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.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block planted on a 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3.5 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. The block was planted 3-4 July 2012 and monitoring ACP with flush inspection and sticky cards commenced 13 August. Sticky cards are monitored for ACP and other common citrus pests and replaced every other week. 4011 psyllids have been found on sticky cards of which greater than 67% are in no-mulch plots while only 15% have been found in plots that receive insecticides and 4% in plots with both mulch and insecticides. All of 79,944 shoots have been examined so far of which 12,316 were infested with ACP and 1759 were infested with Aphids, both predominately in no-mulch plots with more than 65% infestation. In contrast, 1396 ACP and 569 aphid-infested flush were found on insecticide-treated trees and 463, and192 on trees on mulch and receiving insecticide drenches. Leaf samples for HLB testing were collected July 2014 of which there were 55 positive samples: 12 from mulched treatments and 10 from insecticide plots. Only 1 tree was positive in plots with both insecticide and mulch. Trunk area cross section measurements collected on July 1, 2014 showed different results from previous reports. Trees receiving mulch and foliar nutrition are now 20% larger than trees receiving nutrition. Also the no-treatment control with mulch is still larger than without mulch. However, the same is no longer true for the treatments reviving insecticide applications. Treatments receiving insecticide drenches either with and without foliar nutrition and no mulch now have larger trunks than the same treatments with mulch. This may be a result of an irrigation system failure during 13 days in Feb and 15 in May which caused more drought stress to trees on mulch while the unmulched trees benefited more from rainfall (1.53 inches at FAWN SWFREC). HLB samples and growth measurements are scheduled to be collected again late January 2015. The first fruit harvest was completed 9 Dec with 19% more fruit by weight was collected from plots with mulch when compared to plots without mulch. Insecticides increased yield by 17%, foliar nutrition increased yield by 7% compared to the check. Juice analysis was conducted January 14. Normal grove care operations continued which included Kocide sprayed monthly for control of canker, and one application of Intrepid for leafminer control. Delegate was sprayed in all insecticide plots late on October 15 as well as an application of Baythroid to all trees as a dorment season spray January 8. A manuscript entitled “Role of Metalized Reflective Mulch with Nutritional and Insecticidal Treatments in Mitigation of HLB in New Citrus Plantings is in preparation.
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.
Report for period ending 6/30/14 During the period of 4/1/14 to 6/30/14, Mr. Page assisted with the daily activities associated with CRDF funded field trials. These daily activities included (but was not limited to) the following: Worked on standardization of data collection spreadsheets, set up water testing contracts, ordered supplies (soil probes, harvesting bags, etc…) for crop consultants (CC’s), talked with CC’s about trial progress, set up trial sites around the state including southwest, east coast and ridge locations, hounded CC’s for data, worked on details of new proposals, collection of picutres from field trials to add to data base whcih was setup, maintain the organization of the database, conducted rankings from trial photos received from CC’s, perfromed DI ratings, setup locations for thermal therapy trials, provided CC’s with protocols for leaf sampling for pcr, and assembled budgets for trials. Overall, Mr. Page was constantly working on a daily basis with different CC’s to ensure open lines of communication between the CC’s and the CPDC to ensure information was collected and delivered in a timely manner. Additionally, this required frequent travel to study sites around the state which during this period included a visit to trial site in Labelle (4/4/14), trip to trial site in Ft. Pierce (4/9/14), visited trial site in Balm (4/10/14), attended the CPDC meeting (4/17/14), field visit to thermal therapy trial sites with Reza Ehsani (6/18/14), photographed ridge grove site trial (6/19/14), and additional photographs of a ridge trial site (6/26/14).
