Objective 1. Identify key metabolites that are associated with rootstock traits. Accomplishments: Metabolite profiles of rootstock seedlings of ten rootstock cultivars in two separate experiments were assessed for primary metabolites in collaboration with West Coast Metabolomics Center (WCMC), UC-Davis. Results from the original study were published (Albrecht, Fiehn, Bowman. Plant Physiology and Biochemistry 107:33-44). The data obtained are large and complex and are being used for additional detailed analysis. An additional manuscript was submitted to Plant Physiology and Biochemistry in this quarter. An excerpt from this analysis is shown below. Objective 2. Investigate the effect of grafting on metabolite profiles. Accomplishments: Eleven rootstocks were analyzed as greenhouse and field-grown grafted trees in combination with Valencia and other scions. Samples were submitted to WCMC for analysis, and metabolic profiles of leaves and roots of the grafted trees are being compared. These studies will aid in identifying rootstock-scion interactions. The data sets are large and complex, but a publication is expected to be submitted in the next few months. Objective 3. Establish metabolite profiles of trees on different rootstocks in response to HLB. Accomplishments: An experiment consisting of 300 trees of Valencia grafted on ten rootstocks and grown in greenhouses was completed. Trees were inoculated with Las. Leaf and root tissue of Las-infected and uninfected plants were collected at 30 weeks after inoculation. Samples from these experiments were sent to WCMS for GC-TOF-MS analysis of primary metabolites and secondary metabolites in 2017. Partial results have been received, with the remaining results expected soon. Results from these analyses are being used for detailed analysis of metabolite profiles associated with HLB. A publication of the results is being prepared. Additional information is available on request.
Objective 1. (Greenhouse experiment): Another round of PCR samples were taken from every tree and sent to the Southern Gardens diagnostic laboratory to determine HLB status – results are just back and are now being analyzed. Leaf nutritional samples were sent to Waters Lab for analysis, results are back and are now being analyzed and interpreted. Most trees, even though infected, are growing exceptionally well, except for treatment #3, where trees are not showing much growth and are beginning to yellow. Objective 3: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The nutrition treatments have been applied for 2 year now. The third year first quarter applications of all the treatments have been completed and third year tree health parameters have been collected. The fruits at both the locations have been harvested and evaluated. Currently, we are in process of data analysis and interpretation. The results from year 2 nutritional trial will be presented at OJ Break on May 15th, 2018 Ojective 5. (funded by Lee Groves, 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. Positive results will be presented at ASHS meeting this summer, and we initiated preparation of a journal manuscript to present the results to the scientific community.
Objective 1. (Greenhouse experiment): PCR samples were taken from every tree and sent to the Southern Gardens diagnostic laboratory to determine HLB status. Nutritional profile as well as infection status of all grafted plants were analyzed in this quarter. qPCR values indicated that there was no significant differences in the infection rate amongst the different treatments at this time. The Harrell s nursery mix supplemented with Boron has lower overall infection rates as estimated by higher Ct values in all the rootstocks except UFR15, where the nursery mix supplemented with Manganese performed better. It is still too early to see much treatment and rootstock differences. Objective 3: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The total 2nd year fertilizer applications were completed. Year 2 yield and fruit quality data collection will begin this quarter. Ojective 5. (funded by Lee Groves, 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. Potential breakthrough – qPCR was run on each tree – significant bacterial titer differences were found among the treatments. The best treatment was a 4x overdose of manganese, producing an average ct value of 32.75; whereas the control tree average ct value was 23.19 (indicating severe infections). Seven of twelve trees in the high manganese treatment exhibited ct values of 32 or above, and were considered not to have an active infection; whereas no control trees reached 32. Other treatments containing high manganese also did much better than the controls, except for the treatment with both 4x manganese and 4x boron. This level of boron seemed to be antagonistic to the uptake of manganese, as demonstrated by the leaf mn content data. Yield and fruit quality data from these plots are being collected this quarter – results should be quite interesting.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) Analyzed another batch of rootstock transgenic lines (54 Carrizo lines, 7 Swingle lines, and 3 C-MAC lines). Two more Swingle lines and three C-MAC lines accumulated high levels of transgene products. More than half of the Carrizo lines accumulated high levels of transgene products. These rootstock transgenic lines will be analyzed based on the protein levels. (2) Conducted another cage experiment for replicates of the transgenic lines with low numbers of psyllid progenies. (3) Made more replicates of the transgenic lines tolerant to HLB.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). All these propagates (ca. 250 trees) are growing well in greenhouse, and expected to go to field next month. These propagates will be tested by qPCR before going to fields. The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. New propagations of the selections from the the Scott grove seedlings, including 245 Red Ruby and 51 white Marsh plants on three different rootstock and 38 Red Ruby cuttings were planted in our research farm on Nov.15, 2017. Before going to field, samples were taken from these plants, and processed for qPCR detection. We will monitor the disease incidence and Las titers if there is an infection. Meanwhile we will go second round of selection and propagations on Scott seedlings, and then plant them in Scott grove for field evaluation.
