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 have survived and passed a PCR screen, and these have been grafted onto rootstocks. Grafted plants are currently growing, and will be tested for responsiveness to the elf18 ligand for EFR and 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. 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 submitted 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. 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. About 40 of each group are being tested for transgene insertion and level of expression. 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.
Objective 1. Identify key metabolites that are associated with rootstock traits. Summary of accomplishments: Metabolite profiles of greenhouse-grown rootstock seedlings of four standard rootstock cultivars (Cleopatra, Swingle, Sour orange, Ridge Pineapple) with known horticultural traits were assessed for primary metabolites possibly associated with traits. Analysis has been completed; however, additional studies are necessary to corroborate findings. These include physiological assays and confirmation of identified compounds using chemical standards. The team is currently working on establishing these procedures to strengthen research findings. In addition to these four standard rootstock cultivars, profiles of seven additional greenhouse-grown cultivars that are popular with the citrus industry (Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) are currently being analyzed. Extensive additional metabolic data from our experiments were received back from the West Coast Metabolomics Center (WCMC), UC-Davis, at the end of March 2017, and are being used for detailed analysis by the USDA-IFAS team. Objective 2. Investigate the effect of grafting on metabolite profiles. Summary of accomplishments: The four standard rootstocks (Cleopatra, Swingle, Ridge, and Sour orange) and seven additional rootstocks (Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) were analyzed as greenhouse-grown grafted trees in combination with Valencia. Metabolic profiles of leaves and roots of the grafted trees are being compared with those of leaves and roots from rootstock seedlings to assess rootstock effects on the scion and the possible implications for tree performance. Extensive additional metabolic data from our experiments were received back from the West Coast Metabolomics Center (WCMC), UC-Davis, at the end of March 2017, and are being used for detailed analysis by the USDA-IFAS team. Due to the complicated nature of these data sets, this process is expected to take several months until ready for publication. In addition to the study of greenhouse-grown trees, analysis of metabolite profiles has been expanded to grafted trees grown under field conditions. A current data set including young trees with two different scion cultivars (Cara Cara and Hirado) in combination with the four standard rootstocks (Cleopatra, Swingle, Ridge, and Sour orange) was received from WCMC at the end of March and is in the final process of analysis. A publication is expected to be submitted in the next few months. These studies will aid in identifying rootstock-scion interactions and the possible impacts on stress and disease tolerance under commercial conditions. Objective 3. Establish metabolite profiles of trees on different rootstocks in response to HLB. Summary of accomplishments: An experiment consisting of many hundred grafted trees grown in the USDA greenhouses was completed. Trees were composed of Valencia grafted on a diverse array of standard and USDA rootstock cultivars and were either mock-inoculated or inoculated with Las. PCR analysis of Las bacterial titers of leaves and roots for the trees at different time intervals is in process. Leaf and root tissue of Las-infected and Las-uninfected plants were collected at the end of the experiment, and appropriate extractions completed. Samples from these experiments were sent to WCMS for GC-TOF-MS analysis in April 2017, and the resulting metabolic data should be available in August, to be used by our USDA-IFAS team to conduct detailed analysis of metabolite profiles associated with HLB response. Experimental design, data collected, analysis, results, and interpretation are too complex to present here. Additional information is available on request.
