Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. From the screening of random mutants which had previously been performed, it became clear that we needed to screen a larger number of mutant clones to find recognition of elf18-CLas. In order to perform screening on complex EFR mutant libraries required to discover mutants which respond to elf18-CLas we have been developing a FACS based screen. To this end we have generated a number of reporter lines in both suspension cultures and transgenic Arabidopsis plants. The reporter lines are driven by the FRK1, WRKY30 and PER4 promoters. We have tested two PER4 cell suspension lines for responsiveness to elf18 and both of these give clear induction of the reporter gene following treatment. We will also test the WRKY30 and FRK1 cell suspension lines once the liquid cultures are established. In addition we have generated transgenic plants with these reporter constructs and are currently waiting to collect seed from primary transformants. We plan to test the activity of the pPER4:GFP cell suspension lines following protoplast transformation to ensure that the reporter is not activated during. If these tests are clear then we will proceed to FACS screen of mutant EFR libraries. In addition to the mutagenesis approach we are currently setting up to screen the Nordborg collection of Arabidopsis ecotypes for sensitivity to elf18-CLas or reciprocal chimeric peptides of elf18-Ecoli and elf18-CLas, in order to determine whether a natural variant of EFR capable of binding elf18-CLas can be isolated. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. These constructs have been constructed and tested and a manuscript is under revision. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Constructs containing EFR alone or in combination with XA21 or XA21-EFR were provided to the Moore lab, and epicotyl transformation experiments are ongoing (May to July 2014). To date a total of 2,945 ‘Duncan’ grapefruit and 879 sweet orange epicotyl segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). Preliminary GUS histochemical assays were carried out on 6 sweet orange shoots regenerated from transformation experiments with EFR, EFR-XA21 and EFR-XA21-EFRchim. Results indicate that no GUS positive shoots were obtained, however 1 shoot from the EFR-XA21-EFRchim construct was chimeric for GUS.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. We reported cloning six new full-length cDNAs of different citrus genes into the entry vector last quarter. Now we have moved five of these clones into the binary vector pBINplusARS. Citrus transformation with these five clones shall be initiated shortly. In addition, a seventh new full-length cDNA was obtained in the entry vector for further DNA construction followed by plant transformation. In addition, we continue to characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project (#129), although the support of the project has already been terminated.
This quarter we have continued to make progress on our transformation approaches: 1. Stable transformation in citrus using new vectors. We previously found that the original vector system used to create the Bs3 promoter constructs was contributing to low transformation efficiency in citrus, and switched to a pCAMBIA-based vector system. Two ProBs314EBE:avrGf2 transgenic plants created using the new vectors in citrus cultivar Carrizo have now been confirmed by PCR to contain the transgene, and these will be examined for appropriate gene expression by RT-PCR. An ongoing pipeline of transformants are being generated with the new vectors. To date a total of 2,056 putative transgenic shoots of grapefruit, sweet orange and Carrizo were screened for this period. Results show that no GUS positive has been observed for the sweet orange cultivar transformed with any of the constructs analyzed, however, 3 shoots were chimeric for the pCAMBIA2201:NosT:Bs3super::avrGF2 construct. In general, grapefruit had a combined total of 12 and 41 shoots being GUS positive and chimeric for GUS, respectively for all constructs anlayzed while Carrizo citrange had 185 and 180 shoots being GUS positive and chimeric for GUS, respectively. These GUS and chimeric shoots will later be screened via PCR once rooted and transferred to soil for acclimatization. 2. Stable transformation in tomato test system: A tomato test system was previously designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. T1 generation of Bonny Best and Large Red Cherry transformed with ProBs3_14EBE:avrBs4 were screened for pathogenicity reaction with X. euvesicatoria strain (Race 9). Promising resistant transgenics have been selected for T2 generation analysis to confirm that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance.
