HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates were monitored for tree growth and disease incidence, including the portion of the St. Helena project planted with dwarfing selections, and a 40-acre Hamlin/Valencia cooperative rootstock trial with trees planted between 300-500/acre. The latter planting is 3.5 years old, yield data were collected last season, and tree growth (height and trunk diameter) and fruit load estimates were recently made. Seed trees for selected dwarfing rootstocks, already showing good performance, were propagated and some were planted, to support expanded trials in the future. Additional new rootstocks from the CRDF Rootstock Matrix selected for their potential in high density plantings through good tree size control were entered into the DPI Parent Tree Program for cleanup by shoot tip grafting followed by indexing, to provide certified budwood of these rootstocks for commercial nurseries upon their release. Seedlings of some other Flying Dragon hybrids have come through the ‘HLB gauntlet’ screening process (grafted with CLas-infected Valencia budsticks, and then cycled through a hot psyllid house, ending with no obvious HLB symptoms); these will be planted in the field, under a DPI permit for further observation. The McTeer OHS Sugar Belle trial tested 15 tree-size controlling rootstocks. This trial, planted in 2009, is nearly 100% HLB infected, yet about 85% of the trees are still showing a healthy appearance though fruit quality varies among different rootstocks. Visual assessment showed that rootstocks White #4 (UFR-5) and Orange #19 (UFR-4) were the healthiest trees, with no trees needing to be removed. White #4 (UFR-5) appears to be the best candidate for ACPS in this trial, as trees are very uniform in size, and have cropped well 2 consecutive years. Trees on Orange #19 (UFR-4) are larger, but took an extra year to crop. The somatic hybrid rootstock Sour orange+50-7 also showed consistently good health on smaller trees. Unfortunately, this trial was terminated prematurely because the property was sold for development. Seed were extracted from fruit of two new promising ACPS rootstock selections: somatic hybrid of Murcott + Rubideaux trifoliate; and tetrazyg Nova+HBPummelo x Cleo+Swingle. Seed may be used for larger scale trials.
A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified over 1,150 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a valuable pool of potential candidate genes from which true HLB tolerance genes may be identified. Additional candidate genes have recently become available from an RNA-seq experiment using rough lemon and sweet orange in a comparison similar to what was done with the Affymetrix microarray work in our lab (Fan, et al., 2012). This project aims to screen these potential candidate genes using high throughput target capture, massively parallel sequencing of targeted gene regions, and genetic association and linkage analysis to find the most likely candidate gene(s) for HLB tolerance in Poncirus and rough lemon. After several rounds of candidate searches, we have recruited suitable post-doctoral research associates. One post-doc is scheduled to arrive in January 2014 and another to begin shortly thereafter, so it is our expectation that the project will accelerate substantially at that time. In the meantime, we have continued careful analysis of the RNA-seq data, looking at changes in gene expression over time. Recent application of Pathway Studios software has provided more powerful discrimination of critical genes in particular pathways associated with disease responses of plants. We are continuing compilation of a list of nearly 1,300 candidate genes and downloading their sequences for designing a high throughput target capture system that will be used to target sequencing, genetic association and linkage analysis.
An experiment involving ciFT3 transgenic tobacco plants and the effect of plant hormones Ethylene and Gibberellin (GA) as well as a GA pathway inhibitor chemical Paclobutrazol(PBZ) is currently being monitored. The effects of the chemicals are evident due to the variation in phenotype observed. The transgenic line being used is a T2 that has shown to be homozygous for the ciFT3 transgene from previous GUS assay experiments. PBZ produced a short phenotype with succulent leaves and transgenic plants flowered earlier in comparison to the other treatments. Flower buds appeared 39 days after germination. Controls are being monitored for flowering and are expected to flower around the 150 day mark. The outcome of this experiment will be used to determine an effective way of preventing precautious flowering of citrus FT3 transgenic plants in tissue culture stages by perhaps using one of the compounds. RNA will be extracted at three different time points from each treatment to determine the relative expression of ciFT3 when treated with the compounds. New cDNA concentrations from the 12 month collection period of citrus tissue was used to run a new set of quantitative real time PCR amplification curves for the FT1, FT2, FT3 FLD, FLC, ELF5, and AP1 genes. Statistical analysis is being conducted to find any correlation in expression and flowering time. Work has proceeded designing a transcription activator-like effector (TALE) system inducible by methoxyfenozide that will chemically activate the naturally present FT3 gene in citrus. An 18 monomer TALE has been constructed based on the endogenous FT3 promoter region common to ‘Duncan’ Grapefruit, ‘Carrizo’ Citrange, Pummello, and Poncirus citrus. Progress is currently being made to assemble this FT3 TALE into a plasmid with a FMV promoter, a VP16 activation domain and the inducible ecdysone receptor-based expression system. The efficacy of this construct will be evaluated by co-transforming tobacco with the chemically inducible system and with a plasmid that contains the endogenous citrus FT3 promoter followed by a GUS reporter gene.
