This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We have found that three different lines appear to be giving strong tolerance against HLB. We are propagating the plants for more extensive analysis.
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 6 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 six samples. The results showed that the gene expressions were significantly different in resistant vs. susceptible citrus. A total of 686 differentially expressed (DE) genes between two groups using FDR threshold of 0.1 were identified. Among them, 247 genes were up-regulated and 439 were down-regulated in tolerant citrus trees. We performed Gene Ontology (GO) enrichment analysis of DE genes. Genes associated with beta-amyrin synthase, cycloartenol synthase and Camelliol C synthase were significantly up-regulated in the HLB tolerant citrus trees while terpene synthase genes (CiClev10014707, Ciclev10017785) were down-regulated in the tolerant citrus trees. Some PR-protein genes were significantly up-regulated in the resistant citrus trees, including several TIR-NBS-LRR genes. Many cell wall degradation-related genes, such as cellulose synthase/transferase, cellulase and expansins were up-regulated in the susceptible citrus trees. Some glucan hydrolase genes were also up-regulated in the resistant citrus trees. These genes may play important roles in symptom development. The DE genes were also enriched in two classes of RLKs, LRR-RLKs and DUF26-RLKs. We have experimentally verified the expressions of 14 up-regulated genes and 20 down-regulated genes on three HLB-tolerant ‘Jackson’ and three HLB-susceptible ‘Marsh’ trees using real time PCR. 11 of 14 up-regulated genes and 18 of 20 down-regulated genes were validated. We further predicted a protein-protein interaction (PPI) network of citrus using the PPIs of Arabidopsis. There are 1259 proteins and 2298 interactions in our Citrus PPI network. Among 1259 proteins, 42 proteins are differentially expressed between the HLB resistance and susceptible citrus. An interested PPI sub-network includes 14 citrus NPR1-likes proteins and three TGA proteins. There were four NPR1-like genes were significantly up-regulated in HLB resistant citrus trees and one NPR1-like gene up-regulated in HLB sensitive citrus trees. There is also one TGA gene up-regulated in HLB resistant citrus trees. Another interested sub-network includes the RPS2 protein. There were two LRR kinase receptors significantly up-regulated in HLB resistant citrus trees and four LRR kinase receptors significantly up-regulated in sensitive citrus trees. Other interested interactions are between two lipoxygenase genes, LOX2 and EIF4E, a translation initiation factor. Interestingly, the two LOX2 genes were down-regulated in the HLB resistant citrus disease trees.
The Core Citrus Transformation Facility (CCTF) continued to provide service for production of transgenic citrus plants. The work load that included: co-incubation experiments, explants incubation, shoot harvesting and inspection, PCR testing, micro-grafting, and care of plants in the greenhouse was kept at maximum level allowed by the number of holidays within the last quarter of the year. The work done by the CCTF from October through December was concentrated on older orders as five new orders were placed to CCTF from two different clients in late December. Production of transgenic rootstock plants ordered by the CRDF continued at slower pace. One out of 29 plants that were previously growing in the greenhouse died. Nineteen more ‘pot-adapted’ plants were moved from the laboratory to the greenhouse bringing the total number up to 47. The growth of first plants moved to the greenhouse was not as vigorous as expected and they were not cut into explants for propagation. That may be done in the middle of February. In the period covered by this report, CCTF produced plants for the following orders: pNPR1-17 plants, pNPR1-G-five plants, pELP3-G-12 plants, pELP4-G-five plants, pMG36-eight plants, pX7-2- five plants, pHGJ27- one plant. Three orders that are being serviced at this time require detection of transgenes in transgenic plants by microscopy. The methodology for this detection is still being worked out with the EM lab personnel. If this was solved at earlier date, the output of the facility would have been higher. Plants produced in this quarter were mostly Duncan grapefruit and Carrizo citrange with the exception of one Valencia sweet orange plant. As it was anticipated in the previous report, the quality of Duncan fruits and seeds from the CREC’s grove has deteriorated further. Before the end of January, CCTF will contact local grower to get supply of better quality Duncan fruit.