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 is being analyzed. A study of CiFT3 transgenic plants treated with various growth regulators has been performed and all of the data have been collected except for the flowering dates of the nontransgenic control plants that have not yet flowered (they should soon). The growth hormones produced striking and individually different phenotypes in each treatment. The endogenous ciFT3 promoter 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. The complete construct is completed and is being tested in tobacco for a rapid test before citrus experiments are started. This research will be presented at the upcoming ASHS national meeting.
Work on obtaining stable genetic transformation using cell penetrating peptides has not been successful in citrus thus far, using methods that have been successful in at least one other plant species, although that species was a monocot not amenable to Agrobacterium transformation and microspore tissue, different from what we can use, was inoculated. The difficulty in citrus is, not surprisingly, difficulty with getting integration of DNA species into the citrus chromosomes. We are continuing to work on this and very new methods of achieving integration have been developed in other species that we are evaluating. We are having more success with developing a robust transient expression system in citrus with the CPPs. We have shown that we can get locally transient expression of genes and proteins in explants. We are now working with whole plants and other methodology that may allow cargo to move throughout the plant. Finally, we continue our collaboration with the scientists that are working with polymer nanoparticles.
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications; and 3) to create transgenic citrus cultivars with new constructs containing the resistant genes. A total of 25 samples for RNA-Seq, including resistant/tolerant vs. susceptible plants were sequenced and analyzed. We mapped the RNA-Seq data to a reference genome, C. clementina using the bioinformatics program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24,275 genes of the originally predicted genes had been found to be expressed and a total of 10,539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to be expressed. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of ESTs varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 5 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component (PC) analysis study on the expressions of five samples. The results showed that the gene expressions were significantly different in resistant vs. susceptible citrus. Using cuffdiff, a total of 821 genes were identified as difference expressed genes (DE genes) between the two groups using both p-value and an FDR threshold of 0.01. Among them, 306 genes are up-regulated and 515 are down-regulated in resistant citrus. Using the program iAssembler, a total of 53,981 uni-transfrags were obtained. Most of the assembled uni-transfrags should be novel genes, compared with the citrus reference genome. We further identified 3073 DE genes by comparing the gene expression of another group of resistant and sensitive citrus samples using DESeq2 with adjusted P-value (padj) < 0.1. Among these DE genes, 1413 genes were up-regulated in resistant citrus significantly and 1660 genes were down-regulated in resistant citrus. Due to our comparing strategy, the DE genes should most come from the difference between Marsh and Jackson. As all of the 3 resistant citrus came from the offsprings of Jackson and all of the sensitive citrus trees came from that of Marsh. The resistance genes should come from Jackson. A total of 86 DE genes were identified in the tolerant and intolerant citrus group using DESeq2 with adjusted p-value less than 0.01. Among these DE genes, 69 genes were up-regulated and 17 genes were down-regulated in the tolerant samples. We selected top 50 different expressed genes from the up-regulated and down-regulated genes each for the further validation. There are some differences at the isoforms level between the resistant and sensitive samples. Although 205 out of 221 of the different expressed isoforms consist with the expression pattern at gene level, there are 16 different expressed isoforms don't consist at the gene level. For an example, isoform TCONS_00090333, a RING finger and transmembrane domain-containing protein 2, significantly down-regulated in the resistant citrus samples (log2 = -7.17), but the corresponding gene XLOC_025451 have no significantly change between resistant and sensitive citrus samples. We are validating the different expressed genes /isoforms identified by deep sequencing at both gene and isoform levels using RT-PCR, and are making constructs of a few selected genes for citrus transformation.
