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 two activities:(1) Continue treating HLB-tolerant transgenic plants under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours). New shoots that have emerged from the previously treated trees have been tested by ELISA for CTV and by qPCR for CLas. Results have shown that both CTV and CLas do not exist in the new shoots. We have started to graft the budwoods from the new shoots onto rootstocks to generate CTV and CLas free plants, but the number is small. We are waiting for more budwoods. (2) About 50 transgenic progeny plants that have been tested for HLB tolernace were tested by ELISA for CTV and by qPCR for CLas. Results showed that all of these plants are CTV negative and CLas postive. The majority of these plants have no HLB symptoms and three plants show mild HLB symptoms. We will plant these trees into the field in May. The field has been prepared with the help from Dr. Ed Stover in Ft Pierce USDA ARS.
The goal of this project is to generate green fluorescence protein (GFP) labeled Ca. Liberibacter asiaticus (Las), test its application in study of Las movement and distribution in planta, and investigate the control effect of different measurements including heat treatment and antimicrobial treatment. Las and other HLB-associating Liberibacters have not been cultured outside of their hosts in cell-free artificial culture media; therefore, traditional molecular and genetic analyses cannot be applied. This has greatly hampered our efforts to understand the virulence mechanisms of Las. We have been looking for alternative approaches to genetically manipulate Las in vivo. This has been made possible by the large population of Las in psyllid and availability of molecular tools to perform genetic manipulation in vivo. Alternatively, Las can survive for a short time in the media after acquired from psyllid gut and we aim to genetically modify Las with GFP immediately after Las being acquired from psyllids. To achieve the goal of this study, we will pursue the following specific objectives:1) GFP labeling of Candidatus Liberibacter asiaticus. 2) Elucidation of plant-Las interaction through real-time monitoring of Las movement and multiplication in planta using GFP labeled Las. 3) Investigate the effect of different control approaches on the dynamic population of Las in planta using GFP labeled Las. Previously, the reporter plasmid, pBAM1::R-PgyrA-GFP, composed of Tn5 and narrow host-range origin was constructed and therefore the GFP gene can be inserted into the genome of bacteria. However, it was only successfully transferred into a genome of Pseudomonas fluorescence with low transformation efficiency and failed with other bacteria including Escherichia coli DH5a, Sinorhizobium meliloti Rm1021, and Liberibacter crescens BT-1. Recently, pDH3::PgyrA-GFP was constructed which has a wide bacterial host range replicon, repW, but cannot be inserted into a genome. Transformation of E. coli by PEG mediated method with pDH3::PgyrA-GFP showed high transformation efficiency (~2 x 104 CFU/ g of DNA) than with previous reporter plasmid (failed). Following application with L. crescens BT-1 by electroporation was also successful (1.9 x 103 CFU/ g of DNA). Transformants and the GFP expression in L. crescens BT-1 were confirmed by PCR and fluorescent microscopic analysis, respectively. As L. crescens is a phylogenetically closest species to Ca. L. asiaticus, there is a possibility that pDH3::PgyrA-GFP would be useful for GFP labeling of Ca. L. asiaticus. We have further confirmed the Lcr-GFP using western blot. The GFP plasmid is being used to transform Las. To facilitate Las transformation, we have tested multiple novel methods of culturing. Las population was observed to decrease at the beginning, and increase slowly. Repeated experiments show similar pattern which suggest we might be able to acquire enough Las cells for transformation after further optimization. We are testing new methods for culturing Las. Especially, we are testing co-culturing Las with citrus tissue culture and psyllid tissue culture. Currently, we are in the process of establishing a pure psyllid cell culture. We have used two approaches to label L. crescens. Preliminary data showed one approach works for Las in vitro. We are testing whether we can label Las in vivo and observe its movement. 2) We have conducted Las movement and multiplication in planta based on qPCR method. We have tested approaches to prevent Las movement in planta. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript entitled: “Control of Citrus Huanglongbing via Trunk Injection of Plant Defense Activators and Antibiotics” has been published by Phytopathology. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. A manuscript entitled Targeted Early Detection of Citrus Huanglongbing Causal Agent ‘Candidatus Liberibacter asiaticus’ Before Symptom Expression has been published by Phytopathology. We determined the in planta minimum inhibitory concentration of oxytetracycline against Candidatus Liberibacter asiaticus effective for control of citrus Huanglongbing which is being submitted to Phytopathology.
