In the first three months of this project, we started evaluating the performance of a ground penetrating radar (GPR) to map root architecture of HLB-infected citrus trees. The GPR can be used to map tree roots because it is relatively easy to use in the field and it is non-destructive to trees, roots, and the root-soil environment. Numerous tree root scans can be performed and replicated in a short time without interfering with root growth.Although the GPR has been used to detect roots for different plant species, most studies were conducted in controlled environments or in simulations to evaluate signal processing algorithms. Evaluation of the GPR for root detection in an agricultural field setting such as a citrus grove has not yet been conducted. Additionally, many questions remain to regarding the suitability of the technology for studying the impact of diseases on tree root structure and distribution.One objective of this study is to evaluate the performance of a ground penetrating radar to accurately detect citrus tree roots and generate 3D morphology root maps of citrus trees grown in a complex field environment in Florida. To achieve this goal, we are investigating the influence of several limiting factors on the performance of a GPR to accurately detect citrus roots and determine their main structural characteristics. First, single-factor experiments were conducted to evaluate GPR performance. Factors evaluated were: (i) GPR frequency (900 and1,600 MHz); (ii) root diameter; (iii) root moisture level; (iv) root depth; (v) root spacing; (vi) survey angle; and (vii) soil moisture level. Second, two multi-factor field experiments were conducted to evaluate the performance of the GPR in complex citrus grove environments in southwest Florida and to develop 3D morphology root maps. Experiments were conducted at the citrus research grove of the University of Florida Southwest Florida Research and Education Center (SWFREC) in Immokalee, FL, USA. A ground penetrating radar (GPR) (TRU Model, Tree Radar, Inc., USA) mounted to a mobile scanning cart and equipped with: (i) a 1,600 MHz antenna, and (ii) a 900 MHz antenna was utilized to generate a 3D map of the root system. The transmitter of the GPR antenna transmits electromagnetic waves (pulses), and the receiver collects the reflectance when an object is detected beneath the soil surface. The relative distance from the starting point was measured with a wheel recorder. A commercial software (TreeWin Roots and TRU Tree Radar Unit) was used to generate root morphology and root density maps. To present the root layout by location and depth, 3D images were created.Our initial experiments showed that the 1,600 MHz GPR was more accurate in detecting citrus roots and their location than the 900 MHz GPR. Upon target (root) detection, the GPR generated a hyperbola in the radar profile; from the width of the hyperbola the diameter of the root was successfully determined when roots were larger than 0.5 cm in diameter. The GPR also distinguished live from dead roots, which is indispensable for studying the effects of soil-borne and other diseases on the citrus tree root system.
This project produces transgenics, cis/intragenics & subgenics, in agronomically acceptable cultivars, for field testing & potential commercialization.The original proposal was for a 3 year funding period, but the project was only funded for 1 year because the CRDF wanted transgenics made by a company in Brazil. In 2016, after the CRDF realized that logististically, transgenics could not be easily made in Brazil, the mature citrus facility (MCF) was funded 2 more years. In total, 3 proposals were written for this project. Because of instability in funding, similar to what is presently occurring with short-term contracts, it has been difficult to keep good employees & maintain productivity. The significant objectives for the 1st & 2nd funding periods were: Mature plant production as a service using with Agrobacterium harboring vectors with disease resistance genes & molecular analyses to show copy number of the transgenes & gene expression; Plant propagation to form replicates for field testing; Increase micrografting efficiencies, bypass it altogether, or root mature scion; Test different selectable markers & reporters; Develop a biolistics protocol for immature/mature citrus; Introduce new, high yielding cultivars for tests in transformation; Apply for external funding. At the beginning of this funding cycle, new customers were charged a nominal fee for transgenics because previously our services were free. All of the abovementioned objectives have been addressed. Plant production for customers, for technology development, & for increasing efficiency produced ~437 transgenics for this 3 year period (~558 in total since 2014). An additional 400 transgenics were propagated for customers, either through budding or rooting. Mature scion cannot be rooted, micrografting cannot be bypassed, but micrografting efficiencies are stable at 77% by 1 operator. Molecular analyses were conducted for customers (~120 qPCR assays for 1 customer) & for publications, & thousands of endpoint PCRs for the gene of interest conducted. Several grants proposals were submitted & 2 small proposals were funded. Biolistic transformation was developed for immature & mature citrus, & an new selectable marker significantly increased