During the last period we concentrated on the comparative analysis of Liberibacter methabolic enzymes with the goal to find out those present in pathogenic species but absent in a non-pathogen. Two of the identified pathogen-specific genes that are not in the prophage region encode proteins that function in the terpenoid biosynthetic pathway: CLIBASIA_04600 encodes a mevalonate kinase, and CLIBASIA_05065 encodes a geranyltranstransferase. Our initial sequence comparison cutoffs did not detect the Las mevalonate kinase as having an ortholog in L. crescens. However, L. crescens does contain this enzyme fused to the 3-hydroxy-3-methylglutaryl CoA synthase enzyme serving as the first step in the mevalonate pathway (bifunctional enzyme fusion B488_07240). Thus, all of the Liberibacter genomes include the required enzymes of the mevalonate pathway to produce isopentenyl pyrophosphate (IPP). However, only the pathogenic strains possess a geranyltranstransferase capable of elongating the IPP chain. The products of the geranyltransferase enzyme (geranyl-PP and farnesyl-PP) provide the building blocks for monoterpenoid biosynthesis, which is specific to plants) and steroid biosynthesis (mainly in eukaryotes), respectively (from KEGG pathways). For example, the plant terpine limonene that is responsible for the strong smell of oranges and other citrus is formed from cyclization of geranyl-PP. The steroid biosynthetic pathway from farnesyl-PP in plants generates the hormone brassinosteroid as well as other phytosteroids. Modification of this plant metabolic pathway by the pathogens might contribute to pathogenesis.
Putative transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP was obtained. DNA was isolated from 32 of them and PCR test confirmed 28 are positive. Canker test showed two of them greatly increased resistance at the infiltrated concentration of 107CFU/ml. DNA was isolated from 10 chimera transgenic Hamlin and PCR test confirmed 9 of them are positive. They will soon ready for RT-PCR for gene expression. Several transgenic Carrizo with thionin increased canker resistance remarkably with infiltration test at the concentration 107CFU/ml. RNA was isolated from transgenic plants containing chimera and thionin. RT-PCR showed gene expression in the transgenic plants. Further gene expression level was evaluated with RT-qPCR. Our results showed gene expression variation between different transgenic lines, from several fold to 35 fold. Transgenic lines containing D4E1 were evaluated with Xcc infiltration. All the transgenic lines with canker development at 105 CFU/ml while some transgenic lines show less canker development at 104 CFU/ml. Bacterial growth rate in transgenic lines containing D4E1, chimera and thionin was investigated by qPCR. Our results showed some transgenic lines containing chimera and thionin had low Xcc growth rate. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector (collaboration with Duan lab). Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted with eighty Carrizo and ten Hamlin transformants planted in soil. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. 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.
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 three 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. Additional plantings are welcome from the research community.
Evaluation of released cultivars (‘Hamlin’, ‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, and ‘Ruby Red’) for HLB resistance/tolerance is progressing on time. 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 and will continue for another 9 months. All trees exhibited symptoms of HLB and tested positive for CLas. During the 27-month 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. ‘Ruby Red’ also appears to be in significant decline. The highest CLas titer levels were observed in November, December, and January with ‘Sugar Belle’ and ‘Tango’ had the highest titer levels while ‘Fallglo’ and ‘Temple’ had the lowest. 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. Imidacloprid was applied quarterly to a subset of trees and significantly increased stem diameter compared to the non-treated trees but did not have a significant effect on tree height, disease severity, or CLas titer levels. Progress has been made on the antibiotic treatment of HLB infected bud-wood. Bud-wood was treated for nine varieties, 3 HLB-resistant (‘Temple’, GnarlyGlo’, and ‘Nova’) 3 HLB-tolerant (‘Jackson’, FF-5-51-2, and Ftp 6-17-48), and 3 HLB-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. As the scions emerge, standard growth measurements (stem diameter and height), disease severity, and CLas colonization will be evaluated/determined on a quarterly basis. A total of 216 plants out of 320, approximately 60%, were successfully treated and grafted. Development of chimeras is currently underway. One hundred and fifty etiolated seedlings of the trifoliate ‘Rubidoux’ and the sweet orange ‘Hamlin’ has been approach grafted together. After approximately 6 weeks, a horizontal cut will be made through the graft union and treated with plant growth regulators to promote formation of chimeral plants. Generation of new chimeras has been difficult. No chimeras have been produced at this time. To increase the success rate, additional plants will be grafted over the next twelve months. In October 2013, 34 unique genotypes (USDA generated hybrids), some of which appear to have tolerance to HLB, as well as 16 standard and non-standard commercial varieties were exposed to an ACP no-choice feeding trial. Approximately one month after exposure, the trees were transferred to a field plot at Pico’s farm in Ft. Pierce Fl. Growth measurements (stem diameter and height) and disease ratings were initiated in July 2014 and will continue on a monthly basis. At each evaluation period, three leaves will be randomly sampled and CLas titer levels will be quantified using qPCR.