The HLB-tolerant rootstocks US-1279, US-1281, US-1282, US-1283, and US-1284 were released by USDA in September and have now been released by FDACS-DPI as clean sources for commercial use. Material for propagation or establishment of seed trees is available from DPI. Fruit yield of Hamlin trees infected by HLB on these rootstocks is 2-4 times the yield of trees on Swingle, and the trees on these rootstocks also have fruit that is larger in size and higher in sugar content. These promising new rootstock selections, along with other new HLB-tolerant rootstocks from USDA and Univ. of Florida will be used in grower-cooperator field trials with funding provided by the HLB-MAC project in 2015-17. A special permit was obtained from FDACS to establish widespread commercial field trials using clean USDA sources of many new rootstocks, including Supersour selections. Based on this permit, cooperative arrangements are being made with commercial Florida nurseries for large scale vegetative propagation of these promising new rootstocks, and trials are being established with commercial growers. Three new replicated field trials including about 100 Supersour and other promising rootstock selections were field planted this quarter. Nursery trees were prepared for planting of two new field trials with Supersour rootstocks later in 2015. About five thousand new propagations of Supersour rootstocks were prepared for greenhouse testing for disease tolerance and budding for additional field trials in 2015-6. An additional greenhouse was constructed at USDA to propagate Supersour rootstocks for field trials and is already filled with Supersour material. Greenhouse studies continued to assess Supersour tolerance of CTV, calcareous soils, and salinity. Trees were planted into the field to establish seed sources for the most promising Supersour selections. Field trials and greenhouse studies will continue as resources allow. Studies continued to examine the defense gene profiles of trifoliate orange hybrid rootstocks that are highly tolerant to HLB, so as to improve our ability to create and select rootstocks that possess this trait. Studies continued on defense-related gene expression and small RNAs associated with HLB infection, in collaboration with University of Maryland and University of California research groups. A study of localized defense gene expression in shoots and roots provided evidence of striking differences between susceptible and tolerant rootstocks that are a major advance in understanding, and yield strong insights into ways to overcome the disease. New field trials were planted to compare the effects of HLB on the most promising commercial rootstocks under the best management conditions, and to measure defense gene response. Gene expression research to develop HLB-resistant rootstocks will continue as resources allow. Work continued to create and test transgenic citrus with elevated expression of citrus defense genes that appear associated with tolerance to HLB. Three replicated tests with US-942 rootstocks that overexpress the citrus defense gene CtNDR1, are showing significant reduction in Las infection for some of the transformed clones. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Several transgenic rootstock selections showing increased resistance to HLB have been identified from groups transformed with other resistance genes, and are also being prepared for confirmation testing. One hundred new transgenic rootstocks were produced, targeting to increase tolerance to HLB by manipulation of the citrus resistance genes CtMPK4, CtTGA7, CtDIR1, CtERF1, CtFAD7, CtFMO1, CtAZL1, and CtNHL25. Testing of the new transgenic rootstocks will continue as resources allow.
All of the research described in the previous report is being analyzed or active research is being transferred to a new grant from CRB. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype is concluded and analyzed. There were some surprises. RNA levels of FT3, the FT homologue from citrus that other research by us and others indicates is most closely associated with flowering, were low in leaves at bloom time, whereas we expected high levels of expression. This may indicate that high levels of FT protein are produced at that time and sent to apices and mRNA is depleted. A study of CiFT3 transgenic tobacco plants treated with various growth regulators has been performed and the data is now being analyzed. The growth hormones produced striking and individually different phenotypes in each treatment. The data includes plant height and leaf number, size, and area. The endogenous ciFT3 promoter from sweet orange was successfully cloned to be used in the transcription activator-like (TAL) effector system inducible by methoxyfenozide that will hopefully activate the naturally present FT3 gene in citrus. Large numbers of citrus seeds are being germinated for transformation studies with this construct.