Activities are reported by project objectives below. 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. Planted ~2000 seeds from 20 2017 rootstock crosses in calcareous/Phytophthora infested soil (“Gauntlet”), and completed stick grafting of approximately 100 new gauntlet rootstock candidates selected from 2015 crosses. Propagated 5 “super-root” mutants from tissue cultured UFR1, UFR-3, and UFR-6, and begun testes for HLB tolerance. 2. Breeding of HLB tolerant/resistant processing sweet oranges and orange-like hybrids. Recovered ~ 400 plants from interploid crosses targeting HLB tolerant sweet orange/mandarin type hybrids. Planted 203 transgenic plants (73 independent lines, mostly oranges but some Duncan and W. Murcott) containing Valencia-derived BG or LTP at out APHIS approved transgenic site at the SWFREC. 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. Interesting genomic regions have been highlighted that contain putative disease resistance and defense mechanism genes. Worked with Dr. Johnny Ferrarezi to plant the entire population of cybrid grapefruit clones (kumquat cytoplasm) at the IRREC. These include cybrids of Flame, Ruby somaclone N11-11 and White Marsh showing significantly improved canker tolerance in greenhouse assays. The original N11-11 somaclone, derived from Ruby red, has shown a strong recovery response from HLB; thus the cybrids made from this clone are potentially improved for both canker and HLB tolerance. In addition to the original population, propagations of selected clones were also planted, and many were on the rootstocks propagated via the previous CRDF grant with Barrett Gruber. Approximately 340 trees were planted. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. Field planted promising new OLL sweet oranges/new rootstock candidates in 2 locations. Planted 150 E.V. (Early Valencia) trees on promising new rootstocks in a high Diaprpres pressure area, in an effort to identify a rootstock that doesn t drop fruit after HLB infection. Harvested seed from all available UFR seed trees; seed kindly extracted by John Gose @ Lykes, now being distributed to participating licensed nurseries. Other related activities: Scored trees in a high-peel oil lemon selection trial in the IR region, after 7 days of Irma flooding. This will serve as a baseline for future evaluations of flood damage over time. Speaking of Irma, we spent much of September going through all CREC plots and field trials, standing up and staking trees blown over by the storm.
During the period of July, August, and September, 2017, Dr. McNellis submitted a full proposal to the USDA Citrus Specialty Crop program to expand and continue the existing project funded by CRDF, and this full grant proposal was submitted to the USDA on August 18, 2017. This project would be a collaboration between Dr. McNellis and Drs. Ozgur Batuman, Liliana Cano, and Rhuanito Ferrarezi. They have the expertise to do CLas infections and PCR-based quantification of CLas in plant tissues. In addition, Dr. McNellis has been working with Dr. Catherine Hatcher of the CRDF on a pre-proposal for the field testing and greenhouse testing for HLB resistance of the trees produced through this project, in anticipation that the current project will be concluded on December 31, 2017. Dr. McNellis’ lab also continued quantitative analysis of anti-HLB antibody protein expression in the transgenic lines (the FT-scFv fusion protein) by western blotting. It will be the objective and intention of the renewal project to test HLB resistance or tolerance status of ungrafted and grafted trees with the transgenic genotype.
