In this reporting period, we confirmed that overexpression of the P19and H2A gene or repression of the AGO2 and NRPD1 genes could improve Agrobacterium-mediated transformation / gene expression in juvenile citrus tissues but the increases were no higher than 3-fold. We had been trying to repeat the experiments to determine their effects on the mature citrus tissues but due to limited mature plant tissues in winter months, we have not yet produced any meaningful results. However, we did observe that the use of an antimicrobial and an anti-oxidation compound worked reasonably well to reduce contamination and tissue browning problems. We started in planta regeneration of juvenile tissues under non-sterile conditions. We observed that young shoots could be developed from decapitated hypocotyls at a reasonably high efficiency. The shoots produced were not derived from any preexisting meristem tissues because all meristem cells were completely removed. We also tested and confirmed the effects of several chemical regulators on both transient expression and stable transformation efficiencies.
Two Las repressors from Objective 1, and a Wolbachia repressor from Objective 2 were all confirmed as functional transcriptional regulators of Las phage genes and with DNA binding sites within key Las phage promoter regions. These three repressors are therefore potential chemical targets for inhibitors that may control HLB. In addition, a likely Las virulence effector, a secreted peroxiredoxin enzyme, was identified in Objective 3. This enzyme appears to prevent citrus host phloem cells from killing Las and also blocks systemic host responses to Las. This secreted enzyme is also a high value potential chemical target. A high throughput fluorimetric thermal denaturation screen was first used to identify chemicals that bound to the Las peroxiredoxin target. A total of 320 phytochemicals were screened, resulting in the identification of fourteen (14) lead candidates for phytochemical control of HLB. Several of the lead candidates are generally recognized as safe (GRAS) and are not pharmaceutical drugs. The larger library of 1,600 chemicals, including drugs, was then further screened using a direct enzymatic activity inhibition assay to independently verify the results of the fluorimetric assay and also potentially identify additional inhibitors that directly affect the secreted Las peroxiredoxin. Peroxiredoxins react with hydrogen peroxide and both aliphatic and aromatic hydroperoxide substrates. Of 1,600 chemicals screened, 28 exhibited a strong inhibitory effect on the Las peroxiredoxin. Based on possible commercial value as being both GRAS and relatively inexpensive, 7 chemicals were selected for further study. Three of the 7 chemicals were confirmed repeatedly as having a strong inhibitory effect. One of these was confirmed inhibitory by both fluorimetry and direct enzyme inhibition assays. These three chemicals are currently being evaluated for potential direct bactericidal and phtotoxicity effects and for capacity to cure Las infected periwinkle and citrus. Despite repeated attempts, the fluorimetric assay proved unusable for chemical library screening of the three repressor proteins. All three repressors are very small DNA binding proteins with little protential to form folded structures, which is necessary for the thermal denaturation assay. Alternative approaches involving interfering with DNA binding of these represors are currently underway.
