This quarter the South Florida sites were sampled and added to the Indian River and Ridge sites for ImageJ analysis. This data is being compiled and organized for analysis within the neural network software. The merging and comparing of the collected data is still in progress. No new data has been released since the last report since all of the data from the August sampling is still being organized and compiled. Hurricane Irma made landfall in South Florida on September 10, 2017 as a Category 3 storm on the Saffir-Simpson Hurricane Wind Scale. We are still gathering data from all of our sites and do not think that this storm will impair our ability to continue with the study. The sites in South Florida and Indian River region, are flatwood sites, and all experienced flooding of some kind, with a few still flooded weeks after the event. This will influence the health of these trees in the future. This study should offer a unique perspective on HLB status/severity and tree recovery after a hurricane/flooding event. More will be known about all of these locations when we visit all of the sites in November. Objective 1: Leaf nutrient thresholds Samples from the August sampling are still in the process of being analyzed. The August sampling included leaf sampling for ImageJ analysis, qPCR, leaf starch content, and nutrition, as well as tree canopy measurements, SPAD, canopy height and volume. All three locations were sampled around the same time in August and we should finally have one complete data set from all sites across all locations. This data will be added to our very large database for analysis using the neural network software Easy-NN. We will look at the August sampling date as a snapshot in time across all sites and locations for any possible connection or correlation with HLB severity. Objective 2: Determine soil conditions that favor root hair and VAM proliferation i. Soil sampling will be completed in November and we will have a large set of samples for further soil analysis that we would like to work on. We have completed the measurements of permanent wilting coefficient on the first two years of soil data. Soils from the South Florida area will be included into the data set in November and will be measured for all of variables the other two regions have been measured for, including organic matter content, and color analysis. ii. We are investigating a new system for root hair development using Valencia and Murcott seedlings in a nutrient solution. Test runs of seedlings exposed to nutrient fog and hydroponics is underway. Currently a preliminary trial of nutrient solution concentrations is underway for feasibility. Both the nutrient fog and the liquid hydroponics systems are currently working well. In the next quarter, we will choose a method an expand the greenhouse study to include the 3 nutrient solutions, 1. Complete fertilizer, 2. Complete fertilizer minus Phosphorus, with Rock Phosphate (RP) as the source of Phosphorus, and 3. Complete fertilizer minus Phosphorus, with Triple Calcium Phosphate (TCP) as the source of Phosphorus. Currently in the complete fertilizer trial run, we do see the presence of microscopic root hairs, which we hope to promote using the RP and TCP fertilizer solutions.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics survived and passed a PCR screen, and these are currently being tested for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and therefore the putative transformants are being screened by RT-PCR. Eleven PCR-positive shoots have been grafted so far. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis, with several rooted plants obtained so far. Molecular characterization of the putative transformants will be carried out at UC Berkeley. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
1) Assessed use of isolated leaf inoculation, and small plant destructive sampling: Isolated leaf inoculations do not readily distinguish between resistant and susceptible citrus selections, but may prove useful in identifying nearly immune material. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This assay seems to be an efficient way to test transgenics that are expected to kill CLas. Recently we have had delays due to failures in ACP-inoculation and have reinitiated several challenges. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. Rooted cutting of 167 Carrizo plants were obtained. A subset of 67 plants representing 13 independent events and wild types (4-5 replicates each) were inoculated by ACP infestation. All of the plants except 2 were confirmed CLas positive after a 2-week ACP exposure, and the titer between wild type and transgenic groups are similar at two weeks. The plants are maintained in the greenhouse for tests at 3, 9 and 12 months after inoculation. Transgenics expressing AMP D2A21 suppressed canker but not HLB with manuscript accepted for publication. Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 5 below) have also been propagated for testing. 3) Two new chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A group of 100 Carrizo plants were obtained as rooted cuttings and will be used for HLB testing. 4) A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets citrus chloroplasts. Transcription data were obtained by RNA-Seq showing significant alteration in the transgenics. Publication submitted. 5) Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have putative transgenic Carrizo reflecting 69 events from 7 ScFv with verified transgenics ready for testing. These have been replicated by rooting and will be exposed to no-choice CLas+ ACP followed by whole plant destructive assays. 6) To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Paper is published. In-silico analyses are being conducted to develop citrus FLS2 optimized for sensing CLas flagellin. 7) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were downregulated in more tolerant Jackson compared to susceptible Marsh grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in Hamlin , Clementine , Carrizo , rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs aimed to silencing citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so gar) are ongoing. Comparison of reactive oxygen species in transgenic and nontransgenic plants treated with CLas-flg22 are underway, to determine if there is an enhancement of the broad-spectrum PAMP-triggered immunity . With targeted gene expression data, we will propagate selected plants based on the above-mentioned tests for HLB inoculations purpose. 8) Optimizing use of a SCAmpP (small circular amphipathatic peptide) platform, was conducted in collaboration with Dr. Belknap and Dr. Thomson of the Western Regional Research Center of USDA/ARS. SCAmpPs were recently identified and have tissue specific expression, including having the most abundant transcript in citrus phloem. Furthermore, members of the SCAmpP family have highly conserved gene architecture but vary markedly in the ultimate gene product. Variants of a tissue-specific SCAmpP were tested using GUS as a reporter gene: removal of the conserved intron reduced tissue specificity and deletion of non-transcribed 5 region reduced expression. Excellent phloem-specific expression is achieved in citrus when a target gene is substituted for the gene encoding the SCAmpP peptide. Expression of a GUS marker gene was 500 x higher in midribs vs. laminar area. We are using this promoter aggressively in transgenic work 9) Third generation chimeral peptides were designed based on citrus thionins and citrus lipid binding proteins and plants have been transformed. Carrizo transformation of two constructs was completed and regenerated many seedlings. expression. A total of 43 Carrizo regenerations were confirmed being positive by PCR and highly expressed by RT-qPCR. Two constructs with above gene driven by double 35S promoter have 400 explants of Ray Ruby for each. 10) Two constructs with chimeral peptides containing citrus thionin and citrus proteinase were developed with both encoding genes are under by 35S promoter and SCAmpPs promoters. Transformation of those constructs are ongoing.
The goal of this project is to generate green fluorescence protein (GFP) labeled Ca. Liberibacter asiaticus (Las), test its application in study of Las movement and distribution in planta, and investigate the control effect of different measurements including heat treatment and antimicrobial treatment. Las and other HLB-associating Liberibacters have not been cultured outside of their hosts in cell-free artificial culture media; therefore, traditional molecular and genetic analyses cannot be applied. This has greatly hampered our efforts to understand the virulence mechanisms of Las. We have been looking for alternative approaches to genetically manipulate Las in vivo. This has been made possible by the large population of Las in psyllid and availability of molecular tools to perform genetic manipulation in vivo. Alternatively, Las can survive for a short time in the media after acquired from psyllid gut and we aim to genetically modify Las with GFP immediately after Las being acquired from psyllids. To achieve the goal of this study, we will pursue the following specific objectives:1) GFP labeling of Candidatus Liberibacter asiaticus. 