Location: Crop Improvement and Genetics Research2016 Annual Report
1a. Objectives (from AD-416):
The overall goal of the project is to identify DNA elements that support effective strategies for stacking multiple traits within a single locus, removal of unwanted DNA sequences, and predictable expression of each transgene within that locus. These molecular tools will enable improved and precise engineering of complex, multi-gene traits in crop plants. Site-specific recombination systems and gene expression control elements with proven utility will be made available to researchers in the public and private sectors. Objective 1: Develop and deploy in crop plants site-specific recombinase-based systems for (1) targeted transgene integration and gene stacking, and (2) marker gene removal to prevent gene flow to non-genetically engineered crops. Subobjective 1a: Enhance site specific recombination systems for precise integration and excision in crop plant cells. Subobjective 1b: Use Dual RMCE to produce Foundation Lines that will allow transgene stacking via reiterative targeted integration and marker gene removal. Objective 2: Identify and demonstrate the utility of crop-derived gene expression control elements (promoters/enhancers/terminators/insulators) that facilitate trait development in crop plants. Subobjective 2a: Isolate and characterize novel promoters. Subobjective 2b: Isolate and characterize novel transcription terminators.
1b. Approach (from AD-416):
Random mutagenesis will be used to generate site-specific recombinase variants that will be screened for improved integrase and excisionase activities in a recombinase activity assay. Versions with improved catalytic activities in bacterial cells will be tested in plant cells. Mutated recombinases with improved activity will be codon optimized and tested in transgenic plants. In parallel, “target” transgenic plants will be generated by Agrobacterium–mediated transformation of Camelina. “Exchange” T-DNA vectors will be constructed to test four pairs of uni-directional recombinases, and designed so that an incoming gene is integrated at the target site and the selection marker gene is excised in a two-step sequential process. The “exchange” vectors will be transformed into the “target” Camelina transgenic plants. Negative selection will be used to screen for transformants in which the incoming DNA has replaced the original transgenic locus (Recombinase-Mediated Cassette Exchange or RMCE). The resultant transgene structure will be molecularly characterized to demonstrate that cassette exchange and selection marker gene removal have occurred. The efficiencies of different combinations of the unidirectional recombinases in performing RMCE will be compared. Candidate promoters with new cell-type/organ or stress-responsive specificities will be identified from crop plants using gene expression analyses. Emphasis will be on selecting candidates that have potentially useful expression patterns, but are not expressed in the grain. The candidate promoters will be fused to a reporter gene and transformed into rice, wheat, Brachypodium distachyon and/or other plants using Agrobacterium tumefaciens or biolistic transformation methods. Novel transcription terminator sequences will also be isolated from crop plants and fused to a reporter gene. The functionality of these promoter and terminator testing constructs will be examined in transient expression assays and stably transformed transgenic plants. Reporter gene expression levels will be quantitatively measured in major organs and compared to identify the sequences that provide the highest levels of transgene products while preserving promoter expression specificity. Additionally, a screen to identify “insulator” sequences that protect the expression of transgenes from undesirable interactions with nearby enhancers will be performed using a construct containing two copies of the highly active 35S enhancer. A library of crop genomic sequences will be tested for insulation activity using a transient expression assay. Selected candidate insulator sequences will also be tested in stably transformed transgenic plants. The functionality of the candidate insulator sequences will be validated if their insertion between the double 35S enhancer and a test promoter preserves the native specificity of the test promoter.
