The Mature Tissue Transformation Laboratory (MTTL) continued to increase its preparedness for the first incoming orders. Number of rootstock plants available for budding was increased significantly and supply is now at the level necessary for normal operation. In the last three months, seven co-incubation experiments were performed. In three experiments, 2212 explants of Valencia were used. Two co-incubations were done with 497 Hamlin explants. One experiment was done with 240 explants from Pineapple orange plants and one with 701 explant of Ray Ruby. The experiment with Ray Ruby was the first experiment done using the grapefruit explants. The data were analyzed from three experiment performed in the previous reporting period and from four experiments performed in this reporting period. For both binary vectors used pCAMBIA2301 and pTLAB21, transformation rate is about 3%. Because of the problems with the budding success rate, the decision was made to change provider of grafting services again. Within the last 12 months, major efforts were directed towards keeping the facility operational, employees retained, and number of rootstock plants increased to levels needed for performing 9-10 experiments per quarter. Those goals have been achieved. By doing multiple repetitions of transformation experiments with bacterial strains carrying two different binary vectors, proper estimation of transformation success rate was obtained. That rate is at satisfactory level for citrus mature tissue transformation. However, there is a possibly lingering problem that needs to be addressed. Many of the plants produced to be the source of explants in co-incubation experiments have thorns that are one of the major features of juvenility. As a result, at least a half of transgenic plants already produced in the MTTL also have thorns. Within next few months, the oldest transgenic plants in our inventory will reach the age where they should theoretically flower. If they do not flower, protocol used for production of transgenic plants will have to be re-evaluated. Also, we must make sure that the sources of our germ-free certified material for production of ‘mother’ plants are really mature trees.
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. This report marks the end of the first year of the project, during which we have achieved large-scale production of CLas positive ACP. To date on this project, a technician dedicated to the project has been hired, a second career technician has been assigned part-time, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies are being used for caged infestations. A total of 3,583 transgenic plants have passed through the screening program. A total of 71,760 psyllids have been used in no-choice inoculations. 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.
Work continues on the construction and characterization of new FT constructs using cDNA clones. The experiments are underway to compare the new FMVcDNA27 construct, which contains an FT3 cDNA insert in the pCAMBIA2201 vector with a constitutive FMV promoter, with a corresponding genomic clone, which we have been using up to this time. Transformation of Carrizo and tobacco tissue is underway in order to compare the action of these two constructs. The new construct was created as a first step towards the development of a new FT3 construct with an inducible promoter. We have arranged for the materials transfer of two inducible promoter systems from the Danforth Foundation. Both of these promoters are inducible by the chemical methoxyfenozide, a widely-available pesticide, approved for field use on citrus. However, we have not yet received the inducible promotors. One system is driven by the CsMV constitutive promoter, and the other by the RTBV vascular-specific promoter. Once we have verified that the smaller and more manageable cDNA is as effective as the original genomic version of the FT3 gene, we hope to begin development of the inducible promoter constructs. Experiments to determine the behavior of the three genomic clones from citrus when overexpressed in tobacco have been completed and a manuscript is being written. Expression of the genes in mature nonstransgenic citrus plants is being recorded monthly.
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. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. 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 1/16th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. Dr. Grosser removed the unsuccessful trees from the first planting and planted additional transgenics among the promising trees still under trial. Additional plantings are welcome from the research community.