2) Elucidation of plant-Las interaction through real-time monitoring of Las movement and multiplication in planta using GFP labeled Las. 3) Investigate the effect of different control approaches on the dynamic population of Las in planta using GFP labeled Las. Previously, the reporter plasmid, pBAM1::R-PgyrA-GFP, composed of Tn5 and narrow host-range origin was constructed and therefore the GFP gene can be inserted into the genome of bacteria. However, it was only successfully transferred into a genome of Pseudomonas fluorescence with low transformation efficiency and failed with other bacteria including Escherichia coli DH5a, Sinorhizobium meliloti Rm1021, and Liberibacter crescens BT-1. Recently, pDH3::PgyrA-GFP was constructed which has a wide bacterial host range replicon, repW, but cannot be inserted into a genome. Transformation of E. coli by PEG mediated method with pDH3::PgyrA-GFP showed high transformation efficiency (~2 x 104 CFU/ g of DNA) than with previous reporter plasmid (failed). Following application with L. crescens BT-1 by electroporation was also successful (1.9 x 103 CFU/ g of DNA). Transformants and the GFP expression in L. crescens BT-1 were confirmed by PCR and fluorescent microscopic analysis, respectively. As L. crescens is a phylogenetically closest species to Ca. L. asiaticus, there is a possibility that pDH3::PgyrA-GFP would be useful for GFP labeling of Ca. L. asiaticus. We have further confirmed the Lcr-GFP using western blot. The GFP plasmid is being used to transform Las. To facilitate Las transformation, we have tested multiple novel methods of culturing. Las population was observed to decrease at the beginning, and increase slowly. We are in the process of repeating and optimization of the methods. 2) We have conducted Las movement and multiplication in planta based on qPCR method. One manuscript has been submitted. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript has been submitted.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) New replicates of the transgenic lines that have been inoculated by CLas-infected psyllids were maintained in the greenhouse for symptom development. Background: We have generated transgenic Hamlin sweet orange and Duncan grapefruit and screened the transgenic lines for HLB resistance or tolerance. We did not find any resistant line but identified three independent lines (two Hamlin lines and one Duncan line) that exhibit robust tolerance to HLB. This result indicates that the Arabidopsis NPR1 gene is able to create HLB tolerance in citrus. Since we only have HLB-tolerant Hamlin and Duncan , we decided to transform the NPR1 gene into other cultivars including sweet orange Pineapple and Valencia as well as grapefruit Ray Ruby (These plants were generated by the mature tissue transformation lab). The following table shows the new lines that have been inoculated by CLas-infected psyllids and are maintained in the greenhouse for symptom development: Replicate Genotype Transgene Parental line A1 Ray Ruby NPR1 21 81 Pineapple NPR1 15 75 Pineapple NPR1 16 66 Pineapple NPR1 17 78 Pineapple NPR1 18 84 Valencia NPR1 20 76 Hamlin NPR1 8 73 Hamlin NPR1 10 67 Hamlin NPR1 11 70 Hamlin NPR1 12 87 Hamlin NPR1 13 79 Hamlin NPR1 14 (2) Propagated plants from those that have been treated with CTV-FT3 and have produced flowers for later heat treatment to remove CTV and CLas. Background: The three independent lines ( Duncan 57-28, Hamlin 13-3, and Hamlin 13-29) with robust tolerance to HLB have been treated with CTV that carrying the FT3 gene, which promotes conversion from juvenile to mature tissues. The three lines have all developed blooms. The flowering-promoting CTV and HLB bacterial pathogen (CLas) in the transgenic plants need to be removed before producing healthy progenies. The following table shows the new lines received from the transformation lab and will be replicated and screened for HLB responses: Transgenic line Genotype Transgene Maturation Replicates made 57-28 Duncan NPR1 Yes 4 13-3 Hamlin NPR1 Yes 6 13-29 Hamlin NPR1 Yes 3 (3) More transgenic lines received from the transformation lab were transplanted into bigger pots and will be analyzed for the transgenic protein accumulation. Background: We have not obtained multiple independent lines for Valencia and Ray Ruby and have requested more from the mature tissue transformation lab. The following table shows the new lines received from the transformation lab and will be replicated and screened for HLB responses: Transgenic line Genotype Transgene A99 Valencia NPR1 A100 Valencia SuperNPR1(a) A102 Valencia SuperNPR1 A101 Valencia SuperNPR1 A72 Valencia ELP3 (b) A73 Valencia ELP3 A97 Hamlin SupperNPR1 A98 Hamlin SupperNPR1 (a) SupperNPR1 is a more active version of the NPR1 gene. (b) ELP3 encodes a disease resistance regulator, which appears to also provide tolerance to HLB. A manuscript titled Overexpression of the Arabidopsis NPR1 protein in citrus confers tolerance to Huanglongbing has been written and submitted to the Journal of Citrus Pathology in this quarter.
