The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications; and 3) to create transgenic citrus cultivars with new constructs containing the resistant genes. A total of 25 samples for RNA-Seq, including resistant/tolerant vs. susceptible plants were sequenced and analyzed. We mapped the RNA-Seq data to a reference genome, C. clementina using the bioinformatics program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24,275 genes of the originally predicted genes had been found to be expressed and a total of 10,539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to be expressed. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of ESTs varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 5 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component (PC) analysis study on the expressions of five samples. The results showed that the gene expressions were significantly different in resistant vs. susceptible citrus. Using cuffdiff, a total of 821 genes were identified as difference expressed genes (DE genes) between the two groups using both p-value and an FDR threshold of 0.01. Among them, 306 genes are up-regulated and 515 are down-regulated in resistant citrus. Using the program iAssembler, a total of 53,981 uni-transfrags were obtained. Most of the assembled uni-transfrags should be novel genes, compared with the citrus reference genome. We further identified 3073 DE genes by comparing the gene expression of another group of resistant and sensitive citrus samples using DESeq2 with adjusted P-value (padj) < 0.1. Among these DE genes, 1413 genes were up-regulated in resistant citrus significantly and 1660 genes were down-regulated in resistant citrus. Due to our comparing strategy, the DE genes should most come from the difference between Marsh and Jackson. As all of the 3 resistant citrus came from the offsprings of Jackson and all of the sensitive citrus trees came from that of Marsh. The resistance genes should come from Jackson. A total of 86 DE genes were identified in the tolerant and intolerant citrus group using DESeq2 with adjusted p-value less than 0.01. Among these DE genes, 69 genes were up-regulated and 17 genes were down-regulated in the tolerant samples. We selected top 50 different expressed genes from the up-regulated and down-regulated genes each for the further validation. There are some differences at the isoforms level between the resistant and sensitive samples. Although 205 out of 221 of the different expressed isoforms consist with the expression pattern at gene level, there are 16 different expressed isoforms don't consist at the gene level. For an example, isoform TCONS_00090333, a RING finger and transmembrane domain-containing protein 2, significantly down-regulated in the resistant citrus samples (log2 = -7.17), but the corresponding gene XLOC_025451 have no significantly change between resistant and sensitive citrus samples.
St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). Collection of yield and fruit quality data was completed with the assistance of Ingram Grove Service. Overall yield among 6-year old trees was down 18% compared to last year (trees now 75% infected). Data is still being analyzed – we expect to identify rootstocks that held or increased their yield over the past year. The nutrition program has been modified with good results: for the last two applications, the Schumann TigerSul blend has been added to the Harrell’s UF controlled release fertilizer before application. Spring bloom/flush across the entire trial was very good. Greenhouse Experiments – Nutritional study: preliminary results are quite interesting on the growth of the HLB infected Valencia trees on Orange #15 rootstock. The treatment with the 3x overdose of TigerSul manganese is showing the best consistent growth across the entire group of trees (large green fully expanded leaves); control trees look terrible. Other TigerSul product overdoses are also performing well. There appears to be a synergistic interaction between the Harrell’s UF mix and the TigerSul. A few trees (but not all) in the group of trees overdosed with controlled release sodium borate also look good. In the rootstock study, the combination of Harrell’s UF controlled release + biochar has inhibited disease development across all the rootstocks; some symptomatic trees are showing a recovery phenomenon. We will design a remediation strategy for the symptomatic trees based on the results of the nutritional study. Protecting Seed Source Trees: 1. Transgenic Orange #4 plants containing the GNA transgene have been clonally multiplied as rooted cuttings and will be evaluated once they have sized up. Transgenic Orange #16 and Orange #19 tetrazyg plants transformed with GNA are being grown out to provide vegetative material for propagation via rooted cuttings (during the next quarter).