Major accomplishments: 1. The frequency of streptomycin resistance in Liberibacter crescens was determined in the lab. One in 500 million cells are spontaneously resistant to streptomycin. 2. The mutations responsible for spontaneous streptomycin resistance in Liberibacter crescens were identified. They are all present in one of two codons in the rpsL gene, as is typically the case for Gram-negative bacteria. 3. A high-throughput method has been developed to rapidly assess the extent of streptomycin in the field. This was done by simply adapting our 16S rRNA sequencing method to another gene, the rpsL gene. We are now working diligently to develop the database needed to analyze the data. This approach will be used to assess the level of resistance in the CLas and non-target bacteria in the field. 4. No spontaneous mutants of L. cresens resistant to oxytetracycline were recovered despite several attempts with billions of cells. We now know that the frequency of spontaneous resistance to oxytetracycline is less than one cell in 10 billion. This is because no single mutation can provide resistance to oxytetracycline and that L. crescens and CLas lack any gene that could confer oxytetracycline resistance. Such genes are often transferred from one bacterium to another through a process called lateral gene transfer. However, the restricted habitats of CLas, the Asian citrus psyllid and citrus phloem, do not provide little opportunity to acquire tetracycline resistance genes from other bacteria. 5. The spontaneous streptomycin-resistant mutants of L. crescens are not resistant to oxytetracyline. 6. In addition to the discovery of the loci involved in spontaneous streptomycin resistance in L. crescens, whole genome sequencing of the streptomycin resistant mutants also showed that a streptomycin resistant mutant has increased methylation of an efflux protein often implicated in antibiotic resistance. Also, the methylation pattern of L. crescens changes with streptomycin treatment itself. We don’t yet know the implications of these results for the control of HLB. 7. Progress was made toward the culturing of the citrus greening pathogen, Ca. Liberibacter asiaticus (CLas). Using the closest cultured relative of CLas, L. crescens, we discovered Liberibacter prefers citrate as major carbon and energy source. The optimal level of citrate required to culture L. crescens was very similar to the amount of citrate found in citrus phloem and the haemolymph of the Asian citrus psyllid. This discovery suggests a simple, inexpensive, and unregulated means to control HLB by foliar phosphate fertilization. This is described in the discussion section of our recent paper (Cruz-Munoz M, Petrone JR, Cohn AR, Munoz-Beristain A, Killiny N, Drew JC, Triplett EW, Development of chemically defined media reveals citrate as preferred carbon source for Liberibacter growth. Frontiers in Microbiology, 2018; 9:668). In summary, foliar phosphate fertilization is expected to dramatically reduce citrate levels in phloem, thereby starving the pathogen. Plants load citrate in phloem in response to P deficiency. We are pursuing this idea now and seeking external support to test it in the field.
1) Assessed use of isolated leaf inoculation and small plant destructive sampling: Isolated leaf inoculations with ACP do not readily distinguish between resistant and susceptible citrus selections, but prove useful in assessing CLas-killing transgenics. Within a week, such assays have shown marked reductions in CLas in leaves and in ACP. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. 67 plants representing 13 independent events and wild types (4-5 replicates each) were transferred into larger pots and are getting ready for field planting and resistance evaluation. The remaining plants were used for bud-inoculation tests as transgenic root stock and/or scion. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. Another 805 rooted cuttings were created at the end of 2017 from 18 events for future experimentations. Tissue specific constructs of the very promising Mthionin gene have been developed. The phloem-specific variant has been transformed into Carrizo and is currently being transformed into other citrus types including Hamlin and Ray Ruby. The root specific variant is being transformed only into Carrizo. 3) Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 4 below) have been propagated and are in replicated testing. New chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A total of 35 lines of Carrizo with citrus thionin V2-LBP construct, and 20 lines with citrus thionin V1-LBP construct have been generated. A total of 18 independent Carrizo lines, each expressing citrus thionin-EDS and citrus thionin D2A21 chimeras respectively, were produced with confirmation of high level transgene expressions. Using the detached leaf ACP-inoculation assay, it was shown than transgenic Carrizo expressing citrus thionin V1-LBP chimera have significantly less CLas titer after 1 week of ACP feeding than the wild type controls. His-6 affinity tagged variants of citrus thionin-BPI/LBP expressing constructs have been created with C-terminal and N-terminal thionin orientations. These constructs have been transformed into benthamiana for efficient in plantae production and purification of protein for use in detached tissue assays with multiple lines for each construct confirmed as transgenic and currently undergoing analysis for expression levels. 