Objective 1: Leaf nutrient thresholds for HLB+ treesWe implemented the full Diagnosis and Recommendation Integrated System (DRIS) method for leaf nutrient analysis that provides protection from cross-correlation of variables and environmental effects. Reference nutrient data for DRIS was obtained from high-yielding ‘Hamlin’ trees growing in the Ft. Meade area prior to HLB (>700 boxes/acre average). Critical nutrient thresholds from HLB+ trees in this survey study were higher than published values for K, Cu, B; slightly higher for Mn, Zn {Note: these are not yet recommendations}. We obtained additional leaf data from collaborating grower’s commercial grove databases in order to expand our new critical nutrient thresholds to include N, P, S, and Ca. The data that we collected during this project survey did not include enough values in the deficient range for those nutrients, which prevented us from establishing critical nutrient thresholds.The results of this research were presented at grower events: Citrus Nutrition Day, Bartow; Citrus School, Arcadia; Citrus Institute, Avon Park, and published in the April 2019 Citrus Industry magazine. Objective 2: Determine soil conditions that favor root hair and VAM proliferationBased on good results with healthy Carrizo rootstock trees grown hydroponically, we set up a final experiment where infected psyllids were allowed to inoculate some replications of the experiment with CLas. This treatment serves to monitor the impacts and interaction of HLB and nutrient solution treatment, as measured in root hair growth (until about May 2019). The final report will be completed in June. A paper on this research will be presented at the FSHS conference in June.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ending in March 2019) with some added treatments to be evaluated. The added treatments are:1. CRF + Tiger Micronutrients+ Mn 50%2. CRF + Tiger Micronutrients+ Zn 50%3. CRF + Tiger Micronutrients+ Fe 50%4. CRF + Tiger Micronutrients+ B 50%5. CRF + Tiger Micronutrients+ Mn +Zn 20%6. CRF + Tiger Micronutrients+ Mn +Fe 20%7. CRF + Tiger Micronutrients+ Zn +Fe 20%8. CRF + Tiger Micronutrients+ Zn +B 20%9. CRF + Tiger Micronutrients+ Fe + B 20%10. CRF + Tiger Micronutrients+ Mn +Zn 50%11. CRF + Tiger Micronutrients+ Mn +Fe 50%12. CRF + Tiger Micronutrients+ Zn +Fe 50%13. CRF + Tiger Micronutrients+ Zn +B 50%14. CRF + Tiger Micronutrients+ Fe + B 50%The treatment for objective 3: 1.CRF + Foliar Micronutrients + Tiger 90; 2.CRF + Tiger Micronutrients These treatments will be initiated one the block that was being used for hybrid fertilizer trial (comparison of different rate of controlled nitrogen). This treatment willend with harvest in spring 2019. In time duration, December-February, we have surveyed the site, ‘valencia’ on swingle between 11-15 year, set up an experimental layout for the newly added treatments, collected pre-treatment data on tree growth, leaf and soil nutrient analysis. The fertiizer application will be made in spring 2019. after harvest of the existing trial. We have also solicited fertilizer donation from Harrell’s and TIger Sul.
A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. In the last quarter, the most signficant advances have been: 1) A replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 2) Initiation of an experiment on pollen flow from transgenic trees. Data on flowering overlap was collected on transgenic US-802 and non-transgenic FF-5-51-2. The FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. When seed mature in the FF-5-51-2, thousands will be collected and tested for nptII, a transgene in the US-802. Controlled crosses were also made of US-802 pollen onto 36 flowers of FF-5-51-2. This will demonstrate cross-compatibility and provide seed to validate testing protocols. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. 3) Annual BRS inspection of the planting site was conducted. Previously established at the site: 1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit. 2) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection. 3) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer 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. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project. 4) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant. 5) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.). 6) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 7) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo. 9) Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site. New transgenics from Zhonglin Mou of UF, Tim McNellis of PSU will be planted in the next quarter. 10) Availability of the test site for planting continues to be announced to researchers.