320 Hamlin on Carrizo trees in CREC Block 8 were treated with biochar and a mix of Harrell’s UF controlled release fertilizer and the Schumann TigerSul blend (iron/zinc/manganese in sulfate form). These trees are 100% HLB infected and also show blight and Diaprepes damage. Trees were topworked with potentially more HLB-tolerant scions by commercial topworking expert Jeremy Murdock. Topworking was completed in mid-March, and trees will be unwrapped and cut on April 21st. Topworked trees were mapped according to the scion. Scions included: 1. C4-16-12; 37 trees (triploid hybrid orange with 8% trifoliate orange, showing no HLB) 2. OLL-4; 40 trees (only OLL at Alligator still showing no HLB) 3. OLL-6; 34 trees 4. OLL-7; 23 trees 5. OLL-10; 20 trees 6. OLL-3; 29 trees 7. OLL-23; 22 trees 8. OLL-27; 20 trees 9. FG 2014 #1; 11 trees 10. FG 2014 #2: 10 trees 11. FG 2014 #3; 8 trees 12. Vernia C2-4-3; 7 trees 13. Valencia N7-3; 10 trees 14. Valencia N7-11; 8 trees 15. Hamlin T8-49; 10 trees 16. Valencia control; 8 trees; 17. red grapefruit N11-7; 7 trees 18. red grapefruit N11-15; 6 trees 19. red grapefruit N11-29; 1 tree
Transgene Stacking for Long-Term Stable Resistance: PCR analysis of transgenic plants containing the NPR1 gene (best gene in our program for HLB resistance) stacked with the CEME transgene (best gene in our program for canker resistance) resulted in identification of 7 lines that contain both transgenes. Agrobacterium mediated transformations to produce transgenic plants containing other combinations of stacked genes are in progress. Transgenic plants with the PCR positive stacked genes are being clonally propagated for further evaluation. Improving Consumer Acceptance: 1. In efforts to develop an intragenic citrus plant, we have in addition to the binary vector for an inducible cre-lox based marker free selection containing the cre gene driven by a Soybean heat shock gene promoter, a binary vector containing the cre gene driven by a citrus-derived heat shock promoter. Citrus rootstock Carrizo has been transformed with both of these constructs and numerous transgenic plants are being regenerated. 2. Hamlin and W Murcott cells have been transformed with a binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cells. One of the T-DNA contain a grapevine myb gene under the control of a 35s promoter and the other contain T-DNA containing the selectable positive/negative fusion marker cassette. Plants have been regenerated that are purple in color from the anthocyanin production. Pending molecular analysis of the regenerated lines, we speculate one of two possible scenarios: a) The plants contains only the T-DNA of interest or 2) The plant contains both T-DNAs integrated into the genome. Somatic embryos are now being germinated and resulting transgenic plants will be evaluated. Induction of early flowering: The citrus FT gene has been incorporated into Carrizo citrange. Numerous transgenic plants containing the Citrus FT stacked with the citrus AP1 has also been produced for testing. Since previously generated FT and AP1 plants flower in vitro but not as young plants in the greenhouse, we are testing the possibility of a synergy when both are present together. Transgenic plants are growing in the laboratory and will be tested for the presence of the gene when they reach suitable size. Propagation of new transgenics for field testing: ‘ Propagation of LIMA-B (AMP) transgenic plants for further study; ‘ Propagation of Carrizo transgenic lines with LIMA gene to test a potential rootstock effect on non-GMO scion. Efforts to establish a new transgenic field site: Working with Dr. Phil Stansly, we have submitted an addendum to our transgenic field permit with APHIS to add an additional field site which would be located at the UF Immokalee Research and Education Center. We plan to plant 400 new transgenic trees at this site after approval. A few hundred trees have also been prepared for planting at the USDA Picos Farm site.