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.
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.
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