Project rationale and focus:The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Most recent quarter:The 35 day federal government shutdown, and the threat of a possible shutdown on Feb 15, directly disrupted our ability to initiate and conduct experiments using the CLas+ ACP colonies. In addition, considerable rehibilitation of colonies and supporting plants was necessary due to the minimal care that could be provided during the shutdown. Only 2400 CLas+ ACP were used for experiments in this quarter and were used for detached leaf assessments of plants expressing three different transgenic constructs. We anticipate a normal demand in the current quarter. Previously achieved in this project: As of December 21, 2018, a total of 14,111 plants had passed through the inoculation process. A total of 361,255 psyllids from colonies of CLas-infected ACP had been used in inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations were many used to refine inoculation procedures, which provided insight into the success of our inoculation methods and strategies for increasing success. After inoculations, plants were returned to the breeders and subsequently subjected to further inoculations when they are transplanted to the field. In addition to inoculating germplasm, infected psyllids were supplied to other researchers for other purposes. This side of the project grew over time, and detailed records were not maintained on how many were given out until 2018. More than 10,000 infected psyllids were supplied to the research community for an array of experiments during 2018. Recipients included researchers with USDA in Fort Pierce, Ithaca and Beltsville, UF in Gainesville, Cornell in Ithaca, University of California, and University of Nevada.
The objectives of this study are to identify optimal pH range for root function and minimize root turnover on HLB-affected rootstocks and how uneven pH levels in the root zone (e.g. irrigated vs. row middle portions of root system) affect the overall health of the tree. This is being done in a split root system in the greenhouse where pH of different parts of the root system can be controlled an maintained. We are in the final stages of rhizotron construction to build enough for the experiments. Rhizotron construction was slightly delayed because of the late Valencia harvest this year for other projects combined with an unexpected loss of a staff member that will soon be replaced. The Masters student has assisted a member of Tripti Vashisth’s lab with the 2nd repetition of the experiment that created the foundation of this project to become familiar with techniques that will be important for maintaining pH and collecting data. We expect to initiate treatments before the end of May.
All transgenic rootstocks (Swingle and Carrizo) expressing the AtNPR1 gene, developed using mature tissue transformation and juvenile transformation have been evaluated using qPCR. Several of the promising lines have also been evaluated for the trans-protein production using western blotting techniques. Cuttings of several lines have been propagated and budded with non-transgenic ‘Valencia’ sweet orange. Many others are currently being propagated in the mist bed. To produce seed source trees, trees are being produced by budding onto US802 rootstock. These will be planted in a USDA-APHIS approved field site when ready. Transgenic rootstocks expressing other potent plant derived transgene(s) are also being produced using the mature tissue transformation method.
Citrus Transformation Facility (CTF) continued its operation without interruptions. In the first three months of 2019, CTF received nine orders. Five of these orders were for production of transgenic Duncan grapefruit plants and four for production of transgenic Valencia plants. Within this period, CTF produced 77 transgenic citrus plants. Out of this number, 42 were Duncan grapefruit plants, 19 Mex lime plants, 11 Pomelo plants, four Kumquat plants, and one Pineapple sweet orange plant. These plants are result of work on 15 different orders.In February, CTF purchased one bin of Duncan grapefruits and stored them in the cold room for the supply of seeds. For experiments requiring Valencia seedlings, we are picking Valencia fruit from the trees on the CREC property to get seeds. The seeds of other cultivars are obtained through purchase from Lyn Citrus nursery in California or by picking fruit from DPI Arboretum in Winter Haven.One of the employees with the lowest FTE has additionally decreased her attendance and will leave the lab at the end of June.