Objective 1. (Greenhouse experiment): We reached the 6 month period since the stick grafting and first CRF application, so all CRF nutrition treatments were re-applied using all donated products (all trees), PCR samples were taken from every tree and sent to the Southern Gardens diagnostic laboratory to determine HLB status – hopefully we can report results in the next quarterly report. Composite leaf samples from each nutrition/rootstock combination were sent to Waters Lab for nutritional analysis. Overall, most trees are showing reasonably good health, with only minor symptoms appearing on some trees. The greenhouse and the experiment were not impacted by Irma. Objective 3: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The third application of fertilizer treatments for the 2nd year was completed at both sites. There were no tree losses due to Irma, but there was some fruit blown off the trees, apparently fairly consistent across the replications within the trial. Ojective 5. (funded by Lee Groves, 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. Trees still look relatively good, no significant impact by Irma. Photosynthesis rates were measured on all trees, and composite leaf samples were taken and sent to Water’s Lab for nutritional analysis.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) Several propagations were made for the Swingle and Carrizo lines accumulating high levels of the transgenic protein. We noticed the Carrizo line that accumulates the highest level of the transgenic protein does not grow well, indicating that two much of the transgenic protein affects citrus growth and development. This provides a threshold for the transgenic protein. (2) The HLB-tolerant transgenic lines were further propagated. (3) Four TAIL-PCR experiments have been conducted with both right and left T-DNA border sequences to clone the gene suppressing psyllid reproduction on citrus plants. Unfortunately, not dominant PCR bands were obtained. The presence of the T-DNA insertion was confirmed using the kanamycin gene primers. We are determining the truncation sites of the T-DNA insertion and will clone the insertion site. (4) Cage experiment for replicates of the transgenic lines with low numbers of psyllid progenies is ongoing. More replicates will be generated.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics survived and passed a PCR screen, and these are currently being tested for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and therefore the putative transformants are being screened by RT-PCR. Eleven PCR-positive shoots have been grafted so far. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis, with several rooted plants obtained so far. Molecular characterization of the putative transformants will be carried out at UC Berkeley. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). All these propagates (ca. 250 trees) are growing well in greenhouse, and expected to go to field next month. These propagates will be tested by qPCR before going to fields. The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees.
Excellent progress was made in continuing the development of new hybrid rootstocks for the Florida citrus industry. Highest priority is being placed on work to develop hybrid rootstocks that can be made available for release to growers in the next three years, including selections from about 300 Supersour-type rootstock selections. It is expected that two of these Supersour rootstocks will be released in 2018, and at least two more in 2019-20, as further information is collected from ongoing field trials, and clean source material is available from from FDACS-DPI. During this quarter, one new replicated rootstock field trial was planted on East coast flatwoods site. The trial used Valencia scion and included statistically randomized replicates of 72 different rootstocks, with most rootstocks represented by 12 replications. Standard rootstocks included for comparison were Sour orange, Swingle, Cleopatra, and Ridge. Emphasis in the new trial was on SuperSour selections for which preliminary performance information is promising and that are in the DPI clean source program. Trees in the nursery were in the final stages of preparation for planting two additional field trials in the coming quarter. The trials will each include 8-12 replicates of about 60 new rootstocks, along with several standard rootstocks for comparison. Emphasis in these new trials is also on SuperSour selections for which other performance information is promising and that are in the DPI clean source program. Liners were prepared in the nursery by seedage and cuttings for additional field trials to be prepared for future plantings, based on new hybrid rootstock selections that look the best with HLB in preliminary field sites. About 600 additional seed source trees were planted into the field for these new promising selections, so that more seed will be available from the SuperSour rootstocks for commercial use after release. Research was conducted in collaboration with commercial nurseries and tissue culture propagation facilities to study and compare rootstock liners produced from seed, cuttings, and micropropagation. The rapid expansion in the demand for the newest rootstocks following release makes it clear that available seed for these new rootstocks will frequently be inadequate to satisfy commercial demands. Preliminary propagation information suggests that more US-942 rootstock will be propagated by micropagation than seed in the 2017-18 season. It is likely that cuttings and micropropagation of rootstocks will become increasing common. Noted differences in nursery performance of liners by propagation type, have prompted concerns about the influence of propagation type on field performance. We have initiated research to study these issues and address the concerns in a way that provides clear answers to growers and nurseries. One field trial containing rootstocks propagated by the three methods will be planted in the coming quarter. Performance data was collected from several field trials, including tree size, canopy health, and infection status by qPCR. Extensive cropping and fruit quality data will be collected from trials beginning the the next quarter. Analysis of results from previously established trials indicates that HLB introduces more complex variability to trials than that observed without HLB. This means that more than five or six statistical replicates will be needed to provide clear evidence of superior rootstock performance. Based on this observation, new USDA rootstock trials will usually include more than six statistical replications, regardless of the number of trees per replication. Using valid statistical comparisons is essential to develop reliable information about relative rootstock performance in field trials and should be the foundation of grower rootstock selection for new field plantings. 