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants transformed with citGRP1 were test by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some of them and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. DNA was isolated from 32 of them and PCR test confirmed 28 are positive. Canker test showed two of them greatly increased resistance at the infiltrated concentration of 107/CFU. DNA was isolated from 10 chimera transgenic Hamlin and PCR test confirmed 9 of them are positive. They will soon ready for RT-PCR for gene expression. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector (collaboration with Duan lab). Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted with eighty Carrizo and ten Hamlin transformants planted in soil. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slightly canker resistance by infiltration test. A series of transgenic scions produced in the last several years continue to move forward in the testing pipeline. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
Evaluation of standard cultivars (‘Hamlin’, ‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, and ‘Ruby Red’) for HLB resistance/tolerance is underway. All trees exhibited symptoms of HLB and tested positive for CLas. Imidacloprid was applied quarterly to a subset of trees and significantly increased stem diameter compared to the non-treated trees but did not have a significant effect on tree height, disease severity, or Candidatus Liberibacter asiaticus (CLas) titer levels. There were significant differences in disease severity, stem diameter, and CLas titer between the varieties. ‘Fallglo’ had the lowest incidence and ‘Ruby Red’ the highest incidence of distinctive HLB mottling. The highest CLas titer levels in 2012 were observed in November and December, with ‘Sugar Belle and ‘Tango’ displaying the highest titer levels while ‘Fallglo’ and ‘Temple’ had the lowest levels. Despite the high titer CLas levels of ‘SugarBelle’, it had the greatest overall increase in diameter and was among the healthiest in overall appearance. Initial results indicate that compared to ‘Hamlin’, ‘Fallglo’ and ‘Temple’ appear to display field resistance to HLB while ‘SugarBelle’ appears to have significant tolerance. To generate plants with a range of CLas titer levels, bud-wood from nine varieties/genotypes, 3 putatively HLB-resistant (‘Temple’, ‘Gnarly Glo’, and ‘Nova’), 3 HLB-tolerant (‘Jackson’, FF 5-51-2, and Ftp 6-17-48), and 3 HLB-susceptible (‘Flame’, ‘Valencia’, and ‘Murcott’) were treated with various concentrations of penicillin and streptomycin. In April 2014, several treatments were repeated due to high bud mortality in December 2013. Treated buds were grafted onto sour orange rootstock. Once the new shoots are established, HLB symptoms and tree growth will be monitored on a monthly basis for a two-year period and compared to Liberibacter-infected and healthy standard varieties. Quantification of CLas titer levels will be conducted on a quarterly basis using real time PCR. Progress has been made on the development of HLB-resistant chimeras. Under in vitro conditions, etiolated hypocotyls from seedlings of Poncirus trifolata (‘Gainesville’, ‘Winter Haven’, and ‘Sacaton’) and ‘Hamlin’ were approach grafted. After the callus develops, a horizontal cut through the grafting union will be made and treated with growth regulators (gibberellic acid, .-napthaleneacetic acid, and 6-benzylaminopurine). Under greenhouse conditions, seedlings of ‘Carrizo’ were successfully approach grafted to ‘Hamlin’ and treated with growth regulators (as mentioned). The regenerated plants will be examined using LC/MS, which differentiates the parental layers based on unique chemical signatures. A method for the rapid identification of potential sources of HLB resistance is currently being developed. Seedlings of ‘Dancy’, ‘Hamlin’, and ‘Carrizo’ at the 3 to 5-leaf stage were exposed to HLB or non-HLB infected ACP feeding trials. Entire leaves, stem, and roots are being processed at 3, 6, 9, and 12 weeks after the initial feeding period. At the 3 week sample period differences in secondary metabolites of methanol extracts from HLB and non-HLB infected leaves was observed using LC/MS. Using real time PCR CLas was detectable in all parts of the seedlings, with the exception of the roots from ‘Carrizo’. ‘Dancy’ stems and leaves had the highest CLas titer levels.