Experiment described in the last report continue as the main focus of the project. In addition, we have formed a collaboration with researchers from UF’s Department of Chemistry and the IRREC (Mingsheng Chen, Brent Sumerlin, and Zhenli He) who work with nano-particles chemically assembled using amphiphilic polypeptides (polymers). Our groups seek to develop a method using a combination of encapsulating nano-particles and cell penetrating peptides (CPPs) to deliver cargos to selected tissues (such as the phloem). The first step, undertaken this quarter, is to elucidate the potential toxicity effects of different nano-particles using a combination of tissue culture techniques and fluorescence microscopy. Then, we will examine combinations of benign polymers and CPPs that we have found to be effective in transferring cargo to citrus.
In a quest to discover the main factors contributing to pathogenicity of Liberibacter, during this period we compared whole genomes of pathogenic Liberibacter species Candidatus Liberibacter americanus str. Sao Paulo (Lam) and Candidatus Liberibacter asiaticus str. psy62 (Las) against the whole genome of non Liberibacter crescens BT-1 (L. crescens). We found 70 genes unique to the pathogenic strains and performed their in-depth analysis. Consistent with the notion that Liberibacter prophage gene content correlates with pathogenesis, 40% of the genes unique to the pathogens reside within the prophage regions. We have analyzed a few potential disease-related genes in greater depth, including potential secreted factors, lytic cycle regulators, and metabolic enzymes. The examples attempt to highlight the potential relevance of pathogen unique genes to disease as well as reveal the potential drawbacks of the automated prediction methods that need to be addressed manually or by revised prediction cutoffs. Two of the prophage-encoded proteins are predicted to have signal peptides: a putitive guanylate kinase (CLIBASIA_00055) and a hypothetic protein of unknown function with distribution limited to Ca. Liberibacter genomes and the SC1/SC2 prophages (CLIBASIA_05560). The presence of a putative secretion signal might imply these proteins function as host virulence factors. However, the N-terminal sequence region of CLIBASIA_00055 does not likely serve as a secretion signal, as it forms the first hydrophobic strand of the guanylate kinase domain. The Las genome contains an additional core guanylate kinase (Gmk) (CLIBASIA_04045) that is orthologous to the Gmk of Liberibacter crescens and likely functions in purine metabolism. The presence of a second unique GMK encoded by the prophage remains unclear. The prophage contains a pathogen specific Xre-Bro protein pair (encoded by CLIBASIA_05625 and CLIBASIA_00020, respectively) similar to phage repressor-antirepressors that determine lytic state. Interestingly, the Las genome contains an additional gene (CLIBASIA_04440) that has potentially migrated from the prophage with sequence similar to the C-terminus of the CLIBASIA_00020-encoded bro protein. CLIBASIA_04440 appeared to be unique to Las by core gene analysis; however searching the Lam nucleotide records identified a potential open reading frame that encodes the entire Bro domain-containing protein sequence (from an alternate start codon: CUG instead of AUG: Translation below). Additional inspection of the nucleotide sequence in the Las genome upstream of CLIBASIA_04440 yielded a potential missed open reading frame encoding the N-terminal bro domain (translation below). Together, these genes may regulate expression of genes that result in induction of the lytic cycle. For example, the pathogen-specific CLIBASIA_05531 or the core CLIBASIA_05655 (has a similar gene in the unrelated L. crescens prophage: B488_04970), which encode potential haemolysins.
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.
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