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.
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. To facilitate Las transformation, we have tested two 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 two 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. One manuscript has been submitted.
1) Plants were generated after exposure of buds to several antibiotic levels to generate a range of infections and plants were monitored for HLB development, CLas, and growth. Within each genotype there was no correlation between final size after 42 months and the CLas titer. The documented tolerant ‘Jackson’ grapefruit and the grapefruit-like selection Ftp 6-17-48 displayed significantly greater stem diameter than all other genotypes. 2) Trees have been in the ground for over three years in a trial of 50 selections and cultivars 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 quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. 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. It may take 2-3 more years to clearly distinguish tolerant material. 3) 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. 4) Trials are established. Volatiles from sweet-orange-like hybrids were shown to be so similar to sweet orange that likely can be legally designated as sweet orange. 5) RNA-seq compared transcriptome responses in HLB moderately tolerant Sun Chu Sha mandarin and susceptible Duncan grapefruit, to Xcc-flg22 and CLas-flg22 (project initiated with Gloria Moore at University of Florida). Recently data analysis revealed that a group of 86 genes were differentially regulated by CLas-flg22 in Sun Chu Sha mandarin but not in Duncan grapefruit and not associated with differential expression from Xcc-flg22, suggesting they may have roles in HLB tolerance. The 16 genes with highest differential expression were selected for RT-qPCR validation, and 10 genes were consistent with the RNA-seq results. To evaluate if these genes were associated with HLB tolerance, Cleopatra mandarin (similar to Sun Chu Sha ) and Duncan grapefruit plants were inoculated with CLas using psyllid infestation. CLas titer and gene expression were monitored biweekly for 10 weeks after inoculation. High bacterial titer (Ct<30) was observed at 2 weeks in Duncan but not until 6 weeks in Cleopatra . RT-qPCR results indicated that 5 of the studied genes were differentially expressed between the Cleopatra HLB-infected and the uninfected control plants, but not in Duncan . Induction of these genes was detected before bacterial infection was detected. Although not fully annotated in the citrus genomic databases, the function of some of these genes include a peroxidase, gibberellin 2-beta-dioxygenase, glucan endo-1,3-beta-D-glucosidase and an F-box domain containing protein. We will continue to characterize the expression of these genes and their association to HLB tolerance in other citrus genotypes, and determine if they may serve as marker genes for selection of tolerant citrus material. 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 in the spring for field testing of HLB resistance. 7) 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). 8) Data are being collected which will contribute to the objective of assessing economic value of tolerance 9) This objective focuses on rapid HLB-resistance screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees that are exposed to no-choice CLas infected ACP feeding. CLas titer levels, using real time PCR, are easily detectable in most plants at 3 weeks. By nine weeks after exposure, susceptible genotypes can be clearly distinguished from reported resistant material by higher CLas levels in roots. Averaged across genotypes and tissues, total CLas per tree was 5 billion in week 3 after ACP exposure and doubled every 3 weeks through week 12. This should be especially useful for screening anti-Las transgenics. 10) Our putative chimeras have not proven to be successful. We identified a chimera (Satsuma and Poncirus) from the Citrus genebank, arranged its importation, and have diligently communicated with DPI to get it released to us. Shoot-tip-grafting has not been successful, but we finally got permission to accept this material and maintain it in a quarantine death house. 11) Initial cuttings have been made.