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. 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 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 July 7, 2014, a total of 5,824 transgenic plants have passed through inoculation process. A total of 115,175 bacteriliferous psyllids have been used in no-choice inoculations.
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. 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 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 July 7, 2014, a total of 5,824 transgenic plants have passed through inoculation process. A total of 115,175 bacteriliferous psyllids have been used in no-choice inoculations.
The Core Citrus Transformation Facility (CCTF) continued to function as a platform for testing of different genes that could potentially render Citrus plants tolerant to greening disease. Almost all of the efforts in the facility are directed towards a single goal-producing valuable transgenic plants that may survive greening bacteria-induced infection. Due to a present way of functioning, in some periods of time the CCTF becomes, to a certain degree, an extended arm of research labs. As such, we follow developments taking place in those labs. Recently, five vectors that were previously supplied to the CCTF by one lab were modified and are being used in the latest co-incubation experiments. These five vectors could be considered as new orders. There were no other new orders. Within the last three months, CCTF serviced old orders but the work was also done on the recent order placed by the CRDF. The progress was made for all the orders. For the existing and new orders, additional co-incubation experiments were performed with the appropriate plant material and Agrobacterium strains. Selection of putatively transgenic shoots based on the PCR screen continued without interruption. Production of rootstock plants carrying the NPR1 gene requested by the CRDF is continuing as planned. High number of shoots (~800) was tested in the primary PCR and about 120 of them were positive. Because of our effort to accelerate the production of this material, shoots harvested from explants were placed on medium with gibberellic acid (GA3) to promote the elongation prior to primary PCR screen. Elongated shoots are much easier to graft. The treatment with GA3 turned out to be detrimental for many of these shoots and about 30 of them that were positive in the primary PCR screen were lost as their elongated stems did not sustain grafting well. Of those shoots that were positive in the primary screen and survived the grafting, 27 were moved from in vitro environment to pots. Four of those plants were negative in the secondary PCR while 23 were positive. Those 23 plants are growing well on the light bench in the lab. For the last three months, CCTF produced plants for the following orders: pX4- 18 plants, pNah-three plants, pX11- two plants, pHGJ2- two plants, pHGJ10+ pHGJ11-five plants, pNPR1-23 plants, pNPR1-G-three plants, pELP3-G-one plant, pELP4-G- one plant, pMG105- two plants, and pTMN1-seven plants. Within this group of transgenic plants, two were C. macrophylla, three were Valencia orange, four were Swingle citrumelo, 17 were Carrizo citrange, and 41 were Duncan grapefruit.