This quarter we have made good progress on our transformation approaches: 1. Stable transformations in citrus using new vectors. Transformation experiments were carried out in Duncan grapefruit and carrizo with 5 constructs in a preferred vector backbone, including an empty vector control . 52 carrizo transformants were selected and analyzed by PCR. The genes of interest that were screened for included avrGf1, avrGf2, and NPTII. PCR results showed that 5 of 6 transformed with the avrGf1 gene were positive and 9 of 30 plants were positive for the avrGf2 gene. NPTII was detected in 22 of the 52 carrizo plants. 6 out of 7 Duncan plants were positive for NPTII. These results demonstrate improved transformation efficiency using this vector backbone. Further epicotyl transformation experiments were carried out with both ‘Duncan’ grapefruit (624 segments) and ‘Pineapple sweet orange (136 segments) with 6 constructs. These segments were placed on media without selection (kanamycin). This modification was done to see if transformation efficiency would improve. Rooting experiments and transplanting segments are ongoing. Of 1,507 transformants selected so far, 168 putative transgenic shoots of grapefruit, sweet orange and Carrizo were confirmed as transgenic by GUS assay (35S:GUS construct in vector) this period. To date, although no GUS positive or chimeric shoots have been observed for the sweet orange cultivar, Duncan grapefruit and carrizo citrange had totals of 4 and 134 GUS positive shoots, respectively. We will continue to screen and progress positively transformed plants for further analysis. Pathogenicity tests will be carried out as soon as plants reach adequate size. 2. Stable transformation in test systems Tobacco: N. tabacum lines transformed with the 14 EBE promoter fused to GUS, generated at the UC Davis transformation facility, were tested by GUS assay in the presence and absence of the X. citri effector PthA4, delivered as a 35S expression construct via Agrobacterium. Nine of 10 of the lines showed GUS expression only in the presence of PthA4. This system is now established as functioning and will provide a rapid means of testing the ability for individual and combinations of X. citri effectors to induce the promoter construct. We are in the process of harvesting seed for full scale screening. Tomato: A second test system was designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. Positive transformants of Bonny Best and large Red Cherry tomato cultivars were confirmed using DNA dot blot. T0 transgenic tomato were screened for pathogenicity reaction with X. euvesicatoria strains (8510 avrBs3 transconjugant & Race 9) and X. gardneri, and several Bonny Best and large Red Cherry were found to be resistant. This demonstrates that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance. We are harvesting seed from transgenic plants and are in the process of testing seedlings for resistance.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Random mutagenesis of EFR ectodomain to gain elf18-CLas responsiveness did not produce gain-of-function mutants. Using chimeric elf18 peptides of wild-type and CLas sequences, we could determine that both the N- and C-terminal regions of elf18-CLas were responsible for the lack of recognition by EFR suggesting that elf18-CLas is impaired in both binding and activation. In order to identify complex EFR mutants that respond to elf18-CLas, we are developing a FACS-based screen. We fused promoters that are expressed at a low basal level and PAMP inducible to nuclear-localized GFP and tested by Agrobacterium-mediated transient expression in N. benthamiana and in Arabidopsis protoplasts. All constructs gave significant basal expression, which is presumably due to unrestricted expression in these transient systems. Therefore, it is necessary to produce transgenic lines of these reporter construct. Modelling of the EFR/elf18/BAK1 complex has been performed, based on the available FLS2/flg22/BAK1 structure. Sites have been selected for targeted mutagenesis to improve recognition of elf18-CLas. We have also examined the interaction with BAK1 and identified two regions which may be involved in elf18 binding. Mutagenesis constructs of these regions is currently being screened for elf18-CLas response. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Reciprocal XA21 and EFR chimeric receptors have been produced and transformed into Arabidopsis for functional testing. Since it is now known that axYS22-Xac is not the ligand of XA21, we tested the response of the EFR-XA21 chimera to elf18, in order to determine the function of the XA21 cytoplasmic domain in dicots. These constructs performed in a similar manner to wild-type EFR. In addition we tested XA21, EFR, XA21-EFR and EFR-XA21 for pathogen resistance to Pseudomonas syringae pv. tomato DC3000 COR-. Interestingly, all constructs provided an elevated level of resistance, indicating that the chimera of XA21-EFR was functional and that XA21 was capable of perceiving a potential ligand Pseudomonas. A manuscript describing this work is currently being prepared for submission to PNAS. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Constructs including EFR alone or in combination with XA21 or XA21-EFR were provided to the Moore Lab for transformation. These were transformed into E.coli DH5. cells and Agrobacterium tumefaciens strain Agl1. Confirmation of clones was done using PCR (EFR and XA21 designed primers) and restriction analysis (NcoI and SpeI restriction enzymes). Initial transformation experiments have been carried out with a total of 100 ‘Pineapple’ sweet orange segments. Germination rate for sweet orange is 5%, which is extremely low. ‘Pineapple’ sweet orange and Duncan grapefruit seedlings are on germination media and transformation experiments will be initiated in May 2014.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we performed another NAD+ treatment experiment, in which NAD+ was either infiltrated into the citrus leaves or sprayed on the surface. After 24 hours, the NAD+-treated leaves were inoculated with the canker bacterial pathogen Xanthomonas citri subsp. citri. Canker symptoms were analyzed two weeks later. Compared with the Actigard treatment, the NAD+ treatment did not provide strong protection to the plants in this experiment. It is possible that time between NAD+ treatment and canker bacteria inoculation was too short. We are design another experiment in which the NAD+ induction time will be longer. We are also preparing citrus plants for root treatment with NAD+. For objective 2, in last quarter, about 20 independent lines have been generated for each construct. The transgenic seedlings have been growing in the greenhouse. We have performed molecular characterization of the transgenic plants. DNA was extracted from each individual plants and PCR was performed to confirm the presence of the transgene. Only a fraction of the putative transgenic seedlings contain the transgene. We are currently testing the expression levels of the transgene in these plants. We will also generate more transgenic lines using the constructs.