Report for period ending 9/30/14 During the period of 7/1/14 to 9/30/14, Mr. Page assisted with the daily activities associated with CRDF funded field trials. These daily activities included (but was not limited to) the following: Attended routine meetings with Drs. Browning and Syvertsen to provide updates on the status of trials, maintained constant communication with crop consultants (CC’s) to ensure projects are moving forward, setup contracts with soil testing laboratory, sorted out results from testing labs, sorted and submitted invoices, contact product reps for material samples, continue to organize spreadsheets for data analysis, and reschedule submission dates for the CC’s filed trial data. Mr. Page also traveled to field sites and attended meetings to ensure projects were up to date. Travels durign this period included attending the CPDC meeting (7/1/14), went to ridge grove regarding a soil microbial trial (7/8/14), attended a thermal therapy field day at SWFREC (7/10/14), visited ridge grove sites and took photos (7/11/14), grove site visit in Labelle (7/15/14), setup new field trial at ridge grove location (7/22/14), conducted DI ratings at northern ridge grove site and photographed another field trial on ridge (7/25/14), was onsite for a thermal therapy treatment evaluation in a ridge grove trial (7/29/14), visited an east coast field trial site (7/30/14), sampled leaves for pcr testing from thermal therapy trial at a ridge grove trial site (8/7/14), took photographs of two thermal therapy trials at different ridge locations (8/15/14), photographed ridge grove trial site (9/4/14), field trial visit with company representative (9/8/14), field trial visit to Labelle (9/9/14), attended thermal therapy field day (9/10/14), attended CPDC meetings (9/18/14), and took photographs of thermal therapy trial at a ridge trial site (9/24/14).
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.
Overall goal: To engineer Liberibacter asiaticus gene regulation in Sinorhizobium meliloti. Previous results: We established a clone expressing the Liberibacter rpoH La rpoH) gene, and introduced it into Sinorhizobium meliloti strains that contain reporter fusions for 5 key genes we expect might be controlled by rpoH. As controls, we have the cloning vector along, and the vector expressing the native Sinorhizobium meliloti rpoH (Sm rpoH) gene. Results as of December 2014: 1. On each fusion strain, we performed GUS assays ‘ IPTG. We employed LB medium, with no stress conditions. Background GUS levels with vector alone are high, perhaps because strain background is WT for rpoH. ‘ The optimized La rpoH is able to regulate 4 of 5 fusions (groES, hslV, clpB, ibpA): each is induced (compared to empty vector) 2X to 4X, completely dependent on IPTG induction of rpoH transcription. ‘ We were surprised to find no induction of target genes with the Sm rpoH1 plasmid. This is a curious result. Why would optimized La rpoH be better at inducing expression of Sm rpoH target genes? We confirmed by PCR that we did not switch plasmids or strains. Here are possible explanation: (a) perhaps codon optimization of La rpoH allows for better expression in S. meliloti than the native rpoH1; (b) it might be that Sm rpoH1 levels are tightly regulated by proteolysis, but optimized La rpoH is not subject to this regulation; (c) For some unknown reason optimized La rpoH is better than Sm rpoH1 at initiating transcription at these target genes. 2. We plan to retest the La rpoH and Sm rpoH1 activities in RFF231 (.rpoH1H2; that is deleted for both native rpoH genes). We have introduced the 5 uidA fusions into that strain and will conjugate the rpoH constructs into each. 3. We are making a new optimized La rpoH construct with a slightly different insert than the one used above. The reason is to test whether using the RBS already present in the pSRK-Gm vector works better than introducing our own RBS (Mike Kahn ORFeome RBS+spacer) 4. Our next step will be to study more Ca. Liberibacter asiaticus genes for a regulatory role. We have designed optimized coding sequence for these genes: CLIBASIA_01180, 00835, 02900, 02905, 01545, 03950. In the next month, we will order the synthetic DNA segments corresponding to the optimized genes. 5. A note on technique: we anticipate that pSRK-Gm plasmid will work for our purposes. However, we will await the results of experiments in 2 and 3 before cloning the new genes into the vector. Summary of accomplishments: we have successfully expressed the Ca. Liberibacter asiaticus sigma factor RpoH in the heterologous Sinorhizobium meliloti, and we showed that it functions to control promoters for defined S. meliloti genes.
The overall goal of this project is to 1) determine the overall effects of ACPS/open hydroponics growing systems on drought susceptibility and 2) the efficacy of plant growth regulators on mitigating the effects of preharvest fruit drop resulting from HLB. The data are now being analyzed for a formal report/publication. Preharvest fruit drop data from the 4-acre plant growth regulator trial in Lake Alfred are in the final stages of analysis. The anatomical data from citrus trees in the different experimental blocks are also in the final stages of sectioning and analysis. As part of an extension of this project a greenhouse study has been initiated to test the efficacy of 2,4-D and other plant growth regulators on root health in small trees with and without HLB. The plants have been potted, graft inoculated, and their initial root mass measured prior to the first applications of the PGR treatments. These trees will be evaluated regularly over the next six months for changes in growth and physiology in response to the PGRs and HLB.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated. – The second cohort of advanced, tetrazygous citrus rootstock materials for en masse propagation have been identified and are being prepared to have cuttings taken from them. – In addition to the 1st & 2nd cohorts of tetrazygous rootstocks, promosing diploid rootstocks have also been identified and are being prepared to have cuttings taken from them.