For the last period, using comparative genomics computational analysis, we concentrated on finding proteins that may kill Liberibacter and here we provide several candidates. Most Liberibacter strains contain two prophages in their genomes. These prophages are integrated and are in their lysogenic cycle. They are repressed by proteins that bind to certain regions in their DNA. However, antirepressor proteins, when expressed, will remove these repressors and stimulate lytic cycle of the phage, when phage will reproduce and kill the bacterium. We found several candidates for antirepressors in Liberibacter species. The gene CLIBASIA_00020 is inside a prophage region. It encodes a BroN domain. The BroN domain was named for a multigene family of Baculovirus Repeated ORFs (Bro) in Bombyx mori Nucleopolyhedrovirus whose N-terminal domains that correspond to BroN bind DNA. The BroN domain is present in a number of viral and cellular proteomes and is combined with many various domains. Accordingly, CLIBASIA_00020 protein includes the BroN domain followed by a C-terminal domain that is distantly linked to the ANT Phage antirepressor protein KilA-C domain. This C-terminal domain could adopt a winged helix-turn-helix (HTH) fold typical of DNA binding proteins. All these features taken together suggest that CLIBASIA_00020 may be an antirepressor. Liberibacter asiaticus has another gene, CLIBASIA_04440, that encodes the C-terminal ANT Phage antirepressor, yet it lacks the N-terminal BroN domain. This gene is not in the prophage region. The gene closest to it in Liberibacter solanacearum CLso-ZC1 (CKC_00980) possesses the N-terminal BroN domain as well as the C-terminal ANT. In fact, the Liberibacter solanacearum CLso-ZC1 genome encodes 4 BroN antirepressors (CKC_00989, CKC_01075, CKC_05845, and CKC_05945) that all remain within two separate integrated prophage regions. Inspection of the nucleotide sequence upstream of the ANT gene CLIBASIA_04440 in the Liberibacter asiaticus genome revealed a partial BroN domain. Translation of the nucleotide sequence reveals a single stop codon interrupting the potential upstream BroN domain and the ANT domain from CLIBASIA_04440. Thus, these two regions together may form a functional antirepressor that may possibly activate the prophage and kill the bacterium. The CLIBASIA_00020 N-terminal and C-terminal domains are also found in a closely related gene gp08 from a prophage Xfas53 of Xylella fastidiosa, a plant pathogen that causes a number of insect vector mediated diseases, including Pierce’s disease and citrus variegated chlorosis. In both prophage gene neighborhoods, the Bro-N domain-containing gene is surrounded by similar phage components: phage DNA Polymerase, VRR nuclease, DEAD-like helicase SNF2, CRISPR system cas4, and Duf2815. The Xfas53 prophage contains a CI repressor upstream from the conserved gene neighborhood. The Xfas53 CI repressor includes a xenobiotic response element (XRE)-type HTH domain, followed by a S24 LexA-type peptidase. Similar CI repressors are involved in the regulation of the choice of phage lysogenic or lytic life cycle. An XRE-type HTH containing gene (CLIBASIA_05625) is found upstream from the Liberibacter asiaticus conserved BroN neighborhood that might also function as a repressor that controls the phage lytic life cycle.
Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types have been propagated on x639. Replicated trials with standards will be established. Six locations each of all sweet orange-like together and 4 with all mandarins will be established with 6-8 trees of each cultivar at each site. We have identified cooperators (in Ridge, IR and Gulf coast) for complete replicated block plantings at each site. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce Fl. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a monthly basis. As of December 2014, the first HLB symptoms are apparent. Evaluation of existing standard and non-standard cultivars (‘Hamlin’, ‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, and ‘Ruby Red’) for HLB resistance/tolerance is complete. In August 2010, the plants were established at Pico’s farm in Ft. Pierce Fl. Data on the growth rate, disease severity, and Candidatus Liberibacter asiaticus (CLas) titer levels have been collected since April 2012. During the 4-year period, there were significant differences in disease severity, stem diameter, and CLas levels among the varieties. ‘Fallglo’ had the lowest incidence of HLB symptoms, whereas ‘Ruby Red’ had the highest incidence. ‘Ruby Red’ also appears to be in significant decline. Despite the high titer levels found in ‘SugarBelle’, it had the greatest overall increase in diameter and was the healthiest in overall appearance. These results indicate that compared to ‘Hamlin’, ‘Fallglo’ and ‘Temple’ appear to display field resistance to HLB while ‘SugarBelle’ appears to have significant tolerance. Progress has been made on the antibiotic treatment of HLB infected bud-wood to compare growth at different levels of CLas infection. Bud-wood of nine HLB symptomatic varieties, 3 fairly resistant (‘Temple’, GnarlyGlo’, and ‘Nova’) 3 tolerant (‘Jackson’, FF-5-51-2, and Ftp 6-17-48), and 3 susceptible (‘Flame’, ‘Valencia’, and ‘Murcott’). In November 2013 and May 2014, HLB positive bud-wood was treated with various concentrations of penicillin and streptomycin and grafted on sour orange rootstock. Standard growth measurements (stem diameter and height), disease severity were evaluated and leaves were sampled for qPCR analysis. Evaluations and sampling will continue on quarterly basis. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is currently underway. One hundred and fifty etiolated seedlings of the trifoliate ‘Rubidoux’ and the sweet orange ‘Hamlin’ have been approach grafted together. Generation of new chimeras has been difficult. Several adventitious buds have emerged from the treated graft region, with several appearing to be chimeral. The newly emerged plants will be tested using LC/MS to determine the origin of the three layers. To increase the success rate, additional plants will be grafted over the next twelve months. A method for the rapid identification of potential sources of HLB resistance is also being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are evaluated at 3, 6, and 9 weeks Seedlings of Hamlin and Dancy show early CLas proliferation and systemic movement. Only very low levels of CLas have been observed in Carrizo.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over four years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Data and tissue samples were collected from almost every tree in the test site during the last quarter. Additional plantings are welcome from the research community.