We worked on permitting beginning in December 2013 when the contract was signed and the NOA was generated. Extensive documentation was provided by CREF, Inc, and UF-IFAS to SWFWMD in order to complete the permit application. As of this writing, we don’t anticipate problems but await permit approval. The POs have been generated for all necessary components and services. When the permit is approved, we will begin the work.
New harvest data was collected from field trials for the 2013-14 season and confirmed outstanding performance of several new USDA rootstocks in comparison with standard rootstocks under heavy HLB inoculation pressure. The studies included measures of fruit yield, fruit quality, tree survival, and tree size. The rootstocks US-896, US-1279, US-1281, US-1282, US-1283, US-1284, US-1318, US-1319, and US-1321 were outstanding, as compared to the common commercial rootstocks. The new USDA rootstocks will be submitted for commercial release in 2014. These promising selections have been provided to Florida DPI to establish clean budwood sources and have been propagated by USDA to multiply material for further testing. These selections are also being made available for the CRDF Product Development Project to establish large scale commercial field trials. Cooperative arrangements are being made with commercial nurseries for large scale vegetative propagation of the most promising rootstocks, as needed to meet commercial demand. Three new rootstock field trials were planted in spring 2014. About 800 Valencia trees on Supersour rootstocks were prepared for planting field trials in St. Lucie County in summer 2014. About 120 new Supersour rootstock hybrids were created in 2013-14 and established in the greenhouse nursery at Ft. Pierce. Ten thousand propagations of Supersour rootstock selections were completed to prepare trees for controlled testing and additional field trials in 2015. Cooperative work continued with a commercial nursery to multiply promising Supersour rootstocks to produce trees for medium-scale commercial plantings. Greenhouse studies continue to assess Supersour tolerance of CTV, salinity, and calcareous soils. Data was collected from a greenhouse test of Supersour rootstocks to measure quick decline reaction in response to CTV infection. Studies were conducted on defense-related citrus genes associate with HLB infection. Data was collected on localized expression in shoots and roots of trees with different scion/rootstock combinations. Genes studied include those identified by our expression studies as being associated with HLB response, such as RDR1, RAP4, CSD1, and CtCDR1, and also genes identified in collaborative work with a University of Maryland team. Genes that appear particularly promising are used for transformation of citrus to manipulate expression of susceptibility and tolerance. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility has been completed and will be published in 2014. A study on the effect of grafting height in HLB tolerance was initiated in the greenhouse with a similar study being prepared for field planting in 2014. New transgenic citrus rootstock selections are produced at about 50 new transgenics per month in our program. The citrus resistance genes CtSID2, CtSFD1, CtPAD3, and CtCDR1 were primary focus for transformation in this quarter. Following promising preliminary testing, propagations of fifteen new transgenic rootstocks with overexpression of citrus gene CtNDR1 were propagated and entered into a replicated greenhouse test with HLB. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Several transgenic rootstock selections with AMP gene expression have been identified that result in reduced infection, reduced disease population, more vigorous growth, or reduced HLB symptoms in the transgenic plants or grafted sweet orange scions.