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.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not the cross protection using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. In order to define the mechanisms that phage are employing to overcome abiotic stress, we designed and optimized specific primer sets for quantitative reverse transcription PCR (qRT-PCR) for genes within the phage region that were likely regulated by heat treatment and other stress. Because this analyses are based on mRNA transcript level and not on genomic DNA, the upregulation of phage genes reflects the relative level of transcript of active living cells. In order to ensure adequate generation of cDNA from Las, we found it necessary to use individual primers specific for the targeted region instead of generalized primers such as random hexamers. The cDNA that was generated in this fashion was then used as the template for qRT-PCR. The present analysis included three biological samples for each condition and threetechnical replicates for each sample for statistical purposes. Particular genes that were found to be upregulated included: CLIBASIA_5590 encoding an unknown protein, CLIBASIA_5610 encoding a putative phage terminase (large subunit), CLIBASIA_5665 encoding an unknown protein, CLIBASIA_5390, which has the conserved sodium: dicarboxilate symporter family domain, CLIBASIA_5525 encoding a guanylate kinase that catalyzes the reaction ATP + GMP <->ADP + GDP. Phage genes found to be downregulated included: CLIBASIA_00005 encoding an unknown protein, CLIBASIA_00010, which has an NTPase domain of typical DNA-packaging enzyme, CLIBASIA_00030 encoding a putative DNA polymerase of bacteriophage origin, CLIBASIA_5565, which has the conserved domain TolA protein and is thought to be required for the translocation of the phage DNA. This data correlated well with what was seen via our previous RNA-Seq analysis and helps reveal the transcriptional response of the phage to abiotic stress factors. Given that previous studies on thermotherapy showed an overall reduction in Las titer in citrus affected by HLB post heat treatment, harnessing the ability to control these particular genes may allow us to lower the bacterium s ability to handle stress. Based on the variations of Las prophages/phages, we recognized certain molecular mechanisms behind the symptom variations and their association with “mild strains” of Las bacteria and host tolerance/resistance. The titration dynamics between 16S DNA-based and phage gene-based results revealed the association of host tolerance with the dynamics. Construction of a transcriptional reporter system is also currently in progress for the final verification of the genes identified as being involved in stress response to heat in plants subjected to thermotherapy. This system will also allow future experimentation to rapidly identify other catalysts that can produce the same reduction in bacterial numbers as thermo-therapy. To investigate the effects of stress on the genes involved in the phage lytic cycle, we identified several phage genes that were over-expressed in citrus plant after heat treatment. These results indicated that thermotherapy has a direct effect on Las bacteria by actively regulating specific phage genes. To further evaluate genes related to stages of the phage lytic cycle, we further compared Las genes expression profile from two distinct insect vectors, psyllids, and mealybugs. Mealybugs were found to contain much higher titers of Type D when compared with psyllids, indicating that the phage may be more active in the mealybugs than in the psyllids. New results revealed Type D prophage/phage is smaller than the prophage/phage A and B, and the association of Type D with suppression of Las bacterial population and HLB symptom expression is under investigation. Using an enrichment method to acquire RNAseq data, we compared the Las transcriptomes between the two insect vectors, and revealed that psyllids samples contained more than four times reads of the Las 16s rRNA than those of mealybugs samples, indicating much higher bacterial titers in psyllids than in mealybugs. However, mealybug s transcriptome profiling showed much higher expression level of prophage genes than those in psyllids, where expression level of prophage genes was completely absent or extremely low. Interestingly, more than 2/3 of the highly expressed genes in mealybugs were identified as prophage/phage genes. Interestingly, eight genes with the highest level of expression in mealybugs were identified as the highly expressed ones in Las-infected citrus after heat treatment. These results indicates that both stresses caused by thermotherapy and in mealybugs environment triggered similar signaling pathways, and result in the expression of prophage genes that may induce lytic cycle and eventually reduce Las titer in citrus plant treated with heat stress, and maintain low titer in mealybugs. We also revealed another mobile element that co-exist as a high-copy element as the prophage/phage D in mealybugs and periwinkle. How these mobile elements suppress the HLB are under investigation.
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 is ongoing. 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: 106, 107, and 108 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 is being prepared to report the findings.
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.