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. Exposure to canker inoculum showed remarkable resistance in chimera compared to control. RNA was isolated from 16 transgenic Hamlin containing Chimera. RT-qPCR showed 50% of them have relative high gene expression. One of them showed over hundred times higher expression compare to plant expressing the lowest level of chimera. These promising transgenic lines were replicated by grafting for HLB challenge. About 30 Hamlin transformed with thionin also were obtained. Twenty transgenic lines were confirmed containing thionin gene by PCR. They will be tested by RT-PCR and replicated for HLB challenge. A new chimeral peptide from citrus genes only has been developed and is being used to transform citrus. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector 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 for HLB and other diseases. Many putative transformants were generated on the selective media. 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 slight canker resistance by infiltration test. Spray inoculation was tried and some of them show obvious canker resistance. To confirm that high ROS production was not due to variability in Hamlin, we examined l 40 Hamlin seedlings and no or very low level ROS production was detected. In contrast, relative higher ROS production was detected from wild-type Carrizo seeding compared to Hamlin seedlings. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
We continue to work to obtain stably transformed citrus containing the BS3 promoter with added TAL effector binding elements (4 or 14 EBE) fused to a marker or defense response inducing gene. A large transformation experiment has been carried out in Duncan grapefruit and sweet orange with an expanded set of constructs, and 75 putative transgenic grapefruit plants and 79 putative transgenic sweet orange plants have been transferred to soil. These plants will be screened by PCR for the presence of the intact transgenes. A model system has been developed in tomato varieties Bonny Best and Large Red Cherry. Transgenic plants have been generated that carry the BS3 promoter with 14 added TAL effector binding sites fused to the effector AvrBs4 (14EBE:avrBs4) which is known to trigger resistance in tomato. We previously reported that one transgenic line carrying this disease resistance construct showed a reduction in symptoms in response to Xanthomonas euvesicatoria strain 85-10 carrying AvrBs3 in initial tests. Further testing has now shown that a hypersensitive reaction was observed in several T2 lines derived from multiple transformation events upon activation with X. euvesicatoria 85-10 carrying AvrBs3. 85-10 carrying empty vector produced a susceptible reaction (disease symptoms) in all transgenic plants, demonstrating that this resistance cassette and pathogen triggered resistance is working successfully in tomato. Homozygous 14EBE:avrBs4 plants will be generated and tested for disease resistance. Experiments continue to determine whether there is cryptic regulation of the 14EBE construct in plants that may contribute to our low transformation efficiency in citrus. We are testing this through marker gene or resistance gene cassettes in model systems – tobacco, tomato and Carrizo. Transgenic tobacco plants carrying the 14EBE:GUS construct have been generated, and whole seedlings were stained to examine whether the construct is expressed anywhere in the plant. No GUS expression was observed in 48 of 50 seedlings tested, demonstrating that this construct is not expressed in tobacco in the absence of pathogen induction, except in low frequency. The recovery of transgenic tomato plants carrying the 14EBE:avrBs4 construct demonstrates that background expression is not an issue in tomato either. In citrus, four transgenic Carrizo lines have been confirmed to contain an intact 14EBE:GUS construct, and these will be used to examine the regulation of the 14EBE promoter in citrus. We expect to obtain further 14EBE:GUS lines in Duncan grapefruit and sweet orange from the current transformation batch, and these will be useful for examining promoter activity in the absence and presence of pathogen triggers.