efficiency. This was the first report of biolistic transformation of citrus with plant regeneration. This objective will become increasingly important considering new 2018 USDA APHIS guidelines in which APHIS will not oversee field tests for cis/intragenics/subgenics that do not carry plant pest or vector sequences, & these trees can be fast-tracked to growers at reduced expense, essentially similar to cultivars produced with traditional breeding. High yielding cultivars from the Plant Improvement Team were introduced & Agrobacterium & biolistics transformation efficiencies determined. There is one new scion cultivar that has an extermely high Agrobacterium transformation efficiency & another scion cultivar has reproduciby high efficiency. Most cultivars can be transformed using biolistics, although none have exceedingly high transformation efficiency yet. A cisgenic selectable marker, constructed by Drs. Zale & Dutt, is being tested in mature citrus to increase transformation efficiency. An intragenic citrus reporter from Dr. Dutt works well to replace GUS. A UF CREC Initiative during this timeframe was to stack two genes in transgenics to prevent the bacteria from overcoming resistance of one gene. An unfunded UF scientist, complied with the request to make the stacked gene constructs, & the MCF produced ~150 transgenics to meet this UF CREC Initiative. Biolistic-mediated gene editing of the PDS gene was achieved in immature Carrizo & Valencia. One customer’s Agrobacterium vector had a tendency to rearrange, in Agrobacterium prior to transformation. A total of 33 trees were produced for this scientist, but the transgenes rearranged in all but 2 events. This mutation was documented by restriction digests of vector DNA grown in E. coli vs Agrobacterium. A consecutive double budding method was devised so that mature citrus is reinvigorated well prior to experimentation. New services were added that offer biolistic transformation of minimal cis/intragenic expression cassettes. This service, if utilized by scientists, will provide significantly more monetary revenue for the mature citrus facility. However the CRDF should encourage scientists to use bioistics for cis/intragenics for faster & cheaper deregulation. Growth room maintenance is expensive & it is expensive to staff the MCF. A number of publications were generated by this project.
We requested a NCE for this grant to allow proper completion of a promising greenhouse citrus root growth study that was repeated with Carrizo rootstock. This is part of objective 2 (Determine soil conditions that favor root hair and VAM proliferation) and does not affect the subawards and efforts of Vashisth, Wright and Morgan. We don’t anticipate any travel or field work. Objective 1: Leaf nutrient thresholds We 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). The DRIS method has proved very valuable for indicating ranges of critical deficiency for K, Mn, Zn, Fe. Mg, B, and Cu. Some nutrients, including S, N, Ca and P did not correlate well enough using DRIS to pinpoint critical thresholds, but general trends were still useful. These results look promising for publication and ultimately will be used to revise existing IFAS thresholds for citrus. We continue to analyze the large survey dataset from every angle and with new analytical tools since it contains an abundance of important information. Objective 2: Determine soil conditions that favor root hair and VAM proliferation The results so far with Carrizo look much better than the original root study which was using rooted scion (Murcott) material. In order to get the required information from the trial, we captured more SEM images to quantify root hair growth (Dec 2018). Visually the root hair growth was dramatically increased by tricalcium phosphate in equilibrium with the growing solution. We will quantify root hairs from the images in early 2019. When psyllid adults become available in February, we plan to inoculate the hydroponic citrus trees with CLas so that the impacts of infection and nutrient solution treatment can be measured in root hair growth (until about May 2019). The final report will be completed in May/June.
During this reporting period, we have been tested the effectiveness of the gene that can reduce chimeras of mutant and wild-type plants of citrus using mature and juvenal tissues as explants. The gene can be useful when Agrobacterium-mediated transient expression of the Cas9 and sgRNA are used to produce transgene free edited plants. The results from the first set of testing experiment will be available in two weeks. We continue to repeat some previously observed results using the proposed genes and also chemicals we have identified. We have observed some shoots regenerated from calli derived from mature tissues of citrus. We are also using fresh young shoot tissues propagated from mature citrus shoots for regeneration and transformation studies. We have also started in-planta transformation of mature citrus shoots. We have worked with Mr. Phillip Rucks and Ms. Beth Lamb ( Phillip Rucks Citrus Nursery in Frostproof, FL) who have kindly provided us significant amounts of mature citrus shoots for our experiments so that we can work on development of an in-planta transformation method for mature citrus tissues. We have shown that we can use in-planta transform method to transform juvenile tissues of citrus.