A genetic construct from Dr. Mou has been transformed into mature scion of Valencia, Hamlin, Ray Ruby, Pineapple, and mature rootstock of Swingle and Carrizo. A number of PCR positive, putatively transgenic shoots have been micro-grafted onto immature rootstock. There are numerous PCR positive shoots in all varieties, which will be verified by additional molecular methods once the plants are larger. PCR positive Pineapple sweet orange shoots have been secondarily micro-grafted in the growth room and are in soil. We have been able to promote rooting of immature tissues in one week with IBA but not NAA. After clonal propagation of desired transgenics, budding in additional combinations will commence. The capacity of our PCR screening methods has been increased using a direct tissue PCR method, which does not involve performing DNA extractions. Putative PCR positives can also been determined by fluorescence of the PCR reaction in the tube on a UV light, without the need to run a gel, although gel electrophoresis is still mandatory. Additional constructs obtained from other UF scientist(s) have been transformed into the appropriate scion and/or rootstock. Shoots have been regenerated and micro-grafted onto immature rootstock. For some constructs, sequence and maps have still not been obtained and therefore work has not commenced with these constructs. A second-hand laminar flow bench was procured for the growth room. This will become a dedicated micro-grafting station. The numbers of transgenic shoots that survive appear to be greater if micro-grafting is performed first and the shoots are screened later. Since none of our constructs have reporter genes, earlier detection by GFP florescence or GUS staining is not possible. Southern blots of plants transformed with marker genes are underway. The nonradioactive DIG labeling kit is being used, which required a few adjustments (e.g. different membrane, different probe generation) compared to the standard protocol using radioactive labeling. Copy number information and expression data for these transgenics are being compiled for a poster presentation at the annual American Horticultural Society (AHS) meeting in Orlando at the end of July, 2014. Once copy number and gene integration has been demonstrated, it will be possible to determine the number of false positives. The gene gun is operational and is being tested on mature and immature explants of both rootstock and scion. We have been able to obtain calli suitable for biolistics by indirect embryogenesis of immature citrus explants, and plant regeneration should be relatively facile. Mature scion will also be used in biolistics experiments and it is anticipated that plant regeneration will also be improved, without the inhibitory effects of the antibiotics used to prevent Agrobacterium overgrowth after transformation.
Our focus in the last quarter was to develop a greater diversity of candidates that could serve as replacements for the CecB lytic peptide domain of our CAP previously described (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We had successfully used the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. We were able to identify 52 aa CsHAT protein that is highly conserved among citrus. Since the protein is too large to syntheize we have begun making constructs to express these in plants. We designed 3 constructs 1) CsP14a with a secretion sequence and Flag tag, 2) CsP14a ‘ CecB (this is construct 1 expressed as a CAP with the original CecB and 3) CsP14a-CsHAT52 (here construct 1 is linked to the 52 aa CsHAT protein from Citrus). These three constructs are being incorporated into CTV vectors so that they can be used for challenging HLB, this work is in progress. We have made Construct 1 and 3 in binary vectors and incorporated these into Agrobacterium strains and these are being used to transform tobacco, construct 2 is in the process of being made. In order to understand better the functionality of the alpha helical domains of CecB we have developed two new computational tools PAGAL and SCALPEL to better predict the antimicrobial activities in portions of existing proteins. PAGAL (Properties and corresponding graphics of alpha helical structures in proteins) implements previously known and established methods of evaluating the properties of alpha helical structures, providing very useful information of the hydrophobicity and charge moments. We have successfully used PAGAL to search 4000 plant proteins in the PDB database and we now have a database that contains a listing of the properties of each and every alpha helical structure present in all of these proteins and the properties of all of the alpha helical structures present in these 4000 plant proteins. We then developed the second program SCALPEL (Search characteristic alpha helical peptides in the PDB database and locate it in the genome) to search for alpha helical structures of a particular type. Once we obtain a particular hit that has the right properties that we are interested in investigating we then use BLAST to find the corresponding citrus protein. Using these two programs we have focused on our phase 1 search which is currently underway where we are examining all small proteins and will compare them to the N-terminal 21 aa domain of CecB that we call CBNT21 (+ve charge). We determined using these two programs that this domain to be the most active of the two alpha helical domains of CecB. We would predict would be the most active in terms of lytic properties. We identified three small proteins in citrus PPC20 (+ve charge very similar to CBNT21 in its structural properties); CsHAT22 (+ve charge smaller version of the 52 aa CsHAT that is currently being tested in plants), ISS15 (-ve charge, very small protein). We also will test CATH12 smallest definsin (12 aa) to define how small a peptide we can use successfully against our pathogens of interest. All 5 proteins have been synthesized and are currently being tested for their lytic activity against E.coli, Xylella, Xanthomonas and Agrobacterium to determine their range of clearance of these pathogens.
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