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. The first of these trials will be field planted at the USDA Ft. Pierce Farm in the next quarter. 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: Metabolite profiles of greenhouse-grown rootstock seedlings of eleven rootstock cultivars (Cleopatra, Swingle, Sour orange, Ridge Pineapple, Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) with known horticultural traits were assessed for primary metabolites possibly associated with traits. Analysis has been completed on a preliminary dataset. The data from an additional study to corroborate findings were recently received from West Coast Metabolomics Center (WCMC), UC-Davis, and are being used for detailed analysis by the USDA-IFAS team. Additional supporting studies are being conducted, including physiological assays and confirmation of identified compounds using chemical standards. The team is currently working on establishing these procedures to strengthen research findings. 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, 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 eleven rootstocks is in 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 300 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 30 weeks after inoculation, and appropriate extractions completed. Samples from these experiments were sent to WCMS for GC-TOF-MS analysis of primary metabolites in April 2017, and the resulting metabolic data were recently received. A second set of samples from the experiment was recently sent to WCMS for analysis of secondary metabolites. Results from these analyses will be used by our USDA-IFAS team to conduct detailed analysis of metabolite profiles associated with HLB response. Publication of the results in a refereed journal will be completed, along with identification of practical utility of the information. Experimental design, data collected, analysis, results, and interpretation are too complex to present here. Additional information is available on request.
1) Trees have been in the ground for 3.75 years in a trial of 50 selections and cultivars on US-802 following no-choice ACP inoculation and several months in an ACP house. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a semi-annual basis. HLB is now widespread and the trees looking healthiest include a full sib of our best mandarin selection, and several of our best grapefruit-like hybrids. The one true grapefruit is the least healthy selection in the trial. There are eleven selections with a canopy volume 50% greater than Valencia and 28 with canopy volume >2X that of Flame. The best performers include hybrids containing Poncirus, and conventional hybrids which are predominately mandarin or pummelo. It may take 2-3 more years to clearly distinguish tolerant material. These trees are cropping this year, though Irma dropped a lot of fruit, and fruit will be used in a complementary project exploring synthesis of orange-like juice from HLB-tolerant types. 2) In June 2015 a field planting was established of: seedling trees of 133 Fortune x Fairchild hybrids from an earlier mapping study, seedlings of 27 Ponkan-like accessions, budded trees of 10 advanced ARS selections that are predominately mandarin, and budded trees of Fortune, Fairchild and Valencia. Data collection is underway. A NIFA grant proposal was submitted to mobilize tolerance for industry use. It is a collaboration between ARS, UF and UC Riverside. 3) Replicated trials in multiple locations are established of our best sweet-orange-like cultivars and mandarin-types. Volatiles from sweet-orange-like hybrids are so similar to sweet orange that likely can be legally named sweet orange. 4) RNA-seq compared transcriptome responses in HLB moderately tolerant Sun Chu Sha mandarin and susceptible Duncan grapefruit, to Xcc-flg22 and CLas-flg22 (most active epitope from the pathogen flagella; project initiated with Gloria Moore at University of Florida). Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with CLas, suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and CLas treatments. Genes identified awere recently published and are valuable for studying HLB tolerance mechanisms and potential for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy. Using these genes as markers, expression analysis from a group of mandarin hybrid in their responses to CLas-flg22 is underway. Highly and lowly responsive plants will be marked for long term observation of field tolerance. 5)A group of CLas effector (28) was predicted using bioinformatics tools. The study of transcriptional level of these effectors in infected citrus samples was initiated. Many of the effectors were successfully detected by RT-qPCR in citrus RNA, suggesting their roles in pathogen virulence and host response modulation. The expression level and profiling of these effectors were being investigated for its connection with host tolerance level, through comparative analysis between HLB tolerant and susceptible genotypes. 6) Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, are being grown, and will be planted soon for field testing of HLB resistance. 7) Our putative chimeras have not proven to be successful. We identified a chimera (Satsuma and Poncirus) from the Citrus genebank, arranged its importation, and we finally got permission to accept this material and maintain it in a quarantine death house. Cuttings of the chimera and each separate component (Owari and Poncirus) have been rooted and will be challenged by hot ACP feeding in the next quarters.