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. One manuscript has been submitted. 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. This manuscript has been accepted for publication with revision by Phytopathology.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) We have started to treat the three independent transgenic lines ( Duncan 57-28, Hamlin 13-3, and Hamlin 13-29), which have gone through the long-term HLB test and exhibited robust tolerance to HLB disease. The first batch of plants, including two replicates of the transgenic line 57-28, three replicates of the line 13-3, and one replicate of the line 13-29, have been treated under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours) for two months. These plants have generated some new shoots during the treatment. We have tested if heat treatment is able to remove CTV and the CLas bacteria. So far the new shoots are negative for both CTV and CLas, indicating that the treatment is effective. The new shoots will be used for generating citrus trees for field trials. (2) We have screened 28 new transgenic lines against HLB-infected psyllids. These lines were generated by the mature transformation laboratory. The following lines still look great and haven’t shown any HLB symptoms: #82-6 Hamlin, #70-4 Hamlin, #26 Hamlin, #65 Hamlin, #82 Hamlin, #73-5 Hamlin, #11 Pineapple, #33 Pineapple, #73-5 Pineapple, #78 Pineapple. Based on the nymph production phenotype, these plants should have been infected by HLB. We have tested bacterial titers in these plants by qPCR, and indeed, the majority of these plants are CLas positive. The bacterium-free plants (with low CLas titers) will be inoculated again. (3) The eight new transgenic lines (A99, A100, A102, A101, A72, A73, A97, and A98) were irrigated and fertilized regularly. After they reach appropriate size, they will be screened against HLB-infected psyllids. (4) The manuscript titled Overexpression of the Arabidopsis NPR1 protein in citrus confers tolerance to Huanglongbing has been revised and published in the Journal of Citrus Pathology in this quarter.
During this reporting period, we have constructed a gene vector that may reduce chimeras of mutant and wild-type plants of citrus using mature and juvenal tissues as explants. The vector contains a Cas9 gene and a sgRNA gene to target on a citrus gene that may lead to an endogenous chemical resistance. The gene may be useful to reduce wt and mutant chimaera when Agrobacterium-mediated transient expression of the Cas9 and sgRNA are used to produce transgene-free edited plants. We will start testing its efficiency in mature tissues of Valencia and juvenile tissues of Carrizo. We have been continuing to repeat some previously observed results using the proposed genes such as AGO and NPRD genes that showed some positive effects of transformation efficiencies. We have also repeated the experiments for the effects of PAT and SMZ we have previously observed some positive effects on transformation of both juvenile and mature tissues of Valencia and Washington navel. Our goals are to generate sufficient and repeatable for the genes and chemicals we have used results for scientific publications and patent applications. We have been working on production of fresh young shoots from mature citrus tissues and using them directly for regeneration and transformation studies. We have observed that reasonable amount of shoots can be produced from callus tissues derived from mature tissues of Valencia. We observed more shoots produced from these callus tissues if chemicals such as NPA or 5-Aza were used. When using the young shoot tissues derived from nature tissues of Valencia as explants for the transformation experiments, in addition to the genes originally described in the proposal, we will test the effects of the Wus and BBM genes. Over-expression of both genes has been shown very effective in enhancing transformation efficiencies of a large number of plants that are difficult to be transformed. We are designing and constructing Wus and BBM genes for citrus transformation and regeneration.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. Major accomplishments per objective (1) Obtain mature tissues of the best transgenic lines: successfully achieved. The citrus flower-promoting gene FT3 was previously cloned into the CTV vector by the Dawson lab. The CTV-FT3 construct was introduced into Agrobacterium. Tobacco leaves were infiltrated with the resulting Agrobacterium. CTV-FT3 recombinant virions were purified from systemically infected tobacco leaves and bark flap inoculated into C. macrophylla seedlings, which began blooming in about five months. Buds from the matured C. macrophylla were grafted onto the original plants of all transgenic lines (EDS5-Dun-205-9, ELP3-Dun-207-8, ZMSN-Ham-73-1, ZMSN-Dun-137-2, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25). All plants began blooming in 6-18 months. We have successfully achieved this objective and also demonstrated that CTV-FT3 is efficient for converting juvenile tissues to mature tissues. The CTV-FT3-infected C. macrophylla plants that have bloomed can be used as bud source for promoting maturation. The three independent transgenic lines (NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25) that have shown robust tolerance to HLB have been treated under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours) to remove CTV and the CLas bacteria. The resulting clean germplasms will be used to generate trees for field trials. (2) Determine whether transgenics prevent psyllids from being infected: accomplished with negative results. CLas-infected psyllids can transfer the CLas bacteria