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas In order to screen a large EFR mutant population for the gain of responsiveness to elf18-CLas we have been developing reporter lines with GFP under the control of the various PAMP-inducible promoters (FRK1, WRKY30 and PER4) which could be used to screen with FACS. Of these lines PER4p:GFP produces the lowest background and clearest PAMP-induced expression. We have generated lines in both Arabidopsis cell suspension and plants. The cell suspension lines fail to respond after protoplast mediated transformation. We suspect that this is a consequence of the protoplasting procedure and are investigating alternative buffers which may enable detection of the reporter following elf18 treatment. We are currently bulking seed to test the stable transgenic plant lines to determine if protoplasts derived from plant tissue will be more reproducible. These lines have the additional advantage that some have been generated in an efr-1 background so any weak basal induction by elf18-CLas would be eliminated. In addition, we have been also investigating the possibility of targeting other PAMPs. To this end we conducted bioinformatics comparison of known PAMPs with those in C. Liberibacter asiaticus. From these search we identified CSP22 (Felix & Boller, JBC 2003, 278:6201) as a potential candidate, since it is conserved in the sequence required for recognition. After a long delay in production we have now received the CSP22-CLas peptide and are growing plants to test their activity. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. The manuscript relating to the generation of chimeric XA21:EFR receptors has now been accepted in PLOS Pathogens and will be online shortly. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Transformation experiments are ongoing; to date, a total of 10,556 ‘Duncan’ grapefruit, 2,025 sweet orange and 191 Carrizo segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). Regenerated shoots from transformed segments are being screened for GUS expression, and GUS positive plants are transferred to soil. So far, grapefruit plantlets (110) from all 4 constructs and sweet orange plantlets (17) from the 3 constructs EFR, EFR-XA21 and EFR-XA21-EFRchim have been transferred to soil.
A manuscript has been submitted, which summarizes scion (Hamlin, Valencia, Pineapple, and Ray Ruby) introduction into the growth facility by shoot-tip grafting (STG) from Dec, 2011 to July, 2013 (20 mo). Sixty-six mother plants out of 171 STGs introduced from FDACS were determined to be disease-free after micropropagation, budding, and disease indexing. A total of 157 putative transgenics were generated, 66 survived micro-grafting, and 42 expressed the NPTII protein. NPTII immunostrips, ELISAs and Southern blots were used to characterize the transgenics. Similar to previous reports in immature and mature citrus, there were a large number of escapes using kanamycin as the selection agent. The biggest loss during this period was due to micro-grafting as only 66 out of 157 (42%) GUS or GFP positive shoots survived. We have determined that micro-grafting success is dependent on the transformation batch and shoot age; it must occur early after shoot development. Alternately, rooting mature citrus must be established. Several of these transgenics flowered after the T5 fluorescent bulbs were replaced with LED lights, night temperatures decreased, drought stress applied, or if the trees were moved to natural light. Because our facility has no natural lighting, a greenhouse with natural and supplemental lighting is necessary to obtain early flowering and fruiting of desirable events. We continue to produce transgenic mature citrus scion and rootstock using plasmids with disease resistance genes obtained from various scientists. Since most of these constructs have no GUS or GFP markers, we micro-graft all shoots and screen with PCR, which is a more rigorous process than with reporters. Transgenics are double and triple checked with PCR and NPTII immunostrips to ensure they are stable, not chimeric, and expressing the NPTII protein. We have successfully micrografted at least as many putative transgenics. There are currently 3,600 putative transgenics in the pipeline to be screened. For replicated disease screening, the number of transgenics will be increased at least threefold by budding, and expression in vegetative progeny can be determined. For one particular genetic construct, budding with transgenic immature rootstock can begin at any time to facilitate experiments to determine the contribution of each genotype in imparting tolerance. One mature Swingle rootstock tree, transgenic for a disease resistance gene, is over four feet tall and should flower soon for seed production. We continue to optimize biolistics in order to increase our productivity. Thus far, the results are promising and we have recorded 200-300 transient GUS and GFP foci per shot in mature scion and rootstock shoots. If 0.01% of these foci develop into plants, 2 to 3 transgenics might be produced after each shot. During the optimization process, we have determined optimum stage height, gold particle size, and helium pressure. The primary advantage of using biolistics is that it avoids all of the antibiotics used to suppress growth of Agrobacterium, which also suppress shoot growth in scion and rootstock. The growth facility is being certified as a nursery. This will hasten the pace of providing plants to scientists, growers, and industry. Routine disease testing in April will be conducted by FDACS.