In Objectives 1 and 2, we proposed targeting specific regulators of key phage encoded virulence genes (such as the Las LexA-like repressor, LC1, a second downstream repressor, LC2, controlled in part by LC1), and a key exogenous regulator of the (lethal) phage lytic cycle encoded by Wolbachia, an important psyllid endosymbiont that is always found when Las is present. These results have so far resulted in three full length manuscripts and 14 abstracts. LC1, LC2 and the Wolbachia repressor have all been confirmed to be transcriptional repressors. All three are therefore prime targets for chemical interference. Purchase orders to have these three repressors commercially produced by synthesized were placed several months ago and all three have finally now been delivered. Both the LC1 and the Wolbachia repressor were charactereized by the commercial vendors as “difficult” to synthesize. Attempts in our lab to synthesize LC1 and the Wolbachia repressor indicated that both cause E. coli host cells to become quite “sick”, with very slow growth, likely because of the repressors’ effects on E. coli host gene regulation. These proteins were synthesized in vitro for evaluation purposes, but this is expensive for the scale needed for chemical screening purposes and slow delivery of the repressor proteins was not anticipated. Two versions of the Wolbachia repressor were delivered. One version, with a histidine (HIS) tag, failed to repress the Las target promoter, probably due to the presence of the tag. The native version, without the tag, worked as expected from the in vitro results. Chemical screening assays are now underway with all three. The Wolbachia protein has been shipped to both the De La Fuente lab in Auburn, and Duan lab at USDA-Ft. Pierce, our collaborators on a separate culturing project.
During the period of April, May, and June of 2017, most of the progress was related to application for follow-up funding to characterize HLB resistance of plants generated in the present project. With assistance from Dr. Catherine Hatcher (CRDF), Dr. McNellis has coordinated with three faculty at the University of Florida to make arrangements for HLB resistance testing for the existing plants expressing anti-NodT scFv antibody (FT-scFv). These faculty are 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. We plan to submit a pre-proposal to the CRDF for funding to support HLB resistance testing of the existing trees at Fort Pierce, propagated in Dr. Tim Gottwald’s lab at the USDA facility there. This will allow us to infect and perform quantitative bacterial population analysis in the citrus trees. We plan to submit this CRDF pre-proposal by the end of August, 2017. Dr. McNellis also attended and gave a presentation at the May 22-23, 2017, Forum on Citrus Breeding and Transformation for HLB resistance hosted by the National Academies of Sciences, Engineering, and Medicine in Irvine, CA. In addition, Dr. McNellis, along with Drs. Batuman, Cano, Ferrarezi, and Vladimir Orbovic (also University of Florida) submitted a pre-proposal to the USDA Citrus Disease Research and Extension Specialty Crop grant program on May 12, 2017, and were invited to submit a full proposal as of June 28, 2017. This proposal uses the present research results from this CRDF project as the preliminary data for the proposal, and builds upon results from the present work. We plan to submit the full proposal for the August 18, 2017, deadline.
This project continues to make progress. We have grafted the new copies of the LuxI plants and they have grown well. We have collected leaf samples from these plants to test them for the presence of acylated homoserine lactone (AHL). We also have already set up a gas chromatography mass spectrometry (GC-MS) method for the detection of AHL in the LuxI plants and we were able to detect these AHL in spiked leaf samples. Our next step is to test the presence of AHL in LuxI plants. In addition, the LuxI plants are being challenged with Diaphorina citri to determine the effect of expressing of (AHL) on the pathogenicity of Candidatus Liberibacter asiaticus (CLas). Our preliminary observation showed that HLB symptoms are localized in specific areas in the LuxI plants, which means that the presence of AHL prevent the spread of CLas pathogen. In our next step, we will use polymersae chain reaction (PCR) to quantify the level of CLas in CLas-infected LuxI and control plants. This work will be accomplished by December 2017. We look forward to the results of this work hoping that it will provided valuable information to the citrus research community.