to the next generation of psyllids by inoculating the flush area in which the nymphs develop, which allows the next generation of psyllids to continue spreading HLB without the need for another source plant. In our experiments, we noticed that several of the transgenic lines exhibit delayed or reduced levels of CLas after infection. We started to test if the delayed or reduced production of CLas is sufficient to prevent or reduce the infection of the progeny psyllids. CLas bacteria-carrying transgenic plants were placed in cages, and clean psyllids (not infected by CLas) were moved into the cages. The progenies of the psyllids were collected and tested for CLas titers. A total of six rounds of cage experiments with vegetatively propagated plants from the transgenic lines ELP3-Dun-207-8, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25 were conducted. Results showed that none of the transgenes was able to pre-vent psyllids from being infected by CLas. (3) Continue testing generations of vegetative propagation from the best transgenic lines: successfully achieved. Three independent transgenic lines, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25, have gone through at least six rounds of HLB inoculation. Three generations of progenies (18 replicates for NPR1-Ham-13-3, 31 replicates for NPR1-Ham-13-29, and 25 replicates for NPR1-Dun-57-25) were inoculated with CLas-infected psyllids. The inoculation was repeated until all plants were CLas positive based on qPCR. All progeny plants have shown no or minor HLB symptoms. The three transgenic lines are thus highly tolerant to HLB and will be put into the field for field trials.
Objective 1: Leaf nutrient thresholds The complete data from the quarterly field survey over three years in three citrus growing regions have been collated and checked. Final analyses are underway, including the Diagnosis and Recommendation Integrated System (DRIS) method for leaf nutrient analysis that provides protection from cross-correlation of variables and environmental effects. Objective 2: Determine soil conditions that favor root hair and VAM proliferation Starting in July, new seedling Carrizo plants were established for use. Six weeks after germination, 27 uniform seedlings were selected and divided into 3 hydroponic tanks (9 seedlings each). Tank 1, is a control fertilizer mix, including all nutrients and pH adjusted to approximately 7.0. Tank 2, is a reduced Phosphorus fertilizer, similar to Tank 1, except it has no soluble phosphorus and Triple Calcium Phosphate added as the sole source of phosphate. The pH of Tank 2 is approximately 7.2 (unadjusted). Tank 3, is the same as Tank 2, except we have added CaCO3 to the mixture to increase the pH (approximately 7.8) of the solution and reduce the amount of available phosphorus. Tank 3 is a replicate from the first series tanks using Murcott seedlings (successful root hair development). All 3 tanks receive an extra dose, 100 ppm, of calcium (calcium chloride). The added calcium and the pH at approximately 7, should drive the chemical reactions in solution towards the removal of available phosphorus, keeping the available phosphorus in the solution at the concentration that favors root hair development, while still providing enough phosphorus to keep the plants nutrition balanced. The Carrizo seedlings from all 3 tanks are healthy, showing no visual signs of nutrient deficiency, and with visible differences in root hair development. Observing with light microscopy, Tanks 1 and 2 show significant development of root hairs; while Tank 3 has some root hairs, they are in much fewer numbers. We are currently assessing the numbers of root hairs using Scanning Electron Microscopy (SEM). Several roots from each tank, along with roots from untreated Carrizo seedlings, have been selected, and prepared for viewing with the SEM. Currently we are taking images and counting root hairs to asses whether there are significant differences. The density of root hairs on several of the roots is so high, SEM has been difficult. Tanks 1 and 2 had sample prep issues relating to the sputter coating stage and needed to be resampled. Those results should be available soon.
For the Third Quarter of 2018, Project #16-007 conducted or completed the following activities: 1) Revised Plot Plans and “Ground-Truthed” all entries for the three grapefruit plantings (July,2017; Nov. 2017; and May 2018), of the “Scott Seedling Variants” and will submit field maps to both the CRDF Project Manager, and Grower Cooperator (Daniel Scott) after cooperator “Walk-Through” 2) Summarized Breeding Line Entry List for each of the above plantings and prepared those lists/maps for both CRDF Project Manger and Grower Cooperator, after cooperator “Walk-Through”. 3) The USHRL farm crew continues to maintain the grapefruit seedling planted previously on the Picos Farm. The three relevant plantings include: a) Pre-CRDF/NIFA Proposal Planting: Block 2, Rows 48 (planted Feb., 2015), and Rows 50-54 (planted July, 2017); b) “Planting 1A: Block 2, Rows 9-15 planted in Nov., 2017; and c) “Planting 1B: Block 4, Rows 27-32 planted in May, 2018. 4) Inventoried greenhouse materials that belong to Grower Cooperator including the number of rootstocks on which they were budded. As of October 1, 696 of the 750 trees have been budded or grafted onto 3 rootstocks; 5) More rootstock trees have been acquired to complete the budding and grafting operation for the planting on the Scott Grove; 6) Revised budget of Project #16-007, and submitted it to CRDF; 7) Project advisor meet with both RMC and CRDF BOD to answer any questions relative to project 16-007, and provide a verbal report of progress.
