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.
This quarter the South Florida sites were sampled and added to the Indian River and Ridge sites for ImageJ analysis. 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 August sampling is still being organized and compiled. Hurricane Irma made landfall in South Florida on September 10, 2017 as a Category 3 storm on the Saffir-Simpson Hurricane Wind Scale. We are still gathering data from all of our sites and do not think that this storm will impair our ability to continue with the study. The sites in South Florida and Indian River region, are flatwood sites, and all experienced flooding of some kind, with a few still flooded weeks after the event. This will influence the health of these trees in the future. This study should offer a unique perspective on HLB status/severity and tree recovery after a hurricane/flooding event. More will be known about all of these locations when we visit all of the sites in November. Objective 1: Leaf nutrient thresholds Samples from the August sampling are still in the process of being analyzed. The August sampling included leaf sampling for ImageJ analysis, qPCR, leaf starch content, and nutrition, as well as tree canopy measurements, SPAD, canopy height and volume. All three locations were sampled around the same time in August and we should finally have one complete data set from all sites across all locations. This data will be added to our very large database for analysis using the neural network software Easy-NN. We will look at the August 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 will be completed in November and we will have a large set of samples for further soil analysis that we would like to work on. We have completed the measurements of permanent wilting coefficient 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 variables the other two regions have been measured for, including organic matter content, and color analysis. ii. We are investigating a new system for root hair development using Valencia and Murcott seedlings in a nutrient solution. Test runs of seedlings exposed to nutrient fog and hydroponics is underway. Currently a preliminary trial of nutrient solution concentrations is underway for feasibility. Both the nutrient fog and the liquid hydroponics systems are currently working well. In the next quarter, we will choose a method an expand the greenhouse study to include the 3 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. Currently in the complete fertilizer trial run, we do see the presence of microscopic root hairs, which we hope to promote using the RP and TCP fertilizer solutions.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics survived and passed a PCR screen, and these are currently being tested for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and therefore the putative transformants are being screened by RT-PCR. Eleven PCR-positive shoots have been grafted so far. 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 The initial target for gene editing 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 are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis, with several rooted plants obtained so far. Molecular characterization of the putative transformants will be carried out at UC Berkeley. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
1) Assessed use of isolated leaf inoculation, and small plant destructive sampling: Isolated leaf inoculations do not readily distinguish between resistant and susceptible citrus selections, but may prove useful in identifying nearly immune material. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This assay seems to be an efficient way to test transgenics that are expected to kill CLas. Recently we have had delays due to failures in ACP-inoculation and have reinitiated several challenges. 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. Rooted cutting of 167 Carrizo plants were obtained. A subset of 67 plants representing 13 independent events and wild types (4-5 replicates each) were inoculated by ACP infestation. All of the plants except 2 were confirmed CLas positive after a 2-week ACP exposure, and the titer between wild type and transgenic groups are similar at two weeks. The plants are maintained in the greenhouse for tests at 3, 9 and 12 months after inoculation. Transgenics expressing AMP D2A21 suppressed canker but not HLB with manuscript accepted for publication. Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 5 below) have also been propagated for testing. 3) Two new chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A group of 100 Carrizo plants were obtained as rooted cuttings and will be used for HLB testing. 4) A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets citrus chloroplasts. Transcription data were obtained by RNA-Seq showing significant alteration in the transgenics. Publication submitted. 5) Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have putative transgenic Carrizo reflecting 69 events from 7 ScFv with verified transgenics ready for testing. These have been replicated by rooting and will be exposed to no-choice CLas+ ACP followed by whole plant destructive assays. 6) To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Paper is published. In-silico analyses are being conducted to develop citrus FLS2 optimized for sensing CLas flagellin. 7) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were 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 aimed to silencing 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 gar) are ongoing. Comparison of reactive oxygen species in transgenic and nontransgenic plants treated with CLas-flg22 are underway, to determine if there is an enhancement of the broad-spectrum PAMP-triggered immunity . With targeted gene expression data, we will propagate selected plants based on the above-mentioned tests for HLB inoculations purpose. 8) Optimizing use of a SCAmpP (small circular amphipathatic peptide) platform, was conducted in collaboration with Dr. Belknap and Dr. Thomson of the Western Regional Research Center of USDA/ARS. SCAmpPs were recently identified and have tissue specific expression, including having the most abundant transcript in citrus phloem. Furthermore, members of the SCAmpP family have highly conserved gene architecture but vary markedly in the ultimate gene product. Variants of a tissue-specific SCAmpP were tested using GUS as a reporter gene: removal of the conserved intron reduced tissue specificity and deletion of non-transcribed 5 region reduced expression. Excellent phloem-specific expression is achieved in citrus when a target gene is substituted for the gene encoding the SCAmpP peptide. Expression of a GUS marker gene was 500 x higher in midribs vs. laminar area. We are using this promoter aggressively in transgenic work 9) Third generation chimeral peptides were designed based on citrus thionins and citrus lipid binding proteins and plants have been transformed. Carrizo transformation of two constructs was completed and regenerated many seedlings. expression. A total of 43 Carrizo regenerations were confirmed being positive by PCR and highly expressed by RT-qPCR. Two constructs with above gene driven by double 35S promoter have 400 explants of Ray Ruby for each. 10) Two constructs with chimeral peptides containing citrus thionin and citrus proteinase were developed with both encoding genes are under by 35S promoter and SCAmpPs promoters. Transformation of those constructs are ongoing.