The merging and comparing of the collected data is still in progress. The most recent data addition has been from the Indian River location. In August, more new data from the South Florida area will be incorporated. Using neural networks to analyze the data is underway and a few compelling results have been obtained and need further validations. Objective 1: Leaf nutrient thresholds At this point, we have just begun to analyze the combined data using the neural network software Easy-NN, including the Indian River Data set with the Ridge data set, which is complete through May 2017. We are looking at the sample dates as snapshots in time and combined for any possible connection or correlation with HLB severity. Continuing from our previous leads using mean leaf perimeter and leaf area as outputs in generating neural networks, we have found that along with calcium and magnesium, iron and copper are looking like important variables to watch with severity of HLB infection. When we run the neural network with soil parameters from the Ridge and Indian River area, we find that the organic matter content still plays a key role as well as soil pH and soil Mg concentration. Another vital variable to explore further is the L* parameter (lightness in color of the soil). As L* increases, the mean leaf area decreases. However, on its own, this variable is not well suited for predicting HLB incidence or severity; it does help to show the importance of organic matter in tree health. Looking further into the soil parameters as inputs and using leaf thickness (grams of leaf dry mass / meter square leaf area) as an output, we find the most important soil variables to be the soil magnesium content, as well as the lightness of the soil (L*), potassium content, and soil organic matter content. These are only preliminary results and more investigation is necessary as well as increasing the data set over the next few quarters. In the next quarterly report, we hope to be able to include data from the South Florida Region. A preliminary set of samples was just delivered for image analysis and soil samples should be forthcoming. Objective 2: Determine soil conditions that favor root hair and VAM proliferation i. We have discussed further soil analysis that we would like to work on, including data about permanent wilting points as well as possibly quantifying the silicon content of the soil. Soils from the South Florida area will be included into the data set and will be measured for all of variables the other two regions have been measured for, including organic matter content, and color analysis. ii. We are investigating a new system for root hair development using Valencia and Murcott seedlings in a nutrient solution. Test runs of seedlings exposed to nutrient fog is underway.
During the reporting period, we continuously examined the effects of the two proposed genes on transient and stable transformation efficiencies of mature and juvenile citrus plant tissues. The experiments produced mixed results. We are repeating these experiments to obtain more definite conclusions. Two of the many chemicals we have tested consistently display 2-3 fold increases in transient and stable transformation efficiency. Meanwhile, our preliminary results suggest that we can also manipulate and enhance Agrobacterium-mediated transient expression and stable incorporation efficiencies of T-DNA genes. Increase in transient expression of the T-DNA gene expression in citrus tissues could help us to develop a technique to use CRISPR to produce non-transgenic mutants of citrus, which can facilitate the use of CRISPR to produce non-transgenic HLB resistant cultivars of citrus. A manuscript using Agrobacterium-mediated transient expression of CRISPR genes to produce non-transgenic mutants of a model plant species will be submitted for a consideration of publication soon.