4) Antibodies (ScFv) to the CLas invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. Two representative constructs, one targeting each gene, have been challenged by CLas + ACP. At six and nine months post inoculation, transgenic plants are showing consistent and significant decreases in bacterial titer (300x) as measured by qPCR and a much higher incidence of plants with no measurable bacterial DNA amplification. Additional plants representing 21 independent events from all 7 constructs have been replicated as rooted cuttings for ACP challenge of whole plants. A second round of ACP inoculations has been conducted on 150 plants replicated from twelve independent transformation events representing three different ScFv constructs. Additional lines will be inoculated once sufficient mature transgenic material becomes available. 5) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were previously shown to be downregulated in more tolerant Jackson compared to susceptible Marsh grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in Hamlin , Clementine , Carrizo , rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs targeting citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so far) are ongoing. Carrizo plants carrying these constructs with multiple events each were transferred into larger pots to stimulate growth in early 2018 and subsequent propagations 6) Budwood from our best performing Mthionin transgenics and citrus gene chimeras have been sent to DPI for cleanup and then broad field testing.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Transgenic Duncan grapefruit and sweet orange lines carrying either EFR alone or EFR plus an XA21-EFR chimera were tested for canker resistance in the greenhouse. The two most promising Duncan grapefruit lines carrying EFR were selected for further testing in the field in collaboration with Dr. Ed Stover at the USDA ARS. Some new Duncan grapefruit transformants carrying EFR, XA21, or both genes have been produced at the Core Citrus Transformation Facility at UF Lake Alfred, and any that survive will be analyzed for canker resistance. No transformants were obtained for CSPR, and this construct has been abandoned. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement Our gene editing target is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we chose to mutate the native citrus Bs5 alleles while simultaneously introducing the effective resistance allele as a transgene, rather than to attempt precise gene editing. Two editing constructs were created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. The constructs were transformed into Carrizo citrange, and transformants are currently being analyzed at UC Berkeley. To date, we have identified one plant with mutations knocking out both alleles of the native Bs5 gene and several other candidate plants that may also have a loss of function of both alleles. More plants remain to be analyzed. Any plants confirmed to have biallelic loss of function mutations in Bs5 plus the introduced bs5 resistance allele will be propagated and shipped to Dr. Jeff Jones’ lab at UF Gainesville for canker testing.
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. 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.
The driving force for this project (Hall-15-016) 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 breeders at USDA-ARS-USHRL, Fort Pierce Florida continue producing germplasm that needs to be evaluated. The more rapidly germplasm can be evaluated, the sooner breeders can identify HLB-tolerant germplasm for the Florida citrus industry. The purpose of this project is to support a high-throughput facility to evaluate citrus germplasm for HLB resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. The original inoculation program called for individual plants to be caged with 20 infected psyllids for a two-week infestation, and then housed for six months in a greenhouse with an open infestation of infected psyllids. As indicated below, the open infestation step was abandoned. After the caged inoculation step, plants are moved into a psyllid-free greenhouse and evaluated for growth, HLB symptoms and CLas titer, and finally the plants are transplanted to the field where evaluations of resistance continue and additional inoculations by field psyllids occurs. CRDF funds for the inoculation program 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 technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected citron plants. Update: As of March 15, 2018, a total of 11,888 plants have passed through inoculation process. A total of 326,295 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. We have abandoned the greenhouse open-infestation step because of continual problems mainly with invasive pests such as thrips, scales and parasitoids. Research indicated that the no-choice inoculation step should usually average around 74% effective and gets plants back to the breeders faster. The plants are subjected to further inoculations in the field. The no-choice inoculation procedure was evaluated monthly for 12 months, and success in getting seedlings infected was evaluated six months after each monthly infestation. The results indicated a 74% average success rate in getting seedlings infected when flush (immature leaves) was present, with success ranging from 40 to 100%. Success was significantly related to how many ACP on a seedling tested positive for CLas, thus greater success rates would be expected using more than 20 ACP per seedling. Based on the results of the research, an infestation shorter than two weeks would be as effective, which would be advantageous for guarding against excessive ACP damage to seedlings if more than 20 per seedling were used. It remains possible that modifications to the no-choice inoculation procedure would increase success rates and reduce variability, for example 25 to 30 ACP per seedling for a 1 week period, perhaps with larger seedlings in larger cages.