3. Progress Report:
Progress was made toward project objectives in Fiscal Year 2016. In research related to Subobjective 1a, a novel gene stacking system was developed that enables the sequential, modular assembly of plant gene expression constructs in Agrobacteria. Two ‘donor’ plasmid vectors that can carry cargo destined for transformation were designed, constructed and tested. In addition, a uniquely modified Agrobacterium strain which has the genetic elements necessary for the sequential recombinase-mediated integration of cargo into the transfer-DNA (T-DNA) of the native virulence plasmid was generated. The process of recombinase-mediated gene stacking is performed within the bacteria using sequential rounds of transformation and alternating between two different antibiotic selections. To demonstrate the functionality of the system, two genes were sequentially stacked into the Agrobacterium strain and the doubly transformed strain was successfully used to generate transgenic Arabidopsis and potato plants. Towards meeting Subobjective 1b, transgenic lines of tobacco and Arabidopsis containing target sites for Recombinase-Mediated Cassette Exchange (RMCE) were generated and re-transformed with exchange vectors containing various expression cassettes for site-specific recombinases. Site-specific integration has now been validated by polymerase chain reaction (PCR). Rates of integration range from 19 to 35%. Previously generated Camelina target lines have proven to be recalcitrant to re-transformation and this line of research is not being pursued further. Investigation is underway to determine whether the novel Agrobacteria gene stacking system (Subobjective 1a) can be merged with the plant RMCE technology to 1) provide greater amounts of recombinase on a transient basis for plant genome targeting and 2) enlarge the cargo capacity of the plant transformation vectors. Towards meeting Subobjective 2a, research investigating the function of four novel root-specific promoters was performed. These previously uncharacterized organ-specific promoters were introduced into rice and tobacco plants. Three of the promoters exhibited root-specific expression in the transgenic rice plants. These promoters each have unique cell-type specific expression within the root and one promoter also exhibits a similar root-specific expression pattern in transgenic tobacco. Towards meeting Subobjective 2c, screens to identify sequences that function as insulators of gene expression continued. Insulators shield genes from the effects of neighboring DNA sequences, allowing them to function autonomously in their chromosome environments. A library of size-fractionated rice sequences cloned into the insulator test vector was constructed. Experiments screening numerous independent clones were performed and several clones that exhibited insulator function were identified and validated in replicated assays. Candidate insulators with strong reproducible enhancer-blocking activity were further evaluated by DNA sequencing. Each of the candidate insulator sequences was mapped onto the rice genome to determine its location and its predicted function. Several candidate insulators have been chosen for further investigation. In a methodological advance, we are now able to use droplet digital PCR to count the number of transgenes present in genetically engineered plant species including rice, wheat, tomato, citrus, Arabidopsis and Brachypodium. This will enable us to more rapidly characterize transgenic plants, compared to previously used methods. In work funded by a Citrus Research Board (CRB) grant entitled “Utilization of founder lines for improved Citrus biotechnology via RMCE” (2030-21000-020-11T), research has continued toward the development of founder lines for future site-specific transgene integration in various citrus cultivars. Eleven different cultivars have been successfully transformed and confirmed by molecular techniques. These include ‘Carrizo’, ‘Hamlin’, Mexican lime, Cocktail grapefruit, ‘Limoneira 8A’ lemon, ‘Sidi Aissa’ Clementine orange, ‘Valencia’, ‘Troyer’, ‘Bitters’ and Blood orange. Currently these citrus lines are being screened for their capacity to undergo site-specific recombination allowing targeted integration using an approach similar to that employed for the Camelina, tobacco and Arabidopsis RMCE lines (Subobjective 1b). In U.S. Department of Energy funded research entitled, “Expanding the Breeder’s Toolbox” (2030-21000-020-12R), a strategy for reducing transgene flow to neighboring plants is being developed. Novel transformation constructs containing pollen-specific promoters have been transformed into switchgrass. Characterization of these transgenic switchgrass plants and an assessment of their ability to ablate their transgenic pollen are underway. In addition, research on a strategy to prevent all floral development in hybrid transgenic plants has also been pursued. Transgenic Brachypodium plants carrying complementary transgenes have been crossed and hybrid progeny identified. The next step is to evaluate how effectively reproductive development is altered in these hybrids.
5. Significant Activities that Support Special Target Populations:
De Oliveira, M.L.P, Moore, G., Thomson, J.G., Stover, E.W. 2015. Agrobacterium-mediated transformation of Mexican lime (Citrus aurantifolia Swingle) using optimized systems for epicotyls and cotelydons. Advances in Bioscience and Biotechnology. 6:657-668.
De Oliveira, M.L., Thomson, J.G., Stover, E.W. 2016. High-efficiency propagation of mature 'Washington Navel' orange and juvenile "Carrizo" citrange using axillary shoot proliferation. HortTechnology. 26:278-286.
Srivastava, V., Thomson, J.G. 2015. Gene stacking by recombinases. Plant Biotechnology Journal. 14(2):471-482. doi: 10.1111/pbi.12459.
Collier, R.A., Bragg, J., Hernandez, B., Vogel, J., Thilmony, R.L. 2016. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum. Frontiers in Plant Science. doi: 10.3389/fpls.2016.00716.