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. We are in the process of repeating and optimization of the methods. 2) We have conducted Las movement and multiplication in planta based on qPCR method. One manuscript has been submitted. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript has been submitted.
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) New replicates of the transgenic lines that have been inoculated by CLas-infected psyllids were maintained in the greenhouse for symptom development. Background: We have generated transgenic Hamlin sweet orange and Duncan grapefruit and screened the transgenic lines for HLB resistance or tolerance. We did not find any resistant line but identified three independent lines (two Hamlin lines and one Duncan line) that exhibit robust tolerance to HLB. This result indicates that the Arabidopsis NPR1 gene is able to create HLB tolerance in citrus. Since we only have HLB-tolerant Hamlin and Duncan , we decided to transform the NPR1 gene into other cultivars including sweet orange Pineapple and Valencia as well as grapefruit Ray Ruby (These plants were generated by the mature tissue transformation lab). The following table shows the new lines that have been inoculated by CLas-infected psyllids and are maintained in the greenhouse for symptom development: Replicate Genotype Transgene Parental line A1 Ray Ruby NPR1 21 81 Pineapple NPR1 15 75 Pineapple NPR1 16 66 Pineapple NPR1 17 78 Pineapple NPR1 18 84 Valencia NPR1 20 76 Hamlin NPR1 8 73 Hamlin NPR1 10 67 Hamlin NPR1 11 70 Hamlin NPR1 12 87 Hamlin NPR1 13 79 Hamlin NPR1 14 (2) Propagated plants from those that have been treated with CTV-FT3 and have produced flowers for later heat treatment to remove CTV and CLas. Background: The three independent lines ( Duncan 57-28, Hamlin 13-3, and Hamlin 13-29) with robust tolerance to HLB have been treated with CTV that carrying the FT3 gene, which promotes conversion from juvenile to mature tissues. The three lines have all developed blooms. The flowering-promoting CTV and HLB bacterial pathogen (CLas) in the transgenic plants need to be removed before producing healthy progenies. The following table shows the new lines received from the transformation lab and will be replicated and screened for HLB responses: Transgenic line Genotype Transgene Maturation Replicates made 57-28 Duncan NPR1 Yes 4 13-3 Hamlin NPR1 Yes 6 13-29 Hamlin NPR1 Yes 3 (3) More transgenic lines received from the transformation lab were transplanted into bigger pots and will be analyzed for the transgenic protein accumulation. Background: We have not obtained multiple independent lines for Valencia and Ray Ruby and have requested more from the mature tissue transformation lab. The following table shows the new lines received from the transformation lab and will be replicated and screened for HLB responses: Transgenic line Genotype Transgene A99 Valencia NPR1 A100 Valencia SuperNPR1(a) A102 Valencia SuperNPR1 A101 Valencia SuperNPR1 A72 Valencia ELP3 (b) A73 Valencia ELP3 A97 Hamlin SupperNPR1 A98 Hamlin SupperNPR1 (a) SupperNPR1 is a more active version of the NPR1 gene. (b) ELP3 encodes a disease resistance regulator, which appears to also provide tolerance to HLB. A manuscript titled Overexpression of the Arabidopsis NPR1 protein in citrus confers tolerance to Huanglongbing has been written and submitted to the Journal of Citrus Pathology in this quarter.
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