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.
For the third Quarter of 2018, Project #16-007 conducted or completed the following activities: 1) The second phase of the Picos Farm planting was completed in May of 2018, in Block 4 Rows 27-32 and included 322 trees; 2) The PI meet with CRDF Project Manager and the Cooperating Grower to participate in a field and greenhouse progress review. A consensus decision was made to re-calibrate the Scope of Work and work schedule, and accordingly a revised project plan was submitted to the RMC of CRDF; 3) Rootstock liners including approximately: a) 250 US-942’s, b) 250 Sour Orange’s, and c) 200 R7T6 Indian Sour Orange’s were procured or germinated to meet the experimental needs of the Off-Site planting a the Scott Grove; 4) The USHRL farm crew continues to maintain the grapefruit scion variants planted previously on the Picos Farm in Nov, 2017, Block 2, Rows 9-15, and July 2017, Block 2 Rows 48-54 (production practices available upon request); 5) Initiated budding and grafting operation in USHRL greenhouse in anticipation of planting 750 to 800 trees with Cooperating Grower (Scott Groves), with approximately 75% (525 trees) of this task completed by June 30, 2018;
For the third Quarter of 2018, Project #16-007 conducted or completed the following activities: 1) The second phase of the Picos Farm planting was completed in May of 2018, in Block 4 Rows 27-32 and included 322 trees; 2) The PI meet with CRDF Project Manager and the Cooperating Grower to participate in a field and greenhouse progress review. A consensus decision was made to re-calibrate the Scope of Work and work schedule, and accordingly a revised project plan was submitted to the RMC of CRDF; 3) Rootstock liners including approximately: a) 250 US-942’s, b) 250 Sour Orange’s, and c) 200 R7T6 Indian Sour Orange’s were procured or germinated to meet the experimental needs of the Off-Site planting a the Scott Grove; 4) The USHRL farm crew continues to maintain the grapefruit scion variants planted previously on the Picos Farm in Nov, 2017, Block 2, Rows 9-15, and July 2017, Block 2 Rows 48-54 (production practices available upon request); 5) Initiated budding and grafting operation in USHRL greenhouse in anticipation of planting 750 to 800 trees with Cooperating Grower (Scott Groves), with approximately 75% (525 trees) of this task completed by June 30, 2018;
The overall goal of the project was to test three complementary molecular genetic approaches for canker resistance to determine which can contribute to a stacked resistance approach. 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. 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 the Bs5 gene was sequenced from twenty-six transformants. We have identified two plants 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. Promising lines will be propagated and shipped to Dr. Jeff Jones’ lab at UF Gainesville for canker testing.
The overall goal of the project was to test three complementary molecular genetic approaches for canker resistance to determine which can contribute to a stacked resistance approach. 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. 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 the Bs5 gene was sequenced from twenty-six transformants. We have identified two plants 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. Promising lines will be propagated and shipped to Dr. Jeff Jones’ lab at UF Gainesville for canker testing.
Objective 1: Leaf nutrient thresholds We requested a 6-month no cost extension for the project in order to make up for lost time after hurricane Irma in 2017. The collection of survey data is now complete and the remaining months until December 2018 will be spent completing and cross-checking the comprehensive analyses and particularly the validity of results and recommendations for growers. Objective 1: Leaf nutrient thresholds Samples from all the sites have been processed and the first phase data analysis is complete. Using stepwise multiple regression and artificial neural networks, this phase screened the entire survey database of some 119,000 data points collected over three years for significant correlations between measured variables and tree performance. We then used these selected data for more intensive linear regression ANOVA testing, followed by Cate-Nelson analysis whereby the data sets were segregated into responsive and unresponsive groups. At the intersection of the two groups we identified the critical threshold values (CT) for deficiency of the leaf nutrient concentrations. These CTs will be useful in future to guide growers how much fertilizer to apply to specifically the HLB-affected groves. We need to re-analyze and cross-check all the results before we are ready to release the new advisory CTs. Objective 2: Determine soil conditions that favor root hair and VAM proliferation Based on the preliminary successful results from the Murcott seedling experiment, where tricalcium phosphate promoted root hair growth, we decided to repeat the experiment with a rootstock seedling. We have 3 new tanks running with 9 Carrizo 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. Root hair development seems enhanced in the low P, high Ca environment and will be studied closer over the next few weeks, utilizing the microscopy lab to quantify and identify root hair development in each treatment.
Objective 1: Leaf nutrient thresholds We requested a 6-month no cost extension for the project in order to make up for lost time after hurricane Irma in 2017. The collection of survey data is now complete and the remaining months until December 2018 will be spent completing and cross-checking the comprehensive analyses and particularly the validity of results and recommendations for growers. Objective 1: Leaf nutrient thresholds Samples from all the sites have been processed and the first phase data analysis is complete. Using stepwise multiple regression and artificial neural networks, this phase screened the entire survey database of some 119,000 data points collected over three years for significant correlations between measured variables and tree performance. We then used these selected data for more intensive linear regression ANOVA testing, followed by Cate-Nelson analysis whereby the data sets were segregated into responsive and unresponsive groups. At the intersection of the two groups we identified the critical threshold values (CT) for deficiency of the leaf nutrient concentrations. These CTs will be useful in future to guide growers how much fertilizer to apply to specifically the HLB-affected groves. We need to re-analyze and cross-check all the results before we are ready to release the new advisory CTs. Objective 2: Determine soil conditions that favor root hair and VAM proliferation Based on the preliminary successful results from the Murcott seedling experiment, where tricalcium phosphate promoted root hair growth, we decided to repeat the experiment with a rootstock seedling. We have 3 new tanks running with 9 Carrizo 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. Root hair development seems enhanced in the low P, high Ca environment and will be studied closer over the next few weeks, utilizing the microscopy lab to quantify and identify root hair development in each treatment.