Understanding the proliferation and movement of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (citrus greening), within the citrus tree remains a major obstacle in the efforts to undermine the pathogenicity and destructive nature of the disease. CLas still remains an unculturable organism, in part due to the lack of information on the phloem contents and internal environment. Once established in the phloem sieve elements, CLas movement within a tree has been assumed to follow the photoassimilate stream. Our current belief of the CLas transport mechanism is dependent on a presumed bidirectional flow of phloem sap, with basipetal movement from the site of infection, proliferation in the roots followed by systemic distribution through the tree. For CLas movement to occur under the current theory, CLas must be able to move laterally around the stems, trunks, and young roots, and then travel bidirectionally within the phloem sap. However, determinations based on general phloem physiology, our limited understanding of CLas, and on current anatomical studies have exposed serious inconsistencies with the accepted beliefs of CLas phloem transport. For example, based on general phloem anatomy and our current microscopy observations, lateral movement of CLas around a stem appears improbable given the size of cytoplasmic plasmodesmatal connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from infected roots to healthy aerial tissues through the phloem conduits is difficult to reconcile without a reversal of the phloem flow, a condition not demonstrated for any evergreen species. The objective of this study is to define the biochemical properties and transport direction of citrus phloem elements that support CLas proliferation and allow movement within a citrus tree. The data will provide the basis for developing culturing conditions for the bacteria and for mitigating strategies based on movement of the phloem sap. This project is well under way, with all preliminary work completed within this period. To determine phloem movement, two separate devices have been constructed following existing literature. To follow phloem sap movement in young tissue, a device consisting of 2 arm probes has been successfully tested. In one arm, a UV light source and fluorescent sensor are mounted on the tip, whereas the second arm probe contains just fluorescent sensor. An externally applied fluorescent phloem-mobile substance is excited upon passage through the UV and its fluorescence detected by both probes as it moves down within the phloem sap. Time is recorded and velocity can be calculated. For this section of the project, data has been collected from both HLB and healthy greenhouse trees and data is being analyzed. For phloem movement in stem tissue, an electronic device was constructed. The device is based on heat transfer through the plant cortex. It contains 2 heat sensors that will determine time of heat transfer from a centrally located heat supply. The instrument has gone through a series of improving steps including solar energy power installation, miniaturization, test for different bark thicknesses, etc. We are finishing the construction of 10 instruments, 9 to be placed in field trees and one for future greenhouse experiments. Field installation is scheduled for December 10.
Transgenic plants containing our stacked transgenes are being clonally propagated for disease resistance evaluation and the first trees will be challenged for HLB resistance in spring 2015. Improving Consumer Acceptance: Following the successful demonstration of the inducible cre-lox gene system, the plant transformation vector has been modified to contain our NPR1 gene and Agrobacterium mediated citrus transformation is underway to incorporate this gene. Induction of early flowering to reduce juvenility (Carrizo transformed with the FT gene): After numerous attempts, we have finally produced transgenic Carrizo citrange plants expressing the clementine-derived CFT3 gene. Several of the plants have flowered once in the greenhouse in the juvenile state. These plants have been micrografted to standard rootstock and are in the greenhouse for further evaluation and observation. The new transgenic field site at the Southwest Research and Education Center (working with Dr. Phil Stansly) was successfully established, and approximately 320 transgenic citrus plants were planted as follows: Constructs: pCIT 107O (35s-CEMA) Line/Events: 15 Constructs: pCIT 109 (35s-SABP2) Line/Events: 24 (SABP2 is a SAR-inducing gene showing great promise) Constructs: pCIT 109A (AtSUC2-SABP2) Line/Events: 57 Constructs: pCIT105 (35s-CEME) Line/Events: 34 Constructs: pLC 216 (35s-LIMA) Line/Events: 190. Note: most of these are transgenic LIMA rootstocks (Carrizo/Orange 16) with non-transgenic Valencia scion. Plants in our Indoor RES structure have not flowered this year. It is possible greenhouse temperature may have played a role in the flowering process. We will attempt to keep the greenhouse unheated this fall in hopes of initiating flowering in spring 2015. We have achieved rapidly growing transgenic sweet orange trees through thorniness. We are also planning an outdoor RES type structure for transgenics.
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