultural Research Laboratory (USHRL) in Fort Pierce, Florida, on June 10 – 11, 2019. Chad Vosburg, who is from the nearby area, was also able to be present for part of these meetings. The current status of existing trees was determined, and a plan was developed by Drs. McNellis and Gottwald and Mr. Vosburg and Mr. Earl Taylor (Gottwald Lab research assistant) for tree propagations that were needed and setting up a series of HLB resistance tests. It was determined that at least 4 of the FT-scFv lines had sufficient numbers of trees available to set up a test immediately. It was determined that Chad would best work at USHRL as a “guest” to expedite his starting work on the project at the USHRL. We plan to initiate plant propagations and one HLB resistance experiment run during the next reporting period, prior to Chad’s formal beginning of studies. Chad will be able to participate in this work in part due to his close proximity to the USHRL this summer.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. Detached leaf assays, with CLas+ ACP feeding, have been conducted and lines with the most promising results have begun greenhouse studies. These studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic.Data collection from the first round of field plantings with Mthionin transgenic Carrizo (45 plants) rootstock with non-transgenic rough lemon continues. Initial results show transgenics maintaining higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. A large group of Mthionin plants went into the ground in April, including transgenic Carrizo with WT Hamlin scions (81 plants) and transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) with WT/WT controls (16 plants). Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) onto transgenic rootstock are being propagated for future plantings. Mthionin construct transformations have also been completed on 250 Valencia explants to provide sufficient events for this critical variety. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, were conducted on lines representing chimera constructs TPK, PKT, CT-CII, TBL, LBP/’74’, `73′, and `188′. Multiple lines from several constructs were moved forward into greenhouse studies based on these results as noted below. Definitive results for TPK, PKT, CII, and TBL were hindered by low inoculation rates. Assays for these constructs are being repeated to identify which lines of each are best suited for greenhouse studies. Detached leaf feeding assay protocols have also been adjusted to improve sensitivity (See section 4)No-choice caged ACP inoculation has been conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’). Early data from CLas+ plants showed a statistically significant reduction (13x) in CLas titer for transgenics vs WT in the CLas+ plants. However, many plants shown little to no amplification of CLas DNA at 3 and 6 months post inoculation. Amplification by this time would be expected from a successful inoculation, indicating low inoculation efficiency. All plants in this experiment will be re-inoculated by bud grafting with HLB+ rough lemon to allow for continued greenhouse studies. Moving forward, we will be emphasizing parallel field trials for phenotyping efforts and modify the ACP inoculation in greenhouse studies to increase CLas pressure. 475 rooted cuttings were previously made from Hamlin and Carrizo lines expressing constructs `74′ and `188′ and are now of a size appropriate for CLas exposure. 360 of these plants will be grafted with WT scions (for Carrizo lines) or rootstocks (For Hamlin lines) for field trials. The remaining 115 plants will be ACP inoculated for greenhouse studies, with the caged no-choice feeding time period extended from 7 days to 14.Seven new transformations, totaling over 2000 explants, have been completed to generate Valencia, Hamlin and Ray Ruby lines expressing constructs `74′, `188′, and TPK.Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best lines have been used as rootstock for WT Ray Ruby scions and will be moved to the field after necessary permit approvals. An additional 129 rooted cuttings are propagated for follow up plantings.Like the `74′ results discussed for objective 2, the 2nd round of ACP-inoculations of scFv plants (150 plants, 12 lines) had a poor infection rate. The plants are to be re-inoculated by budding with HLB+ rough lemon. The 370 scFv rooted cuttings already propagated for a 3rd round of ACP-inoculations will use the higher pressure 14 day feeding protocol described above. Objective 4, Screening Development and Validation: Details of the high throughput ACP homogenate assay, and its use for selecting lytic peptides for activity against CLas, has been submitted for publication and remains in use for early screening of therapeutics in the lab. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. The use of a phloem specific endogene would allow for samples to be normalized to phloem cells instead of total citrus cells, more accurately evaluating bacterial titer and potential therapeutic effects with the phloem limited CLas. Objective 5, Transgene Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) have been generated and confirmed for transgene expression by RT-qPCR. These plants will be used for generating data on the movement and distribution of transgene products in parallel to antibody based approaches.
The first steps in this project have taken place. As we will be using the very latest DNA sequencing technologies, and we are attempting to produce nearly full-length genome sequences, it is important to begin with the highest quality DNA preparation as starting material for library construction for sequencing. We have collected young leaves and young flush from the five cultivars selected for sequencing. This required multiple samplings because we were not always able to get the ideal tissue types; just slightly more mature vegetative tissue than the optimum did not give us the purity and the quantity of high-molecular weight (HMW) DNA required. We also optimized previous protocols we have used to be able to produce quantity and quality required. Finally, we have been successful and samples have been sent to UC Berkeley Genome Sequencing Laboratory for library construction.
In this quarter, the propagated transgenic lines have been budded up with non-transgenic HLB free Valencia budwood. It has been observed that the bud take frequency is higher on cutting made from juvenile transgenic lines when compared to the mature lines. Seed source trees, budded onto US802 rootstock have been produced for field evaluation and are being sized up. Transgenic NPR1 lines that have been observed to be tolerant to HLB under field conditions are also being propagated to use as interstocks in this study. ELISA protocols are being developed to rapidly test for transgene products in the fruit and juice.A USDA-APHIS permit has been applied for evaluation of these transgenic lines.
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 numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using CLas infected psyllids in a greenhouse environment. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A wide series of promoters driving a reporter gene are being tested in transformed citrus and show very different levels of expression with some being expressed in all tissues and some only in vascular tissue. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. 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 sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic tobacco and will be tested in citrus in next quarter. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) 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. The first trial has been in the field for 21 months and a repeated trial has been in the field for 9 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. These data will be analyzed over the next quarter.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. The first trees have been in place for more than eight months. 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. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. Alphascents 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. Unfortunately, this product had little effect on leaf miner. The decision has been made to apply Admire this fall to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF. These will be planted in the spring of 2011, and monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding.
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 numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and ready for exposure to HLB, using graft inoculations and CLas infected psyllids in a greenhouse environment. A detailed set of AMP activity assays will be initiated next quarter on a replicated set (8 plants of each) of 39 independent Hamlin transformants. Transformed rootstock varieties, with two AMPs (D4E1 and Pyrrhocoricin) and 170 transgenic plants, are being challenged using graft inoculations in two new replicated experiments. A series of promoters are being tested with the GUS gene to see how effective they are. As expected, the three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives consistent and very high GUS activity, even though the mRNA levels are similar to D35S promoter. use of this promoter may reduce the number of independent transformants needed. Liberibacter sequence data were used to target a transmembrane transporter,as a possible transgenic solution for HLB-resistance. Radioisotope permits are finally in place to assess effect of identified peptides on preventing ATP uptake in E. coli expressing the Liberibacter translocase. 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 sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences. Citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and will be tested in citrus scions soon. Genes for anthocyanin production are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) 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. The first trial has been in the field for 24 months and a repeated trial has been in the field for 12 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted.
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