Our transgenic efforts have evolved greatly in the three years of this project. As data accumulated and new ideas came to the forefront, efforts were focused on those aspects of the research proving most valuable, while several of the initial objectives were deemphasized. A new project was approved by CRDF (18-022) that is the next series of steps following up on the successes of project 15-026. To accelerate screening for CLas-killing transgenics, a detached leaf inoculation method was developed via CLas+ ACP no-choice infestation. This high throughput lab based method can test plants at small seedling stage, is non-destructive, and provides guiding information on assessment 6-12 month earlier than greenhouse based tests. To evaluate AMPs for potential use in CLas-killing transgenes, we have developed an in vitro procedure for directly measuring their ability to disrupt CLas cells. A homogeneous CLas suspension is recovered by macerating CLas+ ACP in a specially developed extraction buffer and removing cellular debris through spin filter centrifugation. Homogenates are exposed to AMPs vs controls for 4 hours. CLas cell integrity is determined by use of a photo-reactive DNA binding dye. Small plant destructive inoculation assays, where all plant tissues are weighed and sampled after no-choice CLas+ ACP feeding, now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This method is being used in our transgenic efforts to validate the detached leaf assay results. A modified plant Thionin (Mthionin), was designed by G. Gupta using biophysical modeling. Transgenic expression of this peptide in Carrizo citrange showed a marked and highly statistically significant decrease in symptoms when challenged with Xanthomonas citri, the causal agent of citrus canker. When transgenic plants were challenged through graft inoculation with HLB infected material, both transgenic Carrizo tissues and non-transgenic scion (Rough Lemon) tissue showed reduced CLas titer up to 12 month (latest time-point) post graft when compared to control plants, with root CLas titer 1800 times greater in wild-type Carrizo. We are testing Mthionin transgenics in the field. Several of our best lines are at DPI for cleanup and broader field trialing, ideally head-to-head with transgenics from other programs Newer generations of AMPs, categorized as 2nd and 3rd generation, were designed (also by G. Gupta) using citrus native thionin as the foundation combined with other citrus genes with high affinity for CLas membranes. Numerous lines and events of 2nd and 3rd gen AMP transgenic citrus have been subject to the detached leaf assessments. Some showed promising CLas clearance; indicated by transgenic Carrizo leaves showing significantly lower CLas titer compared to wild type controls after a 7-day ACP infestation. Interestingly, bacterial quantity in the ACP bodies was also lower after feeding on the transgenic leaves, suggesting an uptake of AMPs into the insect body and disruption of gut bacterial cells. Several of our best lines are also at DPI for cleanup and broader field trialing. ScFv sequences targeting CLas outer membrane proteins were developed by J. Hartung and used for the creation of transgenes disrupting the CLas infection process. Transgenic plants are showing a consistent and statistically significant decrease in Clas titer twelve months after no choice CLas+ ACP inoculation (up to 250x reduction, measured by qPCR) and have a much higher incidence of plants with no measurable bacterial DNA amplification. Approximately 120 additional rooted cuttings were propagated for field trials, with the primary focus being rootstocks to protect conventional scions. We sought (with W. Belknap and J. Thomson) to identify highly expressed genes in the citrus phloem, reasoning that their promoters might be useful for transgenics combatting HLB. Through this work, a citrus gene family was identified and characterized encoding a group of Small Cyclic Amphipathic Peptides (SCAmpPs) with highly conserved gene structure, but considerable variation in the ultimate gene products. Variants of a tissue-specific SCAmpP promoter were tested using GUS as a reporter gene and resulted in excellent phloem-specific expression: 500x greater expression in leaf midribs/petioles compared to laminar area and visibly greater GUS gene product activity in midribs and vascular tissue compared to GUS driven by D35S. These citrus promoters are being used in all new transgenics from our program, usually with a parallel set of transgenics driven by D35S, to combat gram-negative pathogens in other citrus tissues.