In November and Early December, all sites across all three regions were visited and sampled. Analysis is in progress of the latest soil samples. This data is being compiled and organized for analysis within the neural network software. The merging and comparing of the collected data is still in progress. No new data has been released since the last report since all of the data from the November sampling is still being analyzed, organized and complied. Objective 1: Leaf nutrient thresholds Samples from the November sampling are still in the process of being analyzed. The November sampling included leaf sampling for ImageJ analysis, and nutrition, as well as tree canopy measurements, SPAD, canopy height and volume. All three locations were sampled around the same time in either late November or Early December adding further to our tri-area data set from all sites across all locations. These data will be added to our comprehensive database for analysis using the neural network software Easy-NN. We will look at the November sampling date as a snapshot in time across all sites and locations for any possible connection or correlation with HLB severity. Objective 2: Determine soil conditions that favor root hair and VAM proliferation i. Soil sampling was completed in November and we will have a large set of samples for further soil analysis that we continue to work on. We have completed the measurements of permanent wilting point on the first two years of soil data. Soils from the South Florida area will be included into the data set in November and will be measured for all of the variables the other two regions have been measured for, including organic matter content, and color analysis. ii. We have selected the liquid nutrient solution over the fog hydroponic system for the seedling study using Murcott seedlings. Currently we have 3 tanks running with 9 Murcott seedlings each. We are utilizing three nutrient solutions, 1. Complete fertilizer, 2. Complete fertilizer minus Phosphorus, with Rock Phosphate (RP) as the source of Phosphorus, and 3. Complete fertilizer minus Phosphorus, with Triple Calcium Phosphate (TCP) as the source of Phosphorus. Our next step in this study is to analyze the roots for any root hair development, and make minor changes to the phosphorus/calcium amounts to accelerate root hair development.
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) Analyzed another batch of rootstock transgenic lines (54 Carrizo lines, 7 Swingle lines, and 3 C-MAC lines). Two more Swingle lines and three C-MAC lines accumulated high levels of transgene products. More than half of the Carrizo lines accumulated high levels of transgene products. These rootstock transgenic lines will be analyzed based on the protein levels. (2) Conducted another cage experiment for replicates of the transgenic lines with low numbers of psyllid progenies. (3) Made more replicates of the transgenic lines tolerant to HLB.
During the reporting period, we conducted additional experiments to determine effects of the two genes that displayed promotional effects on citrus transformation efficiency. Using RNAi constructs, we demonstrated 2-4 fold increases in Agrobacterium-mediated stable transformation in juvenile explants or a 2-3 fold increase in mature Washington Navel tissues. We also observed that two chemicals added to media increased stable transformation efficiencies by 3-fold. We further showed that three chemicals enhanced transient expression of T-DNA genes, which can be important for using CRISPR to create non-transgenic mutants of citrus. We produced additional independent transgenic citrus plants using the root specific iaaM gene described in the proposal. We also conducted two experiments to compare efficiencies of Agrobacterium- and biolistic-mediated transient expression in citrus tissues. The experiment may provide a basis what method should be preferred for using CRISPR to produce non-transgenic mutants 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. 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. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript has been accepted for publication by Phytopathology.
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. 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. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript has been accepted for publication by Phytopathology.
