Location: Crop Improvement and Genetics Research
2018 Annual Report
Objectives
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.
Approach
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.
Progress Report
This is the final report for this project which was replaced on March 26, 2018 by 2030-21220-002-00D, “Molecular Genetic Tools Advancing the Application of Biotechnology for Crop Improvement.” For additional information, see the report for the new project.
Objective 1: A novel gene stacking system was developed. The Gene Assembly in Agrobacterium by Nucleic Acid Transfer using Recombinase technologY (GAANTRY) system enables the sequential and modular assembly of plant transformation constructs. The efficient process of recombinase-mediated gene stacking is performed within bacteria following sequential rounds of bacterial transformation and antibiotic selection. The recombinase-mediated cassette exchange (RMCE) is mediated by the TP901 and A118 recombinases, which sequentially perform targeted integration and the ParA recombinase which performs irreversible excision. The RMCE reaction integrates cargo into the transfer DNA (T-DNA) while simultaneously swapping one bacterial antibiotic resistance marker for another. This strategy allows the efficient sequential stacking of cargo sequences into a T-DNA, and thus can generate complicated stacks of transgene expression cassettes. To demonstrate the functionality of the system, ten genes have been sequentially stacked into a novel Agrobacterium strain and this strain has been successfully used to generate transgenic Arabidopsis and potato plants. The technology has been shown to generate high quality transgenic plants that express all the introduced traits and have a single copy of the introduced sequences at a high frequency.
Novel and innovative strategies for using recombinases for plant genome engineering were also devised. A functional Bxb1 recombinase was shown to be inherited in both transgenic wheat and Arabidopsis, where it precisely excised DNA flanked by its target sequences. The CinH recombinase has also been shown to confer unidirectional marker gene excision from the tobacco genome. To investigate the utility of the recombinases in other crop plants, constructs encoding the Bxb1, PhiC31, CinH and ParA recombinases have been transformed into soybean, citrus, and the biofuel crop, Camelina sativa, to study rates of activity. Bxb1 was shown to perform site-specific integration in tobacco. In switchgrass, the Bxb1 recombinase catalyzed the precise deletion of DNA situated between its two recognition sites. The Bxb1 recombinase can also precisely remove DNA from the tobacco plastid genome. Because the Bxb1 enzyme does not add or delete nucleotides outside its recognition domain, the integrity of the surrounding chloroplast genes is maintained. These results demonstrate that Bxb1 is a unique molecular tool that can be used to remove unwanted antibiotic or herbicide resistance genes after genetic engineering of chloroplast DNA, thus allowing reuse of these selection markers for further rounds of transformation or commercial release of GE plants lacking an antibiotic resistance gene. These experiments demonstrate the utility of the Bxb1 recombinase as a new tool for several aspects of plant genomic engineering including excision of marker genes, resolution of complex insertion structures, integration of targeted DNA, and transgene stacking.
In addition, transgenic lines of tobacco, citrus and Arabidopsis containing target sites for RMCE lines were generated and re-transformed with exchange vectors containing various expression cassettes for site-specific recombination. The system has been modified to improve the amount of ‘cargo’ DNA that can be carried. The system was further modified to increase the amount of effective recombinase enzyme being expressed. Rates of integration now range from 19 to 78 percent in the tobacco lines tested. Site-specific integration has been validated by molecular analyses.
Objective 2: The characterization of the activities of several tissue-specific promoters (conferring leaf-, pollen- and seed-specific expression specificities) in transgenic plants was completed. A patent (12/890,974) for the rice Leaf Panicle2 promoter that confers light-inducible gene expression in green tissues was granted. The wheat Dy10 promoter controls seed endosperm specific expression in wheat and Brachypodium. The rice OsGEX2 promoter confers pollen sperm cell specific expression in rice and the rice Pollen Specific 2 and 3 promoters confer pollen specific expression in rice and switchgrass. Research investigating the function of four novel root-specific promoters was also completed. These promoters confer expression within specific cell types of the roots of transgenic rice and tobacco plants. In addition, two candidate promoters that confer expression in vegetative tissues, but not floral reproductive tissues, were isolated and tested. The characterization of three wheat promoters from genes that exhibit low basal levels of expression in healthy plants but are induced by environmental or disease stresses were tested in transgenic wheat and Brachypodium plants.
In addition, several candidate rice sequences that function as insulators to shield genes from the effects of neighboring DNA sequences in a rapid screening assay were selected for further characterization. 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. These insulator sequences were moved into a plant transformation construct and introduced into tobacco plants. One of the candidate insulators exhibited enhancer-blocking activity in the transgenic tobacco and is available for further characterization and potential use in insulating transgene expression in crop plants.
In a methodological advance, novel use of the droplet digital PCR technique was developed to quantify the number of transgenes present in a diverse array of genetically engineered plants. This technique enables the more efficient characterization of transgenic rice, wheat, tomato, citrus, maize, Arabidopsis, switchgrass and Brachypodium 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”, 58-5325-4-0010, which began in 2013, 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 tobacco and Arabidopsis RMCE lines (Objective 1).
In U.S. Department of Energy funded research entitled, “Expanding the Breeder’s Toolbox for Perennial Grasses, 60-5325-2-0878, active from 2012 through 2017, a strategy for reducing transgene flow to neighboring plants was developed. Plant transformation vectors utilizing several of the pollen-specific promoters previously developed by the project were shown to effectively ablate transgenic pollen in switchgrass and Brachypodium sylvaticum, a model perennial grass. The effectiveness of the system to alter transgene heritability in switchgrass hybrids is currently being evaluated.
Accomplishments
1. