USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 2,750 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. 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. 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 one week, 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. This report marks the end of the first quarter of the project, during which we have established the infrastructure for the screening program. A technician dedicated to the project is being hired, two small greenhouses for rearing psyllids are almost completed, and general supplies including insect cages have been procured. USHRL dedicated an existing conventional greenhouse for the project, erected two new hoop houses for the project, and assigned a support scientist to the screening project. Additional ARS funds were used to increase the bio-security of the existing greenhouse to guard against invasion of parasitoids of the psyllid. This screening program supports two USHRL projects funded by CRDF for transforming citrus.
Initial funding for this project was finally obtained on July 03,2012. McTeer trial – (3-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): 450 trees in this trial were visually scored for HLB symptom severity, and leaf samples from each tree were collected for PCR analysis (being run by the SG diagnostic laboratory c/o Mike Irey). Complex tetraploid rootstock Orange #19 (Nova+HBP x Cleo+Arg.trifoliate orange) showed minimal disease symptoms, whereas Swingle was in the middle of the pack, and the worst average symptoms were observed on Flying Dragon and Rich trifoliate orange. There are no Phytophthora issues in this trial at present (as determined by JH Graham). St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 4.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). 200 trees identified by the CREC scouts as being HLB positive were visually scored for HLB symptom severity. As with the McTeer trial, complex tetraploid rootstock Orange #19 showed the least disease symptom severity. Swingle again placed in the middle, whereas Kuharske and Volk controls scored very high disease symptoms. Complex tetraploid Orange #4, a sibling of Orange #19 also showed minimal HLB symptoms at both sites. Leaf samples from around each HLB positive tree, and from symptomatic areas of each tree were collected and sent to the SG diagnostic laboratory for PCR analysis (again c/o Mike Irey). Percentages of trees diagnosed with HLB differed significantly as well, with Kuharske citrange showing a very high infection rate. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. Initial experiments to transform promising complex tetraploid ‘tetrazyg’ rootstocks Orange #4, Green #7 and Orange #19 with protecting constructs are underway, and 20 transgenic lines of Orange 4 and 7 lines of green 7 have been recovered to date. Progress was also made building two-transgene constructs that contain an antimicrobial gene for HLB resistance combined with an insecticidal gene to protect against psyllids.
Initial funding for this project was finally obtained on June 20, 2012. Construction of the rapid flowering system (pvc pipe scaffolding system) in the greenhouse has been completed. Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks and growing in airpots have been entered into this RES system. The plants have been single stemmed, and some are already approaching 6 feet in height. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. The first experiment combines our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene). The two-transgene Gateway based cloning system was employed to build the 2-gene construct. The NPR1 gene is under control of the rolD promoter while the CEME gene is under control of the d35S promoter. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance. Experiments to combine the NPR-1 gene with other lytic peptide transgenes including CEMA and AttacinE are underway, also using the new Gateway technology.
More than two thousand transgenic lines we have produced thus far, and are planted in field trials (2 locations under permit) or are in greenhouse tests at the CREC and with grower-collaborators; we continue to monitor the HLB and canker resistance or tolerance of these lines over time. These transgenic lines contain various combinations of natural or synthetic genes and promoters. New candidate genes continue to be identified by genome mining as well as from other disease resistance plant research. Citrus-specific promoters, transcription factors, and other genetic elements are being identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests, including some containing a broad spectrum, ancient disease resistance gene from rice; the latter are being propagated for HLB challenge. Data are being collected on the early performance of new advanced selections in trials planted to assess adaptation to advanced citrus production systems. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests. Diverse responses of rootstocks are being noted when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance. Greenhouse experiments are being continued examining interactions of rootstock and nutrients in severity of HLB symptom expression. Hot psyllid greenhouse facilities are now being used routinely to assess performance of transgenic citrus (representing our most advanced constructs with phloem-limited promoters and previously proven genes), as well as hybrids between citrus and Poncirus, for responses to psyllid feeding and HLB development. More than 150 new rootstock candidates preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora, and have been used to produce new trees that were planted into new rootstock trials, or held for pending trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. Additional seedless pummelo-grapefruit hybrids have been identified during the 2011-12 season, some showing field tolerance to canker, good fruit quality, and FC-free, potentially producing grapefruit cultivars that address canker and marketing issues of ordinary grapefruit. Trees were propagated onto 30 new sour orange-like hybrid rootstocks, some already shown to be tolerant of CTV quick decline, and planted in a new field trial. A second demonstration planting of advanced sweet orange selections and newly-released cultivars, selected for high yields and superior juice quality, was established to assess to demonstrate their performance and utility in commercial processing, in collaboration with a major juice processor; these trials allow comparisons to be made between different production regions with the same sweet orange candidate selections. A well-attended field day was held in mid-November at the large CREC field experiment at the St. Helena block in Dundee; this featured Vernia and Valquarius orange trees grown on a number advanced rootstock selections, and highlighted early performance (yield and HLB effects).
The Core Citrus Transformation Facility (CCTF) continued to produce transgenic plants at expected rate of about a 100 per quarter. Plants for the following orders were produced: eight Duncan plants (ELP3 gene); ten Duncan plants (ELP4 gene); 15 Duncan plants (p7 gene); three Duncan plants (p10 gene); three Duncan plants (pWG19-5 vector); three Duncan plants (pWG20-7 vector); five Duncan plants (pWG21-1 vector); 15 Duncan plants (pWG22-1 vector); five Duncan plants (pWG24-13 vector); 12 Duncan plants (pWG25-13 vector); five Duncan plants (pWG27-3 vector); four Hamlin plants (pLC220 vector); eight Duncan plants (p35 gene), 11 Duncan plants (35S-TRX vector), and two Duncan plants (SUC-TRX vector). CCTF received three new orders in this period. The decision was made to replace the soil to which plants get transferred to following successful grafting. Use of soil that would get contaminated occasionally resulted in a loss of plants in previous periods. A special, commercially available, foam product is being used instead of commercial potting soil. The problem of loss of plants due to soil contamination has now been remedied.
The antibody developer, Creative Biolabs, Inc., has nearly completed screening for antibodies against the Candidatus Liberibacter asiaticus NodT protein. They have identified six high-affinity binding antibodies to the 30 amino acid peptide antigen used. The materials will be shipped to Dr. McNellis’ lab at Penn State University within the next few weeks. We anticipate beginning to screen the antibodies for their ability to detect the native NodT protein produced by Candidatus Liberibacter asiaticus starting in mid-August, working through the fall.
Quarterly report for April 2012. Two full genome sequences have been assembled and annotated, and made available to the citrus research community, the haploid Clementine sequenced by the ICGC partners using Sanger technology to produce the highest quality reference genome for all subsequent citrus genomics efforts; and the sweet orange, developed by UF, Roche/454, and JGI using the 454 platform. Both annotated assemblies are available at Phytozome and at citrusgenomedb.org. The Clementine assembly has been improved, assembled into the 9 basic chromosomes; it is designated Clementine v. 1.0, and is being used in comparative genomics studies describing the phylogeny of sweet orange and Clementine. New citrus genome sequences have been generated by the Machado lab in Brazil (Ponkan mandarin, 4x coverage, using 454 technology), the Gmitter lab and UF-ICBR (low-acid pummelo, 25x coverage by Illumina technology), and plans are in place for resequencing other genomes to contribute to the phylogeny study. The ICGC is to produce a high-profile manuscript to highlight the work done and the potential utility of the sequencing projects toward future research objectives. Work proceeds on the other objectives of this project. To demonstrate the potential of the silencing approach described in Enrique et al, 2001, as a rapid genetic tool to address HLB infection, the Gadea and Marano labs characterized the behavior and persistence of silencing signals. A total of 6 candidates were cloned, based on our microarray studies comparing sensitive and tolerant citrus, and >70 plants were inoculated with Agrobacterium during January-April 2012. No altered phenotypes have been observed, and based on the systematic problems we are looking at new strategies. We are identifying miRNAs induced in citrus-pathogen interactions, presumably regulating target genes involved in signaling pathways and metabolic events important for plant resistance. A real time protocol has been established to study miRNA and target expression in Citrus and thus validate computational or high-throughput experiments in a biological context. We have mined, screened, and verified SNPs derived from the BAC end-sequences from Dvorak’s lab and the GoldenGate assay platform for hi-throughput genotyping has been produced. DNA samples were prepared and >150 individuals of a large mapping family have been processed; data analysis is underway to anchor the orange genome to genetic and physical linkage maps, thus improving its quality and utility for HLB-targeted research projects. Analysis of data from microarray studies looking at differences in gene expression over time between sensitive (orange) and tolerant (rough lemon) citrus types has revealed substantial differences in host defense responses, and these are being associated with changes in metabolism and phenotypic responses. The Roose lab submitted DNA of 13 sweet orange x trifoliate orange progeny and two parents to evaluate the potential of genotyping by sequencing (GBS) to Floragenex, a company that performs this technique. The method used targets the non-methylated portion of the genome and is therefore likely to be gene-rich. The parents will be sequenced at 30x depth or greater and the progeny at 15-20x depth. If the results from this preliminary trial are acceptable, we will submit additional DNA samples for analysis.
Quarterly report for July 2012. The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, will soon be publicly available. New citrus sequences were generated by the Machado lab in Brazil (Ponkan mandarin, 454 and Illumina), the Gmitter lab and UF-ICBR (low-acid pummelo, Illumina), and Illumina datasets for Willowleaf/Avana mandarin, W. Murcott, Chandler pummelo, and Seville sour orange have been provided (Morgante, IGA-Italy; Talon, IVIA-Spain; and W. Roose-UCR). Comparative analysis has elucidated the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. The fine-scale characterization of citrus genotypes opens the possibility that ancient C. reticulata/C. maxima admixtures (such as sweet and sour orange) can be recreated by conventional breeding guided by a set of genome-wide markers, enabling incorporation of specific, limited genomic regions from other citrus or relatives to confer disease resistance, yet retaining the essence of marketable fruit phenotypes. A manuscript based on these results is under revision. Work proceeds on the other objectives of this project. We are identifying miRNAs induced in citrus-pathogen interactions, presumably regulating target genes involved in signaling pathways and metabolic events important for plant resistance. In order to set up protocols to validate microRNA expression in plant-pathogen interactions and identify target genes, we have performed a comprehensive analysis of the expression of 7 different citrus miRNAs in the context of 4 different Xanthomonas citri subsp. citri (XC) ‘ Citrus limon interactions. The results obtained so far reveal interesting expression patterns for some of the miRNAs. We have used the GoldenGate assay platform for hi-throughput genotyping of DNA from >150 individuals of a large mapping family; data analysis is continuing to anchor the orange genome to genetic and physical linkage maps, thus improving its quality and utility for HLB-targeted research projects. Analysis of data from microarray studies looking at differences in gene expression over time between sensitive (orange) and tolerant (rough lemon) citrus types has revealed substantial differences in host defense responses, and these are being associated with changes in metabolism and phenotypic responses. More genes were differentially expressed in HLB-affected rough lemon than sweet orange at early stages, but substantially fewer at late time points, possibly underlying differences in sensitivity to CLas. Pathway analysis revealed that stress responses also were distinctively modulated in rough lemon and sweet orange. Remarkably phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The pilot genotyping by sequencing (GBS) project has yielded promising results, and plans are being carried out to genotype a large segregating Citrus x Poncirus family. A large scale RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus, that weren’t seen previously in microarray studies, or to validate those already seen, has progressed; RNA samples have been sequenced using Illumina technology, and the massive data set is currently being analyzed.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in Col-0 background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers. We have been in the process of testing disease resistance of these plants to P. syringae. If a citrus SA gene confers broad disease resistance, we expect to see enhanced resistance to P. syringae when the citrus gene is overexpressed in Col-0 and/or to see the complementation of the phenotypes exhibited by its corresponding mutant. For ctNDR1, we have so far obtained 29 independently transformed US942 transgenic citrus plants carrying ctNDR1 overexpressing construct (US942::d35S::CtNDR1). The presence of the transgene in the plants was confirmed by transgene-specific primers. We performed disease resistance assays with Xanthomonas citri subsp (Xac), using excised leaf discs from 24 US942::d35S::CtNDR1 plants. Twelve leaf discs (that was all we could sacrifice for now) from each independently transformed line were used in the infection. Leaf disks, each from a single leaf, were tested in three infection groups and each infection was conducted independently on a separate day. Our preliminary results indicate that US-942::d35S::ctNDR1 transgenic clones had significantly reduced growth of Xac, as compared to untransformed controls. Therefore, these results suggest that overexpression of ctNDR confers enhanced resistance to citrus canker disease.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in Col-0 background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers. We have been in the process of testing disease resistance of these plants to P. syringae. If a citrus SA gene confers broad disease resistance, we expect to see enhanced resistance to P. syringae when the citrus gene is overexpressed in Col-0 and/or to see the complementation of the phenotypes exhibited by its corresponding mutant. For ctNDR1, we have so far obtained 29 independently transformed US942 transgenic citrus plants carrying ctNDR1 overexpressing construct (US942::d35S::CtNDR1). The presence of the transgene in the plants was confirmed by transgene-specific primers. We performed disease resistance assays with Xanthomonas citri subsp (Xac), using excised leaf discs from 24 US942::d35S::CtNDR1 plants. Twelve leaf discs (that was all we could sacrifice for now) from each independently transformed line were used in the infection. Leaf disks, each from a single leaf, were tested in three infection groups and each infection was conducted independently on a separate day. Our preliminary results indicate that US-942::d35S::ctNDR1 transgenic clones had significantly reduced growth of Xac, as compared to untransformed controls. Therefore, these results suggest that overexpression of ctNDR confers enhanced resistance to citrus canker disease.
1. Monitoring genome-wide response to Candidatus Liberibacter asiaticus infections In response to pathogen attack, multiple defense mechanisms are triggered in the host plants, including basal defense and gene-for-gene resistance. So far, neither basal defense nor gene-for-gene defense mechanism has been reported in association with CLas infection. Because of the intracellular life style and lack of Type III effector genes of CLas bacteria, we hypothesize the basal defense resistance may play a more important role in these breeding lines. We expect that monitoring the gene expression differences among HLB-susceptible and HLB-resistant citrus trees using RNA-Seq will enable us to elucidate the HLB defense mechanism. We have identified the most suitable trees for the study and are now collecting and preparing samples. We have also identified and are co-ordinating with a company to perform the RNA-Seq. 2. Reconstructing and verifying the transcriptomic data After the next generation sequencing data is obtained, the first step will be to construct gene transcripts from these billions of short reads. This step includes a series of bioinformatics studies. Due to the enormous amount of reads, the bioinformatics study requires substantial computational infrastructures. The Palmetto cluster computer in Clemson University, which ranks 96th among the top 500 super computers reported in June 2011 (www.top500.org), can provide sufficient support for our bioinformatics studies. The constructed gene transcripts will be verified selectively using RT-PCR. 3. Identifying differentially expressed gene, gene modules, transcription factors under Candidatus Liberibacter asiaticus infections The transcriptome data provide a global monitor of expression changes related to Candidatus Liberibacter asiaticus infections, which open up opportunities for the elucidation of the gene, gene modules and gene networks responding to Ca. Liberibacter asiaticus infections. Here, we plan to conduct multiple computational analyses of our transcriptome data to identify resistance genes. First, the differentially expressed genes will be identified using statistical methods. Second, the gene co-expression networks and gene co-expression modules will be identified using the RMT-based method. Finally, the transcription factor(s) that directly controls each gene module will be determined. 4. Validating the identified genes under Candidatus Liberibacter asiaticus infections Transcription factors play an important role in defining gene co-expression modules. It is anticipated that transcription factors involved in Candidatus Liberibacter asiaticus infections will be identified by transcriptomic analysis. We will test whether an identified transcription factor indeed performs the physiological function as anticipated as a regulatory component of a co-expression module. 5. Creating transgenics of Carrizo, Hamlin, and Ray Ruby The most promising genes will be used in binary vector constructs to create transgenics of Carrizo, Hamlin, and Ray Ruby. It is anticipated that advances in citrus transformation techniques will verify similar efficacy for the Niedz mature tissue transformation protocol (Marutani-Hert et al., 2011) and the juvenile tissue approach we have been using (slight modifications of those published by Orbovic and Grosser, 2006). If this is correct, we will transition solely to mature tissue transformation techniques.
This report covers the first month of the project funding, and new data during this period is limited. Objective 1. Evaluate existing transformed lines: Experimental lines were generated during the first funding cycle, and one of our principal efforts is analysis and testing of candidate transformed lines of Duncan grapefruit to find stable transgenic lines that correctly express gene constructs. Objective 2. Expand stable transformations: This time of year seeds are poor, but we are planning how to best ramp up our efforts for transformation of Ruby Red grapefruit and sweet orange in September. Objective 3. Refine constructs: We have new genetic elements that we are incorporating into our expression constructs with the aim of improving resistance. Objective 4. Sequence more TAL effectors from additional canker accessions: We have identified several cankers strains that have particular phenotypes or geographies that may reflect differences in TAL effectors. We are working on isolating and sequencing these genes, with the particular goal of testing the effectiveness of our resistance strategy against these strains.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, besides transgenic citrus plants overexpressing the Arabidopsis MKK7 (AtMKK7) and NPR1 genes, we have also generated transgenic citrus plants expressing the Arabidopsis NAC1, MOD1, and EDS5 genes. These three genes have been shown to confer disease resistance in Arabidopsis. The transgenic plants are growing and will be propagated for canker and greening resistance test. In addition, we recently established an Arabidopsis-Xanthomonas citri subsp. citri (Xcc) pathosystem. Using this pathosystem, we have found that several genes of the SA signaling pathway function in nonhost resistance to Xcc. We are using the pathosystem to identify novel genes conferring nonhost resistance against citrus canker. For objective 2, we focused on the direct genetic screen for citrus varieties with increased resistance to citrus greening. More seedlings from gamma ray-irradiated Ray Ruby grapefruit seeds were inoculated with psyllids carrying greening bacteria. The first batch of seedlings inoculated with psyllids carrying greening bacteria have been moved out, and we are monitoring the development of greening symptoms on the seedlings.
The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, has spread to citrus growing regions nearly worldwide and adults transmit phloem-limited bacteria (Candidatus Liberibacter spp.) that are putatively responsible for citrus greening disease (huanglongbing). Host plant resistance ultimately may provide the most effective, economical, environmentally safe, and sustainable method of control. In earlier experiments we identified genotypes of Poncirus trifoliata and xCitroncirus sp. (hybrids of P. trifoliata and another parent species) that were resistant to ACP. We are now trying to identify the genotypic and phenotypic traits that promote resistance. We initially collected volatiles from one genotype of Poncirus trifoliata and two genotypes of xCitroncirus. We have since identified a susceptible genotype of P. trifoliata that is closely related to a resistant genotype of P. trifoliata and two pairs of closely related susceptible/resistant genotypes of xCitroncirus. These genotypes are ideal for identifying differences in volatiles among susceptible and resistant genotypes. Therefore, we collected volatiles using a SPME fiber and aerations from these genotypes and two susceptible control genotypes. The volatiles were analyzed using gas chromatography-mass spectrometry and we found clear differences in the volatile profiles. A chemist-collaborator is currently identifying all volatile compounds collected from the samples. We also plan to use these same genotypes to analyze the amino acids, sugars, flavenoids, carrotenoids, isoprenoids, and sterols in phloem. This will give us information about the underlying reasons why ACP avoid certain genotypes of citrus and help identify genes that can be used in citrus breeding programs to confer resistance to ACP. We have continued to screen grapefruit trees that have been genetically transformed to express Lectin from the snowdrop pea for susceptibility to ACP. We compared rate of oviposition, nymphal development, and lifespan of adult ACP on three varieties of grapefruit that express Lectin and one variety that does not. Expression of Lectin did not deter oviposition by ACP. However, our first two replications indicate that adult ACP likely have a shorter lifespan on the two varieties of grapefruit expressing the highest levels of Lectin. Additionally, our first two replications indicate that nymphs may develop more slowly or die at a higher rate on the variety expressing the highest level of Lectin. However, there has been wide variation in the results from the first two replications, so a third replication is currently in progress. We also used these trees to determine whether Lectin interferes with acquisition or transmission of citrus greening disease. Those results are currently being analyzed. ARS maintained contact with the Fujian Academy of Agricultural Sciences through emails and phone calls. ARS (Duan) is currently visiting FAAS in China and is on-site reviewing their research progress. FAAS concluded some no-choice experiments with Poncirus, xCitroncirus, Murraya, and Citrus accessions. In all cases where the same accessions have been studied, FAAS findings are in agreement with ARS’s concerning colonization by ACP eggs except for xCitroncirus (CRC 3957), which they report was more resistant. Among accessions studied by FAAS but not by ARS, ACP longevity was reduced on the following germplasm: xCitroncitrus (CRC 3881, CRC 3969, CRC 3957, CRC 1459), Citrus aurantium (CRC 3929), Citrus x Tangelo (CRC 3874) and P. trifoliata (CRC 838). These CRC accessions deserve evaluations under Florida conditions. In a free-choice settling experiment, adult ACP largely avoided Rhododendron simsii.
A series of transgenics scions and rootstocks, produced in the last several years, continue to move forward in the testing pipeline. It appears prudent to replicate plants of each transgenic event and conduct challenges that last 10-14 months. Most of these plants in our program have been transformed with AMPs driven by several constitutive and vascular specific promoters. Plants from the initial round of scion transformations are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (D. Hall collaboration) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. Several events grew better than all controls at 14 months after initiating the challenge, with 35% greater trunk-cross-sectional area increase than the overall experimental average and 64% greater growth than the mean of the controls, but do not show immunity to CLas development. These will soon be placed in the field for further evaluation. Forty four AMPs were screened in-vitro, a number of which were synthetics specifically designed to enhance efficacy against alpha-proteobacters. There appeared to be a ceiling of activity which could not be exceeded. The most active AMPs included Tachyplesin 1 from horseshoe crab, SMAP-29 from sheep, D4E1 and D2A21. A series of promoters were tested with the GUS gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than the other phloem-specific promoters citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. D35S produces the highest level of expression but with great variability between events. Anthocyanin regulatory genes, give bright red shoots (UF Gray collaboration) and were tested as a visual marker for transformation, as a component of a citrus-only transgenic system. When antibiotics were left out of regeneration media, almost no red shoots were recovered. However, high anthocyanin apples are reported to have field resistance to bacterial fire-blight. Red citrus transgenics will be tested for HLB, ACP, and canker resistance. CLas sequence data target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. In E. coli expressing the CLas translocase, two exterior epitope-specific peptides suppressed ATP uptake by 60+% and significantly suppressed CLas growth in culture. After verification these will be used to create transgenes. In our program, new constructs and resulting transgenics are in process, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), and a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA).
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