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. 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. One manuscript has been submitted. We are conducting further experiments and revising the manuscript per reviewers’ suggestions. 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. During this study, we have developed a method for targeted early detection of Las before HLB symptom development. This manuscript has been submitted to Phytopathology.
In this reporting period, we have confirmed that “in-planta” regeneration of juvenile tissues from non-meristem tissues under non-sterile conditions can be achieved at reasonably high efficiencies. This method is very different from the tissue culture-based method under sterile conditions. Our preliminary results have also showed that such a method can be used to produce CRISPR-mediated mutants of citrus. However, the mutant citrus plants produced were chimeric and the efficiency was low. We are developing methods to reduce the chimeras and to enhance the efficiencies. More importantly, we are testing whether the “in planta” method can be used to regenerate shoots from mature citrus trees under greenhouse conditions. If that can be done with reasonably high efficiencies, such a non-tissue culture method may provide a useful tool to produce transgenic shoots from mature tissues of citrus or to produce CRISPR mutant plants of citrus. On the sterile tissue culture side, we have been working on repeating some previously observed results using our proposed genes and also chemicals we have identified. Our goals are to generate sufficient and repeatable results for publications and patent applications.
This project was based on the idea that blocking the function of the NodT outer membrane transporter of ‘Candidatus Liberibacter asiaticus’ (CLas) would block pathogenicity or survival of the bacterium within citrus plants. Single-chain, mini-antibodies (scFvs) recognizing a peptide corresponding to the major, predicted extracellular loop of CLas were isolated. The scFv with the strongest binding in a qualitative assay was selected and fused to the C-terminal end of the citrus Flowering Locus T (FT) protein as a gene fusion, encoding an FT-scFv protein. The antibody was fused to FT in order to promoter stability, mobility, and expression of the protein in the phloem. The FT-scFv coding region was placed under the control of the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter and introduced into ‘Duncan’ grapefruit (Citrus paradisi) using Agrobacterium-mediated transformation. Fifteen (15) independent transgenic lines were obtained, most of them expressing high levels of the FT-scFv protein, as determined by protein gel immunoblot analysis. Eight lines are maintained in Florida at the United States Horticultural Laboratory (USHRL) and seven lines are maintained at Penn State. Many of the FT-scFv lines have a precocious blooming phenotype, which could be useful for accelerated citrus breeding purposes. Prior attempts to overproduce FT in citrus have encountered problems with lack of plant survival, while FT-scFv plants survive and can produce fruit. All lines have been propagated vegetatively, and they continue to express FT-scFv after propagation. The HLB resistance or tolerance phenotype of the FT-scFv lines has not yet been tested, however. Graft-transmission of the FT-scFv protein has also not yet been tested. However, the materials need to accomplish these last two goals have been produced and this represents an opportunity for future analysis.
This project was based on the idea that blocking the function of the NodT outer membrane transporter of ‘Candidatus Liberibacter asiaticus’ (CLas) would block pathogenicity or survival of the bacterium within citrus plants. Single-chain, mini-antibodies (scFvs) recognizing a peptide corresponding to the major, predicted extracellular loop of CLas were isolated. The scFv with the strongest binding in a qualitative assay was selected and fused to the C-terminal end of the citrus Flowering Locus T (FT) protein as a gene fusion, encoding an FT-scFv protein. The antibody was fused to FT in order to promoter stability, mobility, and expression of the protein in the phloem. The FT-scFv coding region was placed under the control of the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter and introduced into ‘Duncan’ grapefruit (Citrus paradisi) using Agrobacterium-mediated transformation. Fifteen (15) independent transgenic lines were obtained, most of them expressing high levels of the FT-scFv protein, as determined by protein gel immunoblot analysis. Eight lines are maintained in Florida at the United States Horticultural Laboratory (USHRL) and seven lines are maintained at Penn State. Many of the FT-scFv lines have a precocious blooming phenotype, which could be useful for accelerated citrus breeding purposes. Prior attempts to overproduce FT in citrus have encountered problems with lack of plant survival, while FT-scFv plants survive and can produce fruit. All lines have been propagated vegetatively, and they continue to express FT-scFv after propagation. The HLB resistance or tolerance phenotype of the FT-scFv lines has not yet been tested, however. Graft-transmission of the FT-scFv protein has also not yet been tested. However, the materials need to accomplish these last two goals have been produced and this represents an opportunity for future analysis.
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