USDA-ARS-USHRL, Fort Pierce Florida is 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 two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with 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 Las titer. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. As of April 9, 2014, a total of 5,314 transgenic plants have passed through inoculation process. A total of 106,250 bacteriliferous psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. As an offshoot of the research, damage by western flower thrips was so severe that research was conducted to validate damage by this pest to developing flush and facultative predation on ACP, which led to the following publication: Hall, D. G. 2014. Interference by western flower thrips in rearing Asian citrus psyllid: damage to host plants and facultative predation. Crop Protection. 60: 66-69. A thrips predator, Orius insidiosus, proved to feed aggressively on immature ACP, thus would be incompatible for thrips control in an ACP rearing operation.
This project began with funding from the FCPRAC to support participation by the PI in the International Citrus Genome Consortium (ICGC), to sequence a citrus genome for the benefit of the global citrus research community, primarily to enable broad research targeting HLB and other critical citrus disease threats. CRDF continued to support the project until recently. The ICGC selected a haploid derived from Clementine mandarin, to simplify sequence assembly, gene model prediction, and annotation. The first genome was sequenced using Sanger technology, the platinum standard for quality data, in collaboration with the US DOE Joint Genome Institute (JGI) and its affiliate HudsonAlpha (USA), Genoscope (France), and Istituto Genomica Applicata (IGA; Italy). Support from the citrus community came from researchers in Florida, Brazil, France, Spain, and Italy. As sequencing technology rapidly evolved, we were able to leverage the project to use the first pyrosequencing technology from Roche/454, and a second genome was added to the project, that of Ridge Pineapple sweet orange. Roche/454 contributed substantially to this project, as did UF-ICBR and JGI. Both genomes were made freely available to the public in January of 2011, at JGI’s genomic web portal, Phytozome, and also in the Citrus Genome Database of Tree Fruit GDR (tfGDR), an SCRI-supported project. Since then, scores of papers have been published globally on various aspects of citrus biology and genetics, including many manuscripts focused on HLB research, which would not have been possible without the valuable genomic resources from this project. To illustrate the significant impact of these genomes, since January 1, 2013 alone there have been over 31,000 unique pageviews, and nearly 1600 downloads of the datasets at Phytozome. And in the 3 years since posting, the Citrus Genome Database of tfGDR was visited over 15,000 times by nearly 9000 unique users from 135 countries. Since then, as technology costs decreased even further, the ICGC added 6 more genomes; comparative analysis revealed the underlying genomic structure of sweet orange, pointing the way to new strategies to reconstruct orange with genetic contributions from HLB resistant citrus or relatives by sexual hybridization without the use of GMO strategies, or to potentially modify existing cultivars by engineering with constructs made entirely from citrus DNA. Though CRDF support for the work has terminated, the ICGC now has sequenced more than 30 citrus genomes, and work is underway to analyze these sequences and to provide a very rich resource for disease resistance gene identification and manipulation into the future. The Florida citrus growers who were the very first group in the world to fund citrus genome sequencing are to be commended for their bold foresight and financial contributions; they enabled development of universally valuable research tools for the global citrus community. In addition to developing a high quality reference citrus genome, the project proposed to use the genomic resources for research aimed at understanding further the interaction of various host citrus plants with the presumed causal agent of HLB, Candidatus Liberibacter asiaticus, to identify potential genetic targets for modification, and to identify early signals of infection that might be used to develop early detection methods. No fewer than 12 manuscripts have been published by the PI in collaboration with other labs, all of which have relied on the reference genome sequences. These works have explored and compared metabolic, transcriptomic, and proteomic responses of HLB-tolerant and sensitive plants, providing new insights to the mechanisms of disease and tolerance. New CRDF-funded projects underway to map and potentially identify specific genetic targets for intercession, to develop consumer-friendly HLB resistant cultivars, are feasible because of the genome resources developed. The culmination of this project is a manuscript accepted for publication this year in Nature Biotechnology, which compares the genomes of the fundamental citrus species, and elaborates their evolutionary and taxonomic relationships. Such understanding will support and enable more rapid and targeted improvement of commercial citrus cultivars as new threats to the industry appear.
