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:
The site has been prepared and the contract has been signed to initiate construction.
The Core Citrus Transformation Facility (CCTF) continues to serve the community of researchers exploring ways to improve Citrus plants and make them tolerant/resistant to diseases. CCTF does its service by producing transgenic material. Within the last quarter, the CCTF facility worked on producing transgenic plants of the following combinations: produced the following transgenic citrus plants (transgene in parenthesis): Mexican lime (pHK vector); Duncan grapefruit (ELP3 gene); Duncan (MKK7 gene); Duncan plants (p7 gene); Duncan (p10 gene); Mexican lime and Hamlin (p33 gene); Duncan (SUC-CitNPR1 gene); Duncan (pWG19-5 vector); Duncan plants (pWG20-7 vector); Duncan (pWG21-1 vector); Duncan (pWG22-1 vector); Duncan (pWG24-13 vector); and Duncan (pWG25-13 vector). All of these plants are for researchers funded by CRDF in the battle against HLB and canker.
McTeer trial – (3.5-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): Slow release fertilizer was applied to all trees in January. Unlike sweet oranges, HLB-impacted SugarBelle fruit does not drop from the tree, and the impacted crop is still hanging on the trees, months after the typical harvest date. Rootstock effects on longer-term tree health are being monitored, trees on Orange #19 still look the best at present. St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.0 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). CREC scouts assessed HLB again in March. HLB is spreading much more quickly in the zone with the traditional tree spacing (15×25); whereas it was very low in the highest density spacing (9×20). Big differences continued in HLB infection rates per rootstock, with the control commercial rootstocks continuing to show the highest infection rates, with most >80% infected. Sour orange-like hybrids (now 3.5 years old) are growing off very well with lower than expected HLB infection frequencies (mostly Pummelo x Shekwasha mandarin hybrids). Harrell’s UF mix was applied to all trees in January, and TigerSul was purchased for application in the next quarter (to avoid minor zinc deficiency observed last summer). Yield and fruit quality data collection was completed in March; statistical analysis underway. Greenhouse Experiments – Rootstock liners (test rootstocks and controls) were moved into the HLB-greenhouse and grafting with HLB-infected Valencia budsticks was completed for the rootstock comparison experiment (trees growing on slow release fertilizer). Liners of rootstock Orange #15 for the individual nutrient experiment were moved to the HLB-greenhouse – grafting will begin this quarter. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. 1. Agrobacterium mediated transformation to produce transgenic tetraploid Orange 16 plants containing the Snowdrop Lectin insecticidal gene (GNA) have been conducted. In addition, we have produced several Orange 16 plants containing the GNA gene (snowdrop lectin) fused with a Tobacco PR1b signal peptide for improved extracellular secretion of the GNA protein by plant cells and the GNA gene fused with a HDEL C-terminal extension for retention of the GNA protein in the endoplasmic reticulum. 2. Transgenic Carrizo containing a construct containing the Snowdrop Lectin insecticidal gene stacked with the antimicrobial gene CEMA (AMP) have been produced and are being propagated for analysis.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Several of the transgenic plants have reached the top of the scaffold, and the apical stems have been trained to grow down (expected to encourage early flowering). The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Another rootstock with strong potential to influence juvenility was identified (Nova+HBP x sour orange + Flying Dragon). Seeds have been planted. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. Transgenic sweet orange plants containing a construct with CEME gene (AMP) stacked with the NPR1 (SAR inducer) gene have been evaluated. We have recovered 10 transgenic lines that contain both genes incorporated into the genome. We have also transformed our newly released sweet orange somaclone OLL#8 with this construct. Also, constructs containing the AttacinE gene stacked with the NPR1 gene and the CEMA gene stacked with the NPR1 gene have been produced, and transformation of OLL#8 and Valencia Sweet Orange is currently underway. Correlation of transgene expression with disease resistance response: Western blot analysis for plants containing LIMA and GNA are nearly completed, data is showing a strong correlation between transgene expression and desired phenotype. This supports the dogma that fairly large populations of transgenic plants are necessary (for each transgene/cultivar) to obtain adequate transgene expression while maintaining cultivar integrity. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free or ‘all plant’ consumer-friendly transformation): 1. In efforts to reduce transgene mediated metabolic load on the plant, we have transformed Hamlin suspension cultures with constructs containing our reporter gene (grape anthocyanin gene) driven by either an embryo-specific Carrot DC3 promoter or an embryo-specific Arabidopsis At2S2 promoter. It is expected that plants obtained from these constructs will not produce the reporter protein once a transgenic plant has been selected. Currently putatively transgenic embryos have germinated and are being grown to size for analysis. 2. The binary vector for an inducible cre-lox based marker free selection is under construction. We anticipate transformation experiments with this vector in the following quarter. Targeted transgene expression: ‘ additional transgenic plants of Duncan, Carrizo, Pineapple, Hamlin, and Valencia (produced with Agrobacterium-mediated transformation) containing the LIMA gene (AMP) controlled by AtSUS2 promoter (phloem specific) have been propagated by micrografting. Plant characterization and molecular analysis on these plants will begin the next quarter. In greenhouse evaluation (Southern Gardens w/ Mike Irey) of transgenic plants exposed to HLB positive psyllids, we observed several transgenic LIMA and NPR1 lines driven by a phloem specific AtSUC2 phloem-limited promoter to be HLB tolerant. Most of these lines were negative to qPCR after 2 years of evaluation and did not demonstrate visible disease symptoms.
Several anti-NodT scFv single-chain antibodies are now in hand. The anti-NodT antibody with the highest affinity for the target 30 amino-acid peptide has been selected and has been expressed successfully in E. coli. The plant gene expression construct consisting of the 35S Cauliflower Mosaic Virus promoter driving expression of a fusion of the flowering locus T protein with best-performing anti-NodT scFv protein has been produced. We are now in the process of transferring this 35S::FLT-scFv expression cassette into the pTLAB citrus transformation construct. We have been working on this construct for several months during January – April 2013, but still have not completed this step, which has proven to be unexpectedly difficult. We anticipate that we will be able to complete this step during the next quarter, using some alternate techniques. Once the construct is completed, production of transgenic plants will begin.
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