Activities are reported by project objectives below. 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions: A set of approximately 70 new rootstock candidates, stick-grafted with HLB-infected Valencia, were rotated into the hot psyllid house in cooperation with Dr. Nian Wang. The previous set of 50 rootstock candidates were removed from the hot psyllid house and prepared for field planting at the USDA Picos Farm in Fort Pierce. Twenty five new crosses were made using parents that have demonstrated HLB tolerance or have yielded offspring exhibiting high levels of tolerance. 2. Breeding of HLB tolerant/resistant processing sweet orange-like hybrids: At least 35 crosses were made this spring, to develop HLB-tolerant scion cultivars, using tolerant breeding lines with complementary characteristics. Fruit samples of some HLB-tolerant candidate selections were processed and pasteurized; several were found that produced good juice with no bitterness or off-flavors as typically found in mandarin hybrids. Three Valencia clones identified previously as rather tolerant in germplasm collection screening were observed still to be performing well; new anatomical and molecular studies have been initiated to determine underlying mechanisms of their tolerance; further, they have been entered into the DPI PTP for cleanup, and trees have been propagated directly for further field testing. Additionally, cybrid grapefruit clones (kumquat cytoplasm) of Flame, Ruby somaclone N11-11 and White Marsh showing significantly improved canker tolerance in greenhouse assays, were propagated for anticipated field planting at IRREC (~200 trees). Collected leaf samples from approximately 1000 transgenic trees and 300 gauntlet trees for qPCR to determine HLB infection status. Trees in both categories with good appearance were identified for further close observations. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. We continue to monitor our germplasm collection and breeding families for performance against HLB. We have initiated a genomic selection effort based on phenotypic assessments and using a high density SNP chip for genotyping. This may lead to identification of genetic resources to facilitate HLB-tolerant scion and rootstock breeding. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. We reorganized our field data collection team, to focus on collecting data from our most critical trials. A new planting was made of seedless and easy to peel fresh mandarins, some exhibiting HLB-tolerance, in Pasco County. A large rootstock trial was planned and organized in the Peace River region. Completed yield and fruit quality data collection from the St. Helena Project. Preliminary analysis of the data shows an overall slight increase in lbs. solids over last year, with some rootstocks increasing in yield, and some decreasing. Trees on Swingle have shown a remarkable recovery. Collected yield and fruit quality data from 3 year old trees in the OLL sweet orange clone/rootstock trial near St. Cloud. Completed young tree assessments in the large new Camp Mack and Basinger rootstock trials with Lykes; Hamlin and Valencia trees on ~60 rootstock candidates were assessed for size, fruiting, overall health, and HLB disease symptoms. Other related activities: Minor revisions were made to the Rootstock Selection Guide, which has incidentally had over 389,000 hits in its first 17 months.
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) Conducted two more rounds of cage experiments to further test if the transgenic plants inhibit psyllid reproduction. Results showed that none of the transgenes was able to significantly reduce psyllid progeny numbers in the cage experiment. (2) Newly generated replicates of the transgenic lines were inoculated in the psyllid room and nymph-infested plants were recorded and moved to the immediate greenhouse for symptom development.
Objective 1. (Greenhouse experiment): All liners were treated with the various CRF combinations one month prior to grafting. Stick-grafting of seedlings of the required rootstocks: x639, Swingle, WGFT+50-7, UFR-3 and UFR-15 potted in round citripots with HLB-infected Valencia were completed. 12 trees were grafted per treatment per rootstock. There were only a few failed grafts that are currently being repeated. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The treatments have been applied for one year at both sites. The second year first quarter applications of all the treatments have been completed and second year tree health parameters have been collected. The fruits at both the locations have been harvested and evaluated. Although there were obvious trends suggesting an advantage for applications combining traditional fertilizer with CRF, there was no statistically significant differences (95% confidence level) among the yield of different treatments at either site. There were no statistically significant differences in the fruit quality of fruits from different treatment and growers control at both the Ft. Meade and Arcadia locations. Although at the Arcadia site, treatments 9 and 10 resulted in better juice quality than treatment 6. Overall, no significant differences (95% confidence level) among growers control and treatments were found. This is not unexpected, as we believe it takes time to restore the root systems of impacted trees; thus we expect to see greater differences in the year 2 data. These results were also presented on April 4th, 2017 at Citrus Institute, Avon Park. Ojective 5. (funded by Orie Lee, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Yield data was taken after one year, and all treatments yielded significantly better than the non-treated control except for the 4x polycoated sodium borate treatment.
During the period of January, February, and March of 2017, progress was made in planning for HLB resistance testing of the grapefruit lines expressing the FLT-antiNodT fusion protein. Through ongoing discussions mediated by Dr. Catherine Hatcher, Dr. McNellis is in contact with Dr. Johnny Ferrarezi at the Indian River Research and Education Center at Ft. Pierce, and others, to perform no-choice psyllid feeding tests on ‘Duncan’ grapefruit plants expressing the FLT-antiNodT protein. In addition, we are working with Dr. Ed Stover to get some of the FLT-antiNodT plants available at Ft. Pierce to be included in a field test underway at the Pecos farm at Ft. Pierce. Dr. Gottwald’s lab has successfully propagated all eight of the FLT-antiNodT grapefruit lines at Ft. Pierce, with 6-18 plants available for each line, for a total of 75 rooted FLT-antiNodT plants available at Ft. Pierce. During the next reporting period, we plan to apply for permitting to participate in the field test with the FLT-antiNodT lines and initiate the no-choice feeding HLB inoculation test. Dr. McNellis was also invited to participate in the May 22, 2017, Forum on Citrus Breeding and Transformation for HLB resistance hosted by the National Academies of Sciences, Engineering, and Medicine in Irvine, CA, and he plans to attend in person and present at the forum.