During this reporting time period, we made the following progresses for the proposed objectives: We have been testing the effects of the proposed genes on transformation efficiencies of both juvenile and adult citrus tissues particularly on transient and stable transformation efficiencies. The results have been inconsistent and we are conducting additional testing. We have also testing the effects of selected chemical agents for transient and stable transformation efficiencies of citrus. Our preliminarily results show that some agents may be effective in enhancing transformation efficiencies. We are further testing the effects of over-expressing an auxin biosyntetic gene predominately in roots on success rate of micro-grafting. A manuscript reporting effects of over-expression of an auxin biosynthetic gene on grafting success has been accepted by “Plant Biotechnology Journal”. The auxin gene may be useful for micro-grafting of citrus plants. The relevant genes have been shipped to Dr. Janice Zale, The Mature Citrus Facility (MCF) of the University of Florida.
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.
A test site for material proposed to have enhanced HLB resistance has been maintained at the USDA/ARS USHRL Picos Farm in Ft. Pierce, and has been open to requesting researchers for experimental plantings. As space is needed and experiments conclude, trees are pushed and replaced. Dr. Jude Grosser of UF has provided hundreds of transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has planted an additional 100 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenics in place. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov and he has trees expressing lectins under a USDA-ARS permit. Discussions have been ongoing with Eliezer Louzada of Texas A&M to plant his transgenics which have altered Ca metabolism to target canker, HLB and other diseases. Answers have been provided to numerous questions from regulators to facilitate field testing approval. APHIS BRS has visited the site on several occasions and confirmed that plantings are in compliance. We have provided permit details to assist plantings by other groups. A number of plants have been pushed as the analysis has been completed and new plantings made. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 7 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Ramadugu et al., 2016), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Tolerance to HLB was studied, with citron-derived material showing special promise within the genus Citrus (Miles et al., 2016). A new UF-Gmitter led association mapping study is nearing completion using the same planting, to identify genes associated with HLB- and ACP-resistance. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Yu et. Al., 2015). David Hall led an effort to characterize ACP colonization and while pure Poncirus had much lower colonization, all citranges were intermediate between Citrus and Poncirus. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids continue to grow well, with dense canopies and good fruit set, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population has been planted in a collaboration between UCR and ARS to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. An additional trial, led by ARS Riverside is assessing HLB response in a broad array of Poncirus-derived accessions.
The new Post-Doc to take over the responsibilities of continuing and finishing this project, which had been paralyzed since the departure of the previous pos-doc ,arrived in mid September. Preparations to get the project back on track had already begun in terms of plant material, chemicals, greenhouse space, irrigation, etc. Upon his arrival, we started the objectives listed below: Objective III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. Trees were treated with SL and soil amended with Phytophtora and or fungicide following the established sequence of events. Trees are being monitored and the second SL treatment will be applied soon. Objective IV: Effect of SL on the growth of beneficial mychorriza. Trees have been treated with SL and mycorriza applied to the soil as planned. The second SL treatment will take in early spring. Objective V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic such as organic acids, sugars, amino acids, and few other compounds will be applied on potted trees. Trees are being sprayed bi-weekly with a 10 mM solution of individual components. In the meantime, data are being collected on the above treatments, two manuscripts are being prepared from previous work and we are gearing up to address the remaining objectives.
The new Post-Doc to take over the responsibilities of continuing and finishing this project, which had been paralyzed since the departure of the previous pos-doc ,arrived in mid September. Preparations to get the project back on track had already begun in terms of plant material, chemicals, greenhouse space, irrigation, etc. Upon his arrival, we started the objectives listed below: Objective III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. Trees were treated with SL and soil amended with Phytophtora and or fungicide following the established sequence of events. Trees are being monitored and the second SL treatment will be applied soon. Objective IV: Effect of SL on the growth of beneficial mychorriza. Trees have been treated with SL and mycorriza applied to the soil as planned. The second SL treatment will take in early spring. Objective V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic such as organic acids, sugars, amino acids, and few other compounds will be applied on potted trees. Trees are being sprayed bi-weekly with a 10 mM solution of individual components. In the meantime, data are being collected on the above treatments, two manuscripts are being prepared from previous work and we are gearing up to address the remaining objectives.
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.
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