Evaluation of existing standard cultivars (‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, ‘Hamlin’, and ‘Ruby’) for HLB tolerance/resistance is underway . Trees were planted in 2010, using a randomized complete block design, at Picos Farm, Ft. Pierce, FL. HLB symptom development and tree growth (diameter and height) are being monitored on a monthly basis. All of the cultivars in this trial exhibit symptoms of HLB and have tested positive for Candidatus Liberibacter asiaticus (CLas). Results to date support earlier observations that ‘Temple’ and ‘Fallglo’ are in the most tolerant group. Numerous procedures are underway to elucidate mechanisms of resistances. These methods include light, confocal, fluorescence, scanning, and transmission electron microscopy, Fourier Transform Infrared spectroscopy and metabolite profiling using LC/MS to determine if there are chemical signature differences and or compounds(s) that are responsible for resistance. Another project involves the treatment of various resistant/tolerant citrus accessions and susceptible standards with various concentrations of antibiotics to generate a range of CLas titer levels. There are 9 varieties that will be tested: 3 resistant (‘Temple’, ‘GnarlyGlo’, and ‘Nova’); 3 tolerant (‘Jackson’, FF 5-51-2, and Ftp 6-17-48); and 3 susceptible (‘Flame’, Valencia’, ‘Murcott’). Budwood with various concentrations of CLas, derived from the antibiotic treated plants, will be evaluated for their potential to result in HLB symptoms in disease free material. The budded plants will be evaluated for growth and HLB symptoms development over a 2-year period. Temporal progression and systemic movement of the bacteria in the inoculated plants will be determined along with HLB symptom development, and growth of the plants. Development of periclinal chimera using resistant geneotypes and standard varieties is in progress. In vitro shoots have been established from nodal and internodal explants excised from mature, certified disease free plants of Red Carrizo, Temple, Hamlin, and Valencia. After root formation, chimeras will be generated using a procedure developed by Ohtsu (1994). ‘Carrizo’ and ‘Sweet Pineapple’ have been successfully approached grafted. The graft unions were cut horizontally and treated with hormones to induce callus formation. Adventitious buds are starting to develop on the cut surfaces. A technique using flavanone profiling from extracted leave are currently being developed to the layers of the resulting scions. Fifty unique hybrids (USHRL advanced selections) and standard cultivars have been challenged in an Asian Citrus Psyllid (ACP) feeding trial using CLas infected ACP. HLB symptom development, growth, and titer levels will be monitored oin each plant. Trees initially were exposed to no-choice feeding, but are now in a free-flying ACP environment. ACP feeding preference will also be examined using scanning electron microscope to enumerate the amount of ACP feeding structures. One additional study has been added to the project. Screening and evaluating new scion materials is a lengthy process and require multiple testing locations. Due to the urgency to develop tolerant/resistant material, a shorter evaluation cycle procedure is being investigated. If this screening method is successfully, it may be useful to quickly identify new sources of resistance varieties that may enhance and improve citrus production in Florida.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants with citGRP1 were transferred to soil. Nineteen of them were test by PCR and ten of them are transgenic plants with citGRP1 insertion. They will soon be ready for RNA isolation and RT-PCR to check gene expression. More than thirty kan resistant shoots were obtained from citGRP1 transformed Hamilin. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. Seven transgenic Hamlin with MADS6 were planted in soil. In addition, numerous putative transformants are present on the selective media transformed with different constructs. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many kan resistant transformants were generated on the selective media. About twenty kan resistant shoot are rooted in rooting medium and one of Hamlin transformatn was planted in soil. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and 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 responding to HLB and other diseases. The construct pBinARSplus:nbFLS2 was used to transform Hamlin and Carrizo. Many putative transformants were generated on the selective media. About forty resistant shoots were rooted in rooting medium and ten Hamlin transformats were plant in soil. Other targets identified in genomic analyses are also being pursued. 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.
Our recent progress towards proposed research goals: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. A] Mutagenesis of EFR to produce elf18-CLas responsiveness: Our initial approach of random mutagenesis and screening in tomato was unsuccessful, indicating the necessity to generate multiple mutations for elf18-CLas recognition. Presently we are evaluating phage display for this purpose. To this end, we have defined suitable conditions for specific binding of ectodomain fragments of EFR to biotinylated elf24. Elf24 has been used for these experiments to allow linkage of the biotin group to Lysine 24; this peptide is fully functional in the elicitation of ROS. We are in the process of evaluating binding of biotinylated elf24-CLas to different regions of EFR. Once optimal regions are determined, mutagenesis will be performed on these regions and cloned into phage display vectors for screening. B] Screening for natural variants of EFR: A small selection of Brassicae has been screened for elf18-CLas response, however none of these were positive. Pending the outcome of mutagenic approaches, the screen for elf18-CLas response will be expanded to a large number of Brassicae. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Assessing XA21 function in dicots: Transgenic XA21-EFR, XA21 and EFR lines have been generated in Arabidopsis and are now ready to assess their effectiveness in pathogen defence. We plan to test these lines against Xanthomonas, Pseudomonas and Argobacterium. In addition, we have generated transgenic tomato lines expressing XA21. These plants will be crossed with EFR tomato lines to determine the pathogen resistance conferred by these two genes in a heterologous system. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. We will initiate the construction of appropriate expression vectors of genes for citrus transformation and expression.