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 transgene product (protein) levels in 30 independent rootstock transgenic lines and identified three lines accumulating high levels of the transgene product, one Swingle line and two Carrizo lines. These three rootstock lines will be propagated in the coming quarter. (2) After inoculation of the HLB-tolerant transgenic lines with the flowering promoting gene FT3, all three lines have flowered. We are propagating these lines for field trials. (3) One more cage experiment testing psyllid reproduction on transgenic plants was performed. Results showed that only one line consistently prevents reproduction of psyllids, suggesting a T-DNA insertion mutation in this line. We will clone the citrus gene required for psyllid reproduction in the coming quarter. (4) More replicates were made for transgenic lines with low numbers of progenies and these progeny plants will be tested in the coming quarter.
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 transgene expression in 30 independent rootstock transgenic lines and identified lines expressing high levels of the transgene. Accumulation of the transgene product (protein) will be tested in the coming quarter. Lines accumulating the highest levels of protein will be selected for further usage. (2) More propagation has been made for the HLB-tolerant trangenic line that has flowered. The progenies are growing. For the lines that have not flowered, we reinoculated the plants with CTV carrying the flower-promoting gene FT3. (3) Another cage experiment for testing if transgenics prevent psyllids from being infected has been set up and results will be obtained in the coming quarter. (4) Transgenic lines with low numbers of progenies have been propagated. More progeny plants are growing and will be tested when they are ready.
Field performance information this quarter was collected on more than 300 new rootstocks in 19 different replicated active USDA field trials established prior to 2017, including assessment of tree growth, tree health, fruit yield, fruit quality, and tolerance or resistance to HLB and other diseases. Evaluation of trials usually includes periodic testing of trees for Las infection, using PCR. The trials include rootstocks currently in Florida industry use (such as US-802, US-812, US-897, and US-942), as well as SuperSour hybrids not yet released. Additional trees in the USDA nursery now will be used to plant seven additional replicated field trials in 2017. The combined list of current trials and those that will planted in 2017 is shown in the table below, along with additional details on each trial. The focus of data collection from these trials is on parameters that are most relevant to assessing rootstock effects on tree tolerance to HLB. Our aim is to find new rootstocks that grow healthier trees with more fruit despite HLB, than the best rootstocks currently available. In addition to these 26 trials, I am working with the UF breeding team under two HLB-MAC projects to establish at least 13 additional rootstock field trials over the next 12 months, combining the best advanced new rootstocks from the USDA and UF breeding programs. A comprehensive new NIFA SCRI-funded rootstock breeding project involving UF and my program should begin soon, improving the integration of the rootstock efforts of the two institutions, while also involving other researchers to expand the scope and complexity of the search for plant resistance solutions to the HLB problem. Additional information is available on the USDA rootstock breeding project, on request.
Objective 1. Identify key metabolites that are associated with rootstock traits. Summary of accomplishments: Rootstock seedlings were grown in the USDA greenhouses and included standard rootstock cultivars and rootstocks developed by the USDA breeding program and in great demand by the industry. To establish metabolite profiles on a set of cultivars with known horticultural traits, we used the four rootstocks Cleopatra, Swingle, Sour orange, and Ridge Pineapple, and assessed the metabolite profiles of leaves and roots at two different seedling stages under greenhouse conditions. This allows us to characterize rootstocks based on their metabolite composition, and to assess the stability of metabolite profiles at different developmental stages. For a subset of the data, analysis has been completed and several compounds have been identified that may be associated with rootstock traits. A manuscript has been prepared and submitted to the journal Plant Science. In addition to these four standard cultivars, other rootstock seedlings were sampled and leaf and root extracts were submitted for GC-TOF MS analysis at the West Coast Metabolomics Center (WCMC), UC-Davis. Data were received in March 2017 and are currently being processed and analyzed by the team. Preliminary results of a selected set of the most recent data were presented at the IRCHLB in Orlando in March 2017. Objective 2. Investigate the effect of grafting on metabolite profiles. Summary of accomplishments: The four standard rootstocks (Cleopatra, Swingle, Ridge, and Sour orange) and other rootstocks developed by the USDA breeding program were analyzed as grafted trees in combination with Valencia and other scions. Trees were grown in the greenhouse or under field conditions at different locations. Comparison of the metabolite profiles of roots and leaves in these grafted trees with profiles obtained for leaves and roots of rootstock seedlings allows us to analyze rootstock and scion interactions, and evaluate how they affect tolerance to HLB and other biotic or abiotic stresses. The different field conditions under which the plants are grown will also aid in identifying environmental effects on metabolite profiles. The latest data set from this part of the study was received in March 2017 and is currently being processed and analyzed. Analysis of a subset of the data has been completed and we are in the process of preparing a manuscript for publication. Objective 3. Establish metabolite profiles of trees on different rootstocks in response to HLB. Summary of accomplishments: A paper was published last year describing our initial research on metabolic profiles of rootstocks with different response to HLB (U. Albrecht, O. Fiehn, K.D. Bowman. 2016. Metabolic variations in different citrus rootstock cultivars associated with different responses to huanglongbing. Plant Physiology and Biochemistry 107:33-44). In addition, an experiment was initiated that included many hundred grafted trees grown in the USDA greenhouses. These trees are composed of Valencia grafted on a diverse array of standard and USDA rootstock cultivars. One set of trees was inoculated with Las using graft-inoculation; a second set of trees was mock-inoculated using the same procedure. Trees were analyzed by PCR for presence of Las in leaf and in root tissue at regular time intervals. A subset of trees was selected, and leaf and root tissues of these plants were collected for metabolite analysis. These samples have been extracted and were sent to WCMS in April 2017. Some of the selected plants are being used for continuing infection and metabolic studies. Experimental design, data collected, analysis, results, and interpretation are too complex to present here. Additional information is available on request.
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 have survived and passed a PCR screen, and these have been grafted onto rootstocks. Grafted plants are currently growing, and will be tested for responsiveness to the elf18 ligand for EFR and 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 protocol is being optimized for PCR screening. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs were provided to the Lake Alfred transformation facility, but transformation attempts have so far been unsuccessful. Troubleshooting has indicated that it is likely that the constructs have negative effects in citrus, and therefore work on these constructs has been discontinued. 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. 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 and experiments are underway. 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. 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 (second generation) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. 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 to citrus chloroplasts. Transcription data were obtained by RNA-Seq. 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 almost 400 independent transgenic events and 17 different ScFv, but only 69 events from 7 ScFv produced proven 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. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA was isolated and Las titer was tested. Our preliminary results show that transgenic trees expressing NbFLS2 can reduced Las titer. In-silico analyses are being conducted to develop citrus FLS2 optimized for sensing CLas flagellin. 7) Arabidopsis DMR6 (down 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 exhibit moderate to strong activation of plant defense response genes. 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. We are using this promoter aggressively in transgenic work 9) The third generation chimeral peptides were designed based on citrus thionins and citrus lipid binding proteins and plants have been transformed.
The driving force for this project (Hall-15-016) is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus breeders at USDA-ARS-USHRL, Fort Pierce Florida are producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with 20 infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then 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. 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 January 1, 2017, a total of 9,494 plants have passed through inoculation process. A total of 297,595 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. Research we recently published showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Incidentally, Setamou et al. (2016, J. Econ. Entomol., 109: 1973-1978) published supporting information that transmission rates of CLas are increased when flush is present. The no-choice inoculation step used in our program has been projected to be successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant). We are in the process of analyzing data from research comparing success rates using ACP colonies on lemon versus citron, and using ACP colonies from greenhouses versus walk-in chambers.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. In this quarter, we specifically tested the SAR inducer-activated residual activity in potted citrus seedlings. The seedlings were treated by either root drench or foliar infiltration with the SAR inducer. After the first round of disease resistance test, the plants were cut back. Two months later, residual activity in the new flashes was tested. Results showed statistically significant reduction in the number of canker lesions formed in leaves on the plants pre-treated with the SAR inducer. Reduction in the number of lesions was, on average, from 15% at a low dose (0.25 mM) to 40-50% at higher doses of the SAR inducer (5-10 mM). These results demonstrate that the SAR inducer activates strong residual activity in new flushes at least two months after the pre-treatment. We also confirmed the priming effect of the SAR inducer. Briefly, leaves were treated with 1 mM of the SAR inducer. The treated leaves were infected with citrus canker bacterial pathogens 36 hours later. The infected leaf tissues were collected at 0, 4, 8, and 24 hours later, similarly as in the previous experiments. Expression of PAL1,NPR1, PR5, CM1, ICS1, CM1, CM2, and PLDg was analyzed by real-time qPCR. We confirmed that expression of PAL1, NPR1, PR5, CM1, and ICS1 was significantly enhanced by pre-treatment with the SAR inducer, corroborating that the SAR inducer indeed has strong priming effects in citrus.
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