We continue to produce Agrobacterium-mediated mature transgenics for customers, test different approaches for increasing efficiency, & develop biolistics as a tool in citrus. During this quarter, 11 mature transgenics were produced with Agrobacterium & have been micrografted, & a remaining 42 positive shoots were produced using Agrobacterium & must still be micrografted once/if they get larger. These two genetic constructs had GFP & were transformed into Hamlin (one of our better cultivars for transformation). An inordinate number of transgenics were produced, which is very unusal in mature citrus & must have been the result of cultivar x genetic construct interaction. The GFP reporter makes identification of transgenics easy. A new initiative from the CREC Director is to produce mature citrus transgenics with stacked disease resistance genes using biolistics for faster deregulation. We have been able to produce transgenics for Dr. Mou using a promising, new disease resistance gene & the genetic construct did not rearrange in mature citrus. (Unfortunately it rearranged for Dr. Orbovic in immature citrus). We used a RecA- Agro strain because we suspected there might be a problem. In a couple of transgenics, the gene of interest (GOI) was lost, but the majority of transgenics have the transgene. This is very similar to the deletion of the GOI from Dr. McNellis’ genetic construct using the mature citrus transformation protocol. During that instance, Dr. Orbovic was able to produce immature transgenics that had the GOI, while it rearranged in mature transformation, even using a RecA- Agro strain. One advantage of biolistics over Agro transformation is that the GOI does not rearrange as easily as in Agro. A manuscript was accepted & revised for In Vitro Cellular & Developmental Biology-Plant. This manuscript showed that improved selection with phosphomannose isomerase after biolistics in immature citrus drastically increased transformation efficiency after bombardment (a mean of ~2 transgenics per paired shot). We are now testing phosphomannose isomerase (PMI) as a new selectable marker in mature citrus. Once the optimal mannose/sucrose concentrations have been determined in mature citrus, we will test PMI selection after Agrobacterium-mediated transformation. We are testing a new protocol for precipitating DNA onto gold particles for biolistics that increased efficiency in other species. This protocol will also be useful for gene editing using biolistic transformation. We must buy a new soil sterilizer & a new ultra low -80C freezer. Both are essential to this program. An external review of the two transformation labs was conducted March 28 & 29 & presumably there will be a report issued at some time in the future.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. We have tested the transgenic Duncan grapefruit containing the Bs3-executor transgene for resistance to an array of strains representing a worldwide collection. All of the strains were shown to elicit a hypersensitive reaction when infiltrated with a bacterial suspension of Xanthomona citri into young leaves of the transgenic citrus plant but a susceptible reaction when infiltrated into non-transgenic susceptible Duncan grapefruit leaves. We are continuing further experiments to confirm the resistance in the greenhouse. Furthermore, we will quantify bacterial populations in transgenic and susceptible trees to demonstrate if the transgene is activated without the TAL effector that is predicted to activate this gene. We are also confirming activation of the transgene using a realtime protocol to determine if the transgene is activated properly. During the past three months we have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contained an additional selectable marker that allowed for identifying putative transgenics with a higher success rate. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct was recently sent to Dr. Vladimir Orbovic, who is in the process of transforming grapefruit and sweet orange. We hope to have additional transgenics later this year. We have also grafted our lone transgenic plant onto two rootstocks (812 and Sour Orange) and are in the process of planting in the field at Fort Pierce in collaboration with Dr. Ed Stover. They will be going into the field within the next couple of weeks. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic that contain the Bs3-executor transgene. One of the putatve transgenics has a growth defect and in all likelihood will be of no use in future experiments. The other putative transgenic tree which appears to contain the gene may be useful for future testing. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and are in the process of planting in the field at Fort Pierce in collaboration Dr. Ed Stover. They will be going into the field within the next couple of weeks. We have additional transgenics from plants received from Dr. Vladimir Orbovic that need to be tested for the presence of EFR. Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying bs5. Currently the trees are being grown and then will be grafted onto rootstocks for further testing.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is continuing apace. 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 grafted Hamlin on Carrizo with all combinations of WT and transgenic.The first field plantings with Mthionin transgenic Carrizo (45 plants) have been made with leaves of non-transgenic rough lemon on transgenics showing higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. Plants for follow up field plantings of transgenic Hamlin on WT Carrizo (112 plants), WT Hamlin on transgenic Carrizo (84 plants), WT Ray Ruby on transgenic Carrizo (118 plants) and WT Valencia on transgenic Carrizo (118 plants) with WT controls are being propagated.Seeds for additional scion variety transformations have been collected and germinated. Shoots will be developed enough to yield epicotyl tissue for Mthionin construct transformations in 2 (Hamlin), 4 (Ray Ruby) and 6 (Valencia) weeks. 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’). Three month data has been collected, while many plants are yet to show CLas DNA amplification, there is a statistically significant reduction (13x) in CLas titer for transgenics vs WT in the CLas+ plants. An additional 475 rooted cuttings have been propagated from chimera constructs (6 lines of `188′, 7 lines of `74′ and 12 lines of `73′) for the next round of ACP inoculation trials. 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 at the conclusion of the study. The best lines have been used as rootstock for WT Ray Ruby scions and will be moved to the field once the graft union is strong enough. An additional 129 rooted cuttings are propagated for additional grafts and field plantings. ACP inoculations were conducted on 150 more plants from 12 scFv lines. Data from 3 months post inoculation has been collected, but too few plants are testing positive at this time for a conclusive analysis. An additional 370 rooted cuttings have been propagated from the remaining scFv constructs/lines to be tested and will soon be mature enough for ACP inoculation. 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.