In Objectives 1 and 2, we proposed targeting specific regulators of key phage encoded virulence genes (such as the Las LexA-like repressor, LC1, a second downstream repressor, LC2, controlled in part by LC1), and a key exogenous regulator of the (lethal) phage lytic cycle encoded by Wolbachia, an important psyllid endosymbiont that is always found when Las is present. These results have so far resulted in three full length manuscripts and numerous. LC1, LC2 and the Wolbachia repressor have all been confirmed to be transcriptional repressors. All three are therefore prime targets for chemical interference. The Wolbachia protein has been shipped to both the De La Fuente lab in Auburn, and Duan lab at USDA-Ft. Pierce, our collaborators on a separate culturing project. In an attempt to duplicate a method previously used to identify a drug (tolefemic acid) that interfered with binding of a Las regulatory protein (PrbB) (Gardner et al., 2016), purified Wolbachia, C2 and C1 repressor proteins were subject to thermal denaturation analyses using two different florescent markers: SYPRO Orange and Nile Red. Commercially obtained citrate synthase was used as the control. The assays were performed using 5, 20 and 40uM purified protein in 14 different buffers. However, except for the citrate synthase control, none of the target Las repressor proteins yielded thermal shifts amenable for high throughput chemical screens, despite multiple attempts under differing conditions. A newly developed alternative to thermal denaturation screens that we are now evaluating is Protein Induced Fluorescence Enhancement (PIFE). This is also a high throughput method to identify chemicals that interfere with DNA binding proteins. The 3 regulatory protein targets were previously demonstrated to bind specific promoter DNAs. In the PIFE method, the promoter DNAs are labeled by covalently attaching the fluorescent tag Cy3 to one end and biotin at the other. The biotin tag is used to anchor the DNA to 96 well microtiter plates, and the bound DNA fluoresces due to the Cy3 tag. The unlabeled target protein is then applied, and binding to the target promoter should result in a decrease in fluorescence. Binding of an interfering chemical to the target protein can result in a change in fluorescence (usually, an increase) in fluorescence. These experiments are well underway and should be completed in a few months. We have identified a new secreted Las virulence factor that is likely critical for Las pathogenicity in citrus: peroxiredoxin CLIBASIA_00485. Expression of this gene occurs only in the citrus host and is nearly undetectable in psyllids. This pattern of expression is similar to the phage SC2 peroxidase. CLIBASIA_00485 and a second, non-secreted peroxidase, CLIBASIA_00980, were both functionally confirmed as active in the cultured Las proxy, L. crescens (Lcr). Lcr cells expressing CLIBASIA_00980 driven by the lacZ promoter were 65-fold more resistant to hydrogen peroxide. Lcr carries genes with ca. 50% predicted protein similarity to CLIBASIA_00980 and CLIBASIA_00485. Importantly, Lcr with CLIBASIA_00485 were 36-fold more resistant to hydrogen peroxide but a striking 214-fold more resistant to tert-butyl hydroperoxide (tBOOH, an organic peroxide). Transient over-expression of CLIBASIA_00485 in tobacco suppressed (a) H2O2-induced activation of RbohB, the key gatekeeper of plant defense signaling cascade, and (b) tBOOH-induced lipid peroxidation of plant cell membranes. Suppression of lipid peroxidation prevents biosynthesis of antimicrobial oxylipins, and subsequent transcriptional activation of plant defense genes. This likely essential pathogenicity enzyme is also being evaluated as a potential control target.
During the period of July, August, and September, 2017, Dr. McNellis submitted a full proposal to the USDA Citrus Specialty Crop program to expand and continue the existing project funded by CRDF, and this full grant proposal was submitted to the USDA on August 18, 2017. This project would be a collaboration between Dr. McNellis and Drs. Ozgur Batuman, Liliana Cano, and Rhuanito Ferrarezi. They have the expertise to do CLas infections and PCR-based quantification of CLas in plant tissues. In addition, Dr. McNellis has been working with Dr. Catherine Hatcher of the CRDF on a pre-proposal for the field testing and greenhouse testing for HLB resistance of the trees produced through this project, in anticipation that the current project will be concluded on December 31, 2017. Dr. McNellis’ lab also continued quantitative analysis of anti-HLB antibody protein expression in the transgenic lines (the FT-scFv fusion protein) by western blotting. It will be the objective and intention of the renewal project to test HLB resistance or tolerance status of ungrafted and grafted trees with the transgenic genotype.
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) Several propagations were made for the Swingle and Carrizo lines accumulating high levels of the transgenic protein. We noticed the Carrizo line that accumulates the highest level of the transgenic protein does not grow well, indicating that two much of the transgenic protein affects citrus growth and development. This provides a threshold for the transgenic protein. (2) The HLB-tolerant transgenic lines were further propagated. (3) Four TAIL-PCR experiments have been conducted with both right and left T-DNA border sequences to clone the gene suppressing psyllid reproduction on citrus plants. Unfortunately, not dominant PCR bands were obtained. The presence of the T-DNA insertion was confirmed using the kanamycin gene primers. We are determining the truncation sites of the T-DNA insertion and will clone the insertion site. (4) Cage experiment for replicates of the transgenic lines with low numbers of psyllid progenies is ongoing. More replicates will be generated.
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