We have concluded the remaining activities of objective 1 of our proposal which have focused around finding a native citrus protein replacement for cecropin B the C-terminal component of the chimeric antimicrobial (CAP) protein. We had identified CsHAT52 using one set of bioinformatics tools and confirmed antimicrobial activity with a portion of this protein that we designated CsHAT22. Bioassay of CsHAT22 revealed a minimum inhibitory concentration (MIC) of 50 uM with Xanthomonas, 100 uM with Xylella and 300 uM with Liberibacteria crescens (Lc). As mentioned in our last report we used two additional bioinformatics programs, PAGAL and SCAPEL and have successfully identified and tested 2 additional proteins, CsPPC20 and CsCHITI25 that were compared to CB and the N-terminal 21 amino acids of CB designated CBNT-21. Among the test strains used Xanthomonas was most susceptible to the peptides with CB and CBNT21 showing and MIC values 25 uM and the MIC values for CsPPC20 and CsCHITI25 were 50uM and 100uM respectively. Both Xylella and the BT-1 strain of Lc gave MIC values of 200 uM for CBNT21 against both Xylella and Lc BT-1. CsPPC20 was more active than BNT21 against Xylella giving an MIC value of150 uM and as active against Lc BT-1 giving an MIC value of 200uM. CsCHITI25 was as active as CsPPC20 against Xylella but not as active against Lc. The CsISS15 peptide displayed no activity and this was an expected result based on the PAGAL predictions. Based on these results we have decided to include CsPPC20 as an additional construct for testing in planta. As mentioned in earlier reports we have CsP14a as a replacement for neutrophil elastase (NE). CTV vectors for expressing CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and currently being used to infect citrus plants. Binary vectors for expression of CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and have been used to generate transgenic tobacco and transgenic Carizo citrus plants. The construction of both CTV and binary vectors for the expression of CsP14a-CsPPC20 are currently under way. We have also used as a positive control NE-CB to develop plants with CTV based delivery and transgenic tobacco and transgenic Charrizo citrus that can be used to validate the efficacy of the citrus derived CAP proteins against HLB.
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, and finally the plants are transplanted to the field where evaluations of resistance continue. 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. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) 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. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of December 12, 2014, a total of 6,402 transgenic plants have passed through inoculation process. A total of 124,795 bacteriliferous psyllids have been used in no-choice inoculations. Additionally, since our last report we have exposed 664 plants to a total of 14,060 infected psyllids in no-choice situations to answer questions about our inoculation procedures. For example, does the presence of flush enhance transmission? In a colony of bacteriliferous psyllids, why are there sometimes large fluctuations over time in percentages of psyllids that test PCR-positive for the pathogen? Are lemon and citron equally suitable for maintaining colonies of infected psyllids? How effective is our inoculation program? Some of these questions are being answered based on transgenic material that has already been passed through the inoculation process.
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, and finally the plants are transplanted to the field where evaluations of resistance continue. 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. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) 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. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of March 31, 2015, a total of 7,066 plants have passed through inoculation process. A total of 138,855 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. As reported in December 2014, we initiated a series of experiments during fall 2014 specifically to evaluate inoculation success and to investigate different parameters related to the inoculation process. For example, does the presence of flush enhance transmission? In a colony of bacteriliferous psyllids, why are there sometimes large fluctuations over time in percentages of psyllids that test PCR-positive for the pathogen? Are lemon and citron equally suitable for maintaining colonies of infected psyllids? How effective is our inoculation program? Some of these questions are being answered based on transgenic material that has already been passed through the inoculation process. Recent feedback from inoculations of rootstock material gives some insight. Eleven groups of rootstock material (3,105 plants total) were passed through the inoculation program during 2011-2014. The percentage of success was 62% for assays conducted 12 to 19 months after inoculations. There was no difference in the success rates for transformed and non-transformed seedlings.
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