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 (Mthionin) are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. Non-grafted Mthionin are in the field. A propagated group of 200 Mthionin transgenic Carrizo are potted up and grafted with wild type Hamlin to be planted in field next year. More Mthionin Carrizo are propagated and will be used to graft with Valencia and Ray ruby scions for field planting. In greenhouse a group of grafted plants with wt/transgenic and transgenic/wt rookstock and scion combinations were created and subjected to ACP inoculation. The 3-month post inoculation samples were recently collected for titer analysis. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. The 3-month sampling was just completed. Tissue specific constructs of the very promising Mthionin gene have been developed, with 10 Carrizo confirmed. The root specific variant was transformed only into Carrizo, and 17 plantlets were regenerated. A CTV-expression vector for Mthionin was created by Dr. W. Dawson, has been verified, and will be graft inoculated into a range of plants (both infected and healthy for challenging) next quarter. 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. A group of Carrizo transgenics expressing two variants of citrus thionin chimera (code 73 and 74) are being tested side-by-side with modified thionin transgenics. These plants were inoculated by ACP no-choice inoculation and reached 3 month mark for the first disease test in July. Using the detached leaf ACP-inoculation assay, it was shown that transgenic Carrizo expressing citrus thionin V1-LBP chimera has significantly less CLas titer after 1 week of ACP feeding than the wild type controls. Psyllid fed on detached leaves of the citrus thionin V1-LBP chimera Carrizo had lower CLas titer in bodies compared to the ones fed on the wide type leaves.. Comparison among individual lines from modified thionin transgenics were conducted using detached leaves and discovered variations in antimicrobial ability between them, suggesting this protocol allows us to screen better performing lines for further tests. 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 all time points measured after inoculation, transgenic plants are showing consistent and statistically significant decreases in bacterial titer (as much as 400x) when 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. Thirty ScFv-transgenic Carrizo plants (10 each from the 3 best performing constructs in greenhouse studies) are being grafted with Ray Ruby scions in parallel with non-transgenic controls for initial field studies. Approximately 120 additional rooted cuttings for a follow up trial are being propagated. 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. CRISPR constructs with guide RNA targeting DMR6 and DLO1 were made and transformed into Carrizo, generating 3 and 1 confirmed transgenic line respectively. Mutation at the target locus has been confirmed in the first of these plants after heat shock treatment. The remaining plants are being sequenced to detect mutations and new transformations into Hamlin are in preparation. 6) Budwood from our best performing Mthionin, citrus gene chimeras and ScFv antigen binding fragment expressing transgenics have been sent to DPI for cleanup and then broad field testing.
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 (Mthionin) are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. Non-grafted Mthionin are in the field. A propagated group of 200 Mthionin transgenic Carrizo are potted up and grafted with wild type Hamlin to be planted in field next year. More Mthionin Carrizo are propagated and will be used to graft with Valencia and Ray ruby scions for field planting. In greenhouse a group of grafted plants with wt/transgenic and transgenic/wt rookstock and scion combinations were created and subjected to ACP inoculation. The 3-month post inoculation samples were recently collected for titer analysis. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. The 3-month sampling was just completed. Tissue specific constructs of the very promising Mthionin gene have been developed, with 10 Carrizo confirmed. The root specific variant was transformed only into Carrizo, and 17 plantlets were regenerated. A CTV-expression vector for Mthionin was created by Dr. W. Dawson, has been verified, and will be graft inoculated into a range of plants (both infected and healthy for challenging) next quarter. 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. A group of Carrizo transgenics expressing two variants of citrus thionin chimera (code 73 and 74) are being tested side-by-side with modified thionin transgenics. These plants were inoculated by ACP no-choice inoculation and reached 3 month mark for the first disease test in July. Using the detached leaf ACP-inoculation assay, it was shown that transgenic Carrizo expressing citrus thionin V1-LBP chimera has significantly less CLas titer after 1 week of ACP feeding than the wild type controls. Psyllid fed on detached leaves of the citrus thionin V1-LBP chimera Carrizo had lower CLas titer in bodies compared to the ones fed on the wide type leaves.. Comparison among individual lines from modified thionin transgenics were conducted using detached leaves and discovered variations in antimicrobial ability between them, suggesting this protocol allows us to screen better performing lines for further tests. 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 all time points measured after inoculation, transgenic plants are showing consistent and statistically significant decreases in bacterial titer (as much as 400x) when 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. Thirty ScFv-transgenic Carrizo plants (10 each from the 3 best performing constructs in greenhouse studies) are being grafted with Ray Ruby scions in parallel with non-transgenic controls for initial field studies. Approximately 120 additional rooted cuttings for a follow up trial are being propagated. 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. CRISPR constructs with guide RNA targeting DMR6 and DLO1 were made and transformed into Carrizo, generating 3 and 1 confirmed transgenic line respectively. Mutation at the target locus has been confirmed in the first of these plants after heat shock treatment. The remaining plants are being sequenced to detect mutations and new transformations into Hamlin are in preparation. 6) Budwood from our best performing Mthionin, citrus gene chimeras and ScFv antigen binding fragment expressing transgenics have been sent to DPI for cleanup and then broad field 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. 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.
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. 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.
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