During the reporting period, we continuously examined the effects of the proposed genes on transient and stable transformation efficiencies of mature and juvenile citrus plant tissues. We now conclude that two of the four genes examined cannot significantly enhance the transformation efficiencies of citrus tissues consistently. The other two genes are still under testing. On other hand, we tested effects of several chemicals on citrus transformation efficiencies, three of the five chemicals tested show 2-3 fold increases in transient and stable transformation efficiency. We are currently testing the effects of their combinations and developing a protocol of incorporating these chemicals in the culture media at various stages. Increases in stable transformation efficiencies of citrus will facilitate the use of transgenic technologies to create HLB resistance traits. Increase in transient expression of the T-DNA genes can help production of non-transgenic CRISPR-mediated mutants using Agrobacterium.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We tested 27 antibacterial compound producing bacteria including Bacillus cereus, B. licheniformis, Paenibacillus polymyxa, Pseudomonas spp., Streptomyces aureofaciens, Streptomyces fradiae, Streptomyces fradiae, Streptomyces garyphalus, Streptomyces griseus, Streptomyces kanamyceticus, Streptomyces niveus, Streptomyces pristinaespiralis, Streptomyces virginiae, Streptomyces ribosidificus, Streptomyces venezuelae, Streptomyces vinaceuse, and Streptomyces capreolus. We have isolated 327 bacteria from Florida citrus groves. The antagonistic activity against Agrobacterium, Sinorhizobium meliloti, L. crescens and Xanthomonas citri pv. citri was determined. 21 strains including bacteria belonging to Paenibacillus, Burkholderia, Bacillus, and Streptomyces showed good antagonistic activity. Those isolated bacteria showing high antimicrobial activities have been sequenced to help us understand the mechanism and for identification purpose. Currently, the genome sequencing was finished and genome analysis was completed. Because Las infection also affects host resistance to Phytophthora, one common citrus pathogen in Florida, we tested the antimicrobial activity of the bacterial isolates against Phytophthora nicotinae and P. palmivora, multiple bacterial isolates showed antimicrobial activities against Phytophthora spp. Four bacterial strains: two Burkholderia, one Pseudomonas geniculata, and one Rhodococcus strains have been tested for their activity in controlling citrus HLB and canker and all showed induced plant defenses and control effect against infection by Xanthomonas citri. The HLB result is shown below. To further study the antimicrobial producing bacteria, tow Burkholderia strains have been labeled with GFP tag. Seven other strains are being labeled with GFP or RFP tag. We also investigated the antibiotic genes in the 21 antimicrobial producing bacteria that we isolated previously. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi). In a separate experiment, two Burkholderia strains were tested and up to 10E5 cfu/g soil was recovered at five days post inoculation. Four antimicrobial producing bacterial strains belonging to Paenibacillus, Bacillus sp., and Pseudomonas geniculata were tested in field trial via a soil drench method applied every two months for one year. The treated trees were divided into the following categories based on the disease index of 0-5: 1) No symptoms or few symptoms (0-2); 2) Trees with severe HLB symptoms (3-5). One gallon of bacterial culture was applied per tree at the three concentrations: 10E6, 10E7, and 10E8 CFU/ml. Water treatment was used as negative control. The result demonstrated the applied bacteria survived better in rhizosphere soil than applied via irrigation, but the overall survival in the soil is still limited and the bacteria did not establish high population on the root surface. Application of beneficial bacteria slowed down the disease index increase and Las titers for the asymptomatic or trees showing few symptoms compared to the control, but it did not prevent the disease index and Las titers from increasing. For the trees showing severe symptoms, the applied bacteria did not show any effect on disease index and Las titers. One manuscript has been submitted to Frontiers in Microbiology. One more manuscript is being prepared.
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) Trees have been in the ground for 3.5 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 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 is in preparation to map genes associated with tolerance. 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 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) 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. 6) Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance is completed, has revealed potentially valuable tolerance and indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. In August 2010, the plants were established at Pico s farm in Ft. Pierce FL. Despite the high incidence of mottle in SugarBelle / SourOrange, it had the greatest overall increase in diameter. ‘SugarBelle’ and ‘Tango’ (which were not on the same stock as ‘Hamlin’ and so results should be viewed as comparing cultivar/rootstock combinations) were the healthiest in overall appearance in 10/15 and had the most fruit (88 per tree). 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 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.
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