1) Assessed use of isolated leaf inoculation, and small plant destructive sampling: Isolated leaf inoculations do not readily distinguish between resistant and susceptible citrus selections, but may prove useful in identifying nearly immune material. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This assay seems to be an efficient way to test transgenics that are expected to kill CLas. Recently we have had delays due to failures in ACP-inoculation and have reinitiated several challenges. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. Rooted cutting of 167 Carrizo plants were obtained. A subset of 67 plants representing 13 independent events and wild types (4-5 replicates each) were inoculated by ACP infestation. All of the plants except 2 were confirmed CLas positive after a 2-week ACP exposure, and the titer between wild type and transgenic groups are similar at two weeks. The plants are maintained in the greenhouse for tests at 3, 9 and 12 months after inoculation. Transgenics expressing AMP D2A21 suppressed canker but not HLB with manuscript accepted for publication. Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 5 below) have also been propagated for testing. 3) Two new chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A group of 100 Carrizo plants were obtained as rooted cuttings and will be used for HLB testing. 4) A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets citrus chloroplasts. Transcription data were obtained by RNA-Seq showing significant alteration in the transgenics. Publication submitted. 5) Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have putative transgenic Carrizo reflecting 69 events from 7 ScFv with verified transgenics ready for testing. These have been replicated by rooting and will be exposed to no-choice CLas+ ACP followed by whole plant destructive assays. 6) To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Paper is published. In-silico analyses are being conducted to develop citrus FLS2 optimized for sensing CLas flagellin. 7) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were downregulated in more tolerant Jackson compared to susceptible Marsh grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in Hamlin , Clementine , Carrizo , rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs aimed to silencing citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so gar) are ongoing. Comparison of reactive oxygen species in transgenic and nontransgenic plants treated with CLas-flg22 are underway, to determine if there is an enhancement of the broad-spectrum PAMP-triggered immunity . With targeted gene expression data, we will propagate selected plants based on the above-mentioned tests for HLB inoculations purpose. 8) Optimizing use of a SCAmpP (small circular amphipathatic peptide) platform, was conducted in collaboration with Dr. Belknap and Dr. Thomson of the Western Regional Research Center of USDA/ARS. SCAmpPs were recently identified and have tissue specific expression, including having the most abundant transcript in citrus phloem. Furthermore, members of the SCAmpP family have highly conserved gene architecture but vary markedly in the ultimate gene product. Variants of a tissue-specific SCAmpP were tested using GUS as a reporter gene: removal of the conserved intron reduced tissue specificity and deletion of non-transcribed 5 region reduced expression. Excellent phloem-specific expression is achieved in citrus when a target gene is substituted for the gene encoding the SCAmpP peptide. Expression of a GUS marker gene was 500 x higher in midribs vs. laminar area. We are using this promoter aggressively in transgenic work 9) Third generation chimeral peptides were designed based on citrus thionins and citrus lipid binding proteins and plants have been transformed. Carrizo transformation of two constructs was completed and regenerated many seedlings. expression. A total of 43 Carrizo regenerations were confirmed being positive by PCR and highly expressed by RT-qPCR. Two constructs with above gene driven by double 35S promoter have 400 explants of Ray Ruby for each. 10) Two constructs with chimeral peptides containing citrus thionin and citrus proteinase were developed with both encoding genes are under by 35S promoter and SCAmpPs promoters. Transformation of those constructs are ongoing.
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