Data from multiple rootstock trials affected by HLB were analyzed for identification of promising advanced rootstock selections. Some selections appear to perform better than standard rootstocks in locations being affected by HLB. The most promising new USDA rootstocks were identified for a cooperative effort with CRDF, to place new rootstocks with HLB tolerance into larger scale commercial plantings. These promising rootstocks include US-896, US-1279, US-1281, US-1516, US-1282, US-1284, US-1283, US-1305, US-1297, US-1311, US-1280, US-1287, US-1271, and US-1298. The rootstock selection US-896 has been tested at multiple locations over many years, has been established as a clean budwood source with Florida DPI, and will be submitted for commercial release within the coming year. The other promising selections were propagated to establish multiple seed source trees, multiply material for larger scale testing, and enter material into the Florida DPI clean budwood program. Seed trees of the promising new rootstocks mentioned above are being propagated for planting into the new screenhouse under construction at the Whitmore Foundation Farm. Budded nursery trees were grown off in preparation for planting in three new rootstock field trials in spring 2014. Two thousand propagations of supersour rootstocks at USHRL are being budded with Valencia for use in field trials to be planted in summer 2014. Cooperative work continued with a commercial nursery to multiply promising supersour rootstocks to prepare trees for medium-scale commercial plantings. Rootstock liners were supplied to another commercial nursery for propagation of trees for three cooperative rootstock trials, including supersour selections. Work continues to assess supersour tolerance of CTV, salinity, and calcareous soils. A new greenhouse test was budded to measure quick decline reaction in response to CTV infection of grafted trees. Specific defense-related citrus genes were investigated in detail, including genes identified by expression studies as being associated with HLB response, such as RDR1, RAP4, CSD1, and CtCDR1. In collaborative work with a University of Maryland team, constructs designed to alter expression of a series of citrus defense genes are being used to transform citrus for improvement of HLB tolerance and resistance, and derived transgenics will be tested using the pathogen. In a collaborative study with a University of California team and funded by CRB, we compared gene expression for trees infected with HLB to those infected with CTV. There are some common elements to the two different diseases that help us more fully understand the citrus defense response to HLB. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility has been completed and will be published later this year. A preliminary study to examine the effect of HLB tolerant rootstock grafting height on tree response to HLB was completed. A second, more thorough study of grafting height was initiated. More than 100 new transgenic rootstock selections with potential resistance to HLB were produced this quarter, including the citrus resistance genes CtNHL1, CtJAR1, CtMOD1, CtACD1, or CtEDS1. Fourteen new transgenic rootstocks with selected antimicrobial genes were propagated and entered into a replicated greenhouse test with ACP inoculation to assess tolerance to HLB. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Transgenic selections that appear to exhibit tolerance or resistance to HLB are additionally propagated and scheduled for further greenhouse and field testing.
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 such as RT-PCR; and 3) to create transgenic citrus cultivars with new constructs containing the resistant gene(s). The first group of 5 samples for RNA-Seq, including resistant/tolerant vs. susceptible plants are nearly complete. The second group of 10 samples is in the sequencing process. 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 plot of the first PC against the second PC showed that R2017 and R20T18 clustered together (the resistance group) and R19T23, R19T24 and R20T24 clustered together (the susceptible group). This result 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. To reveal the differences in resistance, we also identified the exon variations (SNP/INDEL). A total of 612,618 SNP/INDELs were identified using the mpileup method employed in samtools. We focused on two types of mutations that could contribute to the resistance difference. The first type of mutation is a mutation in the genes of susceptible citrus leading to pseudogenes (Type1) and the other type of mutation is a mutation in resistant citrus genes that may gain a new function (Type2). Type1 mutations should have a homozygote mutation genotype in the susceptible citrus. We identified 146 candidate genes having Type1 mutations, which produced high impact variations such as frame shifts, splice site acceptors, splice site donors, a start lost, a stop gain or stop lost, and 3,578 genes with Type2 mutations. We expect that as the number of libraries being sequencing increases, the number of candidates will be reduced to a reasonable number allowing for further validation. We identified a few LRR-PKs genes for further comparative study based on the RNA-Seq data. The results indicated sequence variations of these genes in different varieties are indeed due to SNP/indels, and some of them were annotated as putative pseudogenes because of a truncation. Further verification is underway.