The productivity of the Core Citrus Transformation Facility (CCTF) in the time between January and April of 2014 was higher than it was in the previous quarter. We have continued to produce transgenic plants for different research groups from the state of Florida and beyond. The work done within this quarter concentrated mostly on old orders. In communication with one research group, which is the biggest client at this time, the decision was made to prioritize the work on some of the orders they previously placed so CCTF had to shift the efforts towards specific orders and disregard time when the orders were placed. Also, through the communication with the CREC Director, CCTF received direct order from CRDF to produce some rootstock plants transformed with the NPR1 gene. These efforts are being coordinated with the Mature Tissue Transformation Lab (MTTL). Considering the importance of this project, CCTF started working on it immediately. Initial co-incubation experiments were done just a few days upon receipt of this order with the plant material obtained from MTTL. However, the quality of seedlings obtained from the MTTL was not good and results from that series of experiments had to be discarded. Rootstock cultivars of Carrizo, Swingle, and Citrus macrophylla are being used in this project. C. macrophylla seeds used for production of seedlings, that are the source of explants for co-incubation experiments, seem to be carrying some endophyte as most of the material obtained from these seeds ended up being contaminated. Because of this observation, the decision was made to put the emphasis on the Carrizo and Swingle cultivars. The work on this order is continuing at high pace. Plants produced by the CCTF within this three months period belong to the following orders: pNah-10 plants, pN9-seven plants, pN18-six plants, pX11- eight plants, pW14- three plants, pX20- one plant, pX28- one plant, pN7- one plant, pHGJ4- one plant, pMed16- one plant, pMed14- two plants, pELP3-G-nine plants, pELP4-G- three plants, pTMN1-five plants, and pMG105- two plants. Altogether 60 plants were produced and they were all Duncan grapefruit.
In this proposal our objective is to find citrus versions for the two proteins that make up the functional components of a chimeric antimicrobial protein (CAP) previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have successfully identified a suitable replacement for the first component, the human neutrophil elastase (HNE). We obtained a close match with the tomato PR14a protein that is highly conserved in both Citrus sinensis (Cs) and Citrus clementina (Cc) genomes. We have completed the construction of two synthetic genes that encode this protein, one that just contains the coding region of CsP14a and the other is a chimeric version that contains the CsP14a coding region linked to the CecropinB (CecB) protein. We have also completed the construction constructing two CaMV35S expression cassettes to express both these synthetic genes so that the expressed proteins can be secreted. We have used CLASP to identify a citrus replacement component for CecB. Since CB has no enzymatic activity, we could not use a well-constrained motif like an active site. We chose instead the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. We have used an approach similar to that described earlier to identify a replacement for HNE to identify a replacement component for CB. However, instead of comparing the reactive atoms as was done for the HNE matching algorithm, in this case we match for the C-alpha atoms of specific residues based on the overall shape of CB. Thus, we adopted a slightly different computational flow in this case. First, we did a keyword search, ‘plants’ in http://www.pdb.org/ that yielded about 1000 proteins. Each pdb was expanded on basis of each chain. For example, PDB X.pdb with chains A and B resulted in PDB files XA.pdb and XB.pdb. This list was filtered based on a 80% similarity redundancy. Then proteins larger than 60 aa in length were ignored. APBS (Adaptive Poisson-Boltzmann Solver) software developed to examine electrostatic properties of proteins at a nanoscale) was run on each pdb (the ones that failed electrostatic analyses, a few, were excluded). We were able to extract 200 proteins which we then analyzed further using CB motif. The CB structure is characterized by two helices. We chose specific residues from these two helices such that the residues were polar, and had stereochemical matches. For example, Lys can be replaced by either Arg or His, and thus constitutes a good candidate. We chose, Lys10, Lys11, Lys16 and Lys29 as the input motif from CB, allowing Lys to be matched by Lys, Arg or His. Since the 52 aa H+ATPase (HAT) is large so we cannot have it cheaply synthesized as a protein. We are developing additional computational tools to better evaluate individual alpha helical domains so we can identify smaller proteins that can be more cheaply synthesized for testing. We have also begun making a CAP construct where CB is replaced by HAT. This will be expressed in plants to evaluate antimicrobial properties.
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