Three field trials were initiated in two orchards near Frostproof FL. Both had 15-month-old Valencia trees on Kuharski rootstock planted at 10×20 foot spacing. Trees exhibit variable growth and health and are infested by sting nematode. The first trial to determine the effect of perennial peanut in middles with or without oxamyl in rows was designed as a randomized complete block within four rows. Treatments comprise 4 adjacent trees replicated 4 times (complete factorial arrangement of untreated, peanut, oxamyl, peanut + oxamyl). We purchased the peanut as sod that was installed on 5 March and is being watered by hand 3 times weekly until established. The second trial compares the efficacy against sting nematode of oxamyl, aldicarb, fluensulfone, fluopyram, fluazaindolizine, and an experimental compound (Syngenta) in a randomized complete block design with eight, four-tree replicates. All plots were plumbed across 16 rows to be treated by injecting the materials into a single, central manifold feeding the appropriate microjets. Two products per day are being applied with the irrigation cylcle and the first treatments of all products will be completed this week. The third trial is in a nearby orchard in which sunn hemp has been sown in tree middles in such a way that eight, four-tree replicates will be arranged in a randomized complete block design testing effects on sting nematode and tree health of the complete factorial treatments of untreated, oxamyl treated, sunn hemp treated, and oxamyl + sunn hemp. Sunn hemp was planted on 7 March. Trunk girth of all experimental trees in all trials was measured on 6-7 March.
The research agreement contract was finalized in January, 2019, and funds became available in February, 2019. Dr. McNellis initiated a search for a graduate student to perform the work through the Department of Plant Pathology and Environmental Microbiology graduate program and the Plant Biology graduate program at Penn State. A suitable candidate was interviewed in February, 2019, and an offer of admission was made in March, 2019. A rotation student in the Intercollege Program in Plant Biology at Penn State also expressed interest in the project and began a rotation in Dr. McNellis’ lab on March 5, 2019, which will continue for at least the remainder of the spring semester. It is likely that one or both these students may become available to work on the project. The grapefruit trees expressing the FT-scFv anti-HLB antibody protein were continuously maintained at Penn State and at the USHRL in Ft. Pierce, FL, and continue to grow normally and are ready for analysis.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars. This is the 1st year, 1st quarter progress report; our grant started December 1, 2018. In the last three months, we have processed all the paperwork needed to establish the grant and begin spending funds at ARS. We have identified a qualified and interested post-doctoral researcher, Dr. Tamara D. Collum, who we will be hiring with grant funding. However, all ARS hiring actions, even those using soft funds, are currently on hold at the Department of Agriculture level. Now that the department has a full-year budget, we hope this hold will be lifted shortly so we can bring Tami on board in the next couple weeks. Objective 1 (development of transgenic constructs) is close to completion and work has begun in the Stover lab on objective 2 (production of transgenic citrus lines). For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) inserts have been assembled and sent to Dr. Dawson’s lab for inclusion in CTV vectors and subsequent introduction into citrus.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