This is a new project that only just was funded, so there is not yet much to report. However, we do have some preliminary results. Cell penetrating peptides (CPPs) are small protein fragments that have been shown to be able to pass through the cell membrane that surrounds mammalian cells. More significantly, when the CPPs translocate in this manner they can also escort ‘cargoes’ across the membrane. Cargoes include proteins, plasmid or linear DNA, RNA, and antibodies that cannot enter the cell or blood-brain barrier without the presence of CPPs. CPPs have also been shown by others to work to introduce cargoes into plant cells. We have determined what CPPs work effectively in citrus for the import of proteins and nucleic acids. Imported DNA clones transiently express marker proteins; experiments on stable transformation have begun.
The objective of this project was to discover citrus germplasm with resistance to the Asian citrus psyllid. Research during the project showed that germplasm in the following major groups of Rutaceae did not have any significant resistance to infestations of the Asian citrus psyllid (ACP): sweet oranges, citrons, pomelos, limes, lemons, sour oranges, papedas, mandarins and hybrids among these groups including grapefruit. However, within the trifoliate group, most (but not all) accessions of Poncirus trifoliata as well as a number of its hybrids (.Citroncirus) were shown to have natural resistance to ACP. Two types of resistance have been identified. One of these resistance types (antixenosis) greatly reduces infestation levels of the psyllid, a resistance trait that may be related to differences in volatiles used by the psyllid to find and infest plants or the presence of a volatile that repels the psyllid. The other type of resistance (antibiosis) results in reduced longevity of psyllids, possibly related to the presence of toxic secondary plant metabolites. P. trifoliata can be crossed with Citrus species using traditional breeding methods, thus it may be possible to identify traits conferring resistance and to breed these directly into Citrus using traditional breeding or other methods. Additionally, volatiles imparting antixenosis may have value for managing or monitoring ACP. We have already discovered clear differences in the volatile profiles of susceptible and resistant plants. Poncirus trifoliata accessions (CRC code at the National Clonal Germplasm Repository, University of California, Riverside) identified as having antixenosis resistance to ACP: 838, 1498, 1717, 2552, 2554, 2861, 2862, 3151, 3206, 3207, 3209, 3210, 3212, 3213, 3215, 3217, 3218, 3219, 3330A, 3330B, 3338, 3411, 3412, 3484, 3485, 3486, 3547, 3548, 3549, 3571, 3586, 3588, 3882, 3888, 3938, 3939, 4006, 4007, 4009, 4017, and 4138 (41 of a total of 48 accessions evaluated). .Citroncirus (trifoliate hybrids) accessions with antixenosis resistance: 275, 1438, 1447, 1448, 1459, 1463, 2618, 3205, 3348, 3771, and 3881 (11 of a total of 34 accessions evaluated). Poncirus trifoliata and .Citroncirus accessions with antibiosis resistance: 275, 276, 1438, 1447, 1448, 1463, 3330B, 3348, 3881, 3889, and 3969 (11 of a total of 17 accessions evaluated). Two publications have thus far been published from this research project: Westbrook, C. J., D. G. Hall, E. W. Stover, Y. P. Duan and R. F. Lee. 2011. Colonization of Citrus and Citrus’related germplasm by Diaphorina citri (Hemiptera: Psyllidae). HortScience. 46 (7): 997-1005. Richardson, M. L., and D. G. Hall. 2013. Resistance of Poncirus and Citrus x Poncirus germplasm to the Asian citrus psyllid. Crop Science. 53: 183-188. Although this project is terminating, we are concluding antixenosis testing of a number of additional Poncirus and hybrid accessions, and an evaluation is being conducted on antibiosis of Poncirus to psyllid nymphs. Results of these experiments will be published. It is hoped that traits conferring antibiosis or antixenosis can be identified in the near future.
The transformation construct for expressing the FLT-antiNodT fusion protein in citrus is nearing completion. We encountered major difficulties cloning the FLT-antiNodT expression cassette into the pTLAB21 citrus transformation vector. The FLT-antiNodT cassette DNA appeared to be unstable in E. coli when cloned into pTLAB21, which stymied our cloning efforts for several months. The instability of the FLT-antiNodT cassette in pTLAB21 was surprising, since the FLT-antiNodT cassette was stable in E. coli when cloned into non-transformation vectors such as pBluescript. For reasons unknown, the FLT-antiNodT cassette was specifically unstable in pTLAB21. However, we serendipitously discovered that the inclusion of an additional segment of DNA next to the FLT-antiNodT cassette in pTLAB21 actually stabilized the FLT-antiNodT cassette in pTLAB21. This piece of DNA was derived from the original FLT-antiNodT cassette cloning vector, pBluescript. This additional piece of DNA should not cause a problem for transformation of citrus or expression of the FLT-antiNodT antibody in transgenic citrus. We are now in the process of sequencing and verifying the pTLAB21-FLT-antiNodT transformation vector. Once this process is complete, we will commence citrus transformations. We anticipate that transformations will begin within the next reporting period.
In the period between April and July of 2013, Citrus Core Transformation Facility (CCTF) experienced significant changes in personnel. These changes reflected negatively on the productivity and as result, the goal of 100 transgenic plants per quarter was not reached. The experimental efforts were directed towards orders that were placed within previous six months. Produced transgenic plants were all Duncan grapefruit. They belong to following orders: MED16-11 plants; X20-four plants; X16-three plants; X19-one plant; X4-one plant; Bi121-six plants; N5-31 plants; N7-nine plants; HGJ3-one plant; and W14-one plant. As opposed to a few previous quarters when the number of placed orders was unusually high, facility received three orders during this time. The ‘genes’ of interest in those vectors were: gMOD1, ELP3-CIV, and ELP4-CIV. All three orders require transformation of Duncan grapefruit plants. Actually, two more orders were placed but after initial attempts to mobilize binary vectors into appropriate Agrobacterium strains the quality of cultures was reviewed and client decided to withdraw them. Within the first year (14 months) of funding of this project, CCTF received 35 orders for production of transgenic plants. Altogether, about 430 plants were produced. They belong to 23 orders received within this funding period and eight orders from previous period. Transgenic plants that were requested from clients in new orders were mostly Duncan grapefruit, but there were some requests for Valencia oranges, Carrizo citranges, and Mexican limes. Ratio of produced plants reflects well the ratio of requested plants in placed orders. CCTF remained a reliable producer of transgenic material. High influx of orders speaks of the important role that CCTF plays in efforts directed towards disease tolerance improvement of Citrus cultivars. Researchers that have well developed ideas or initial encouraging data about the beneficial activity of certain genes know that CCTF can help them test their hypotheses by producing citrus plants that carry those genes in them. While waiting for transgenic material from CCTF, those research groups can direct their efforts towards development of appropriate challenge tests that will be performed on transgenic plants or work on other aspects of protection against citrus diseases.
This project was initiated May 1 2013 and we have focused our efforts on Activity 1. In this activity we are using a rational design strategy to develop and rapidly test individually the two components of a chimeric antimicrobial protein (CAP) based therapeutic that could provide broad spectrum resistance to young citrus plantings against a range of biotrophic bacterial pathogens (HLB and CBCD). Gene mining bioinformatics tools are being used to search existing citrus genomic DNA sequence information available in Phytosome (http://www.phytozome.net) to identify structurally relevant candidate citrus components that can be fashioned into a CAP molecule. The current and highly functional CAP design described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725) is being used as a scaffold/guide to search for the citrus components. Working with the developer of a recently described bioinformatic tool CLASP we have successfully searched using the active site architecture (3D shape) of the forst component HNE and have found a citrus PR14a sequence whose shape is similar. We have determined the similarity of the citrus protein shape to that of the tomato PR14 and grapevine PR14a proteins. We used the tomato sequence as the protein 3D structure has been determined and could thus be used to thread the citrus and grapevine protein sequences whose structure is yet to be determined. We have also been working on the finding the citrus DNA sequence to replace CecropinB component. We have identified several candidates and are evalusting the similarity in their structure with respect to the known structure of Cecropin B. We have begun the process of designing the vector system to express the citrus PR14a sequence that we have identified. This vector system will be used for the plant based expression system described in our Activity 2. Once designed and constructed will be expressed in Nicotiana benthamiana to produce active protein that can be tested for activity. We have also submitted an application to APHIS-PPQ for the movement of the recently cultured Leberibacter crescens (Lcre). Once we get permission then later this year we can test these proteins for their efficacy using the Lcre to evaluate growth and mortality.
We completed predictive computations on all proteins from the Citrus sinensis genome and prepared all web-pages needed for manual, expert-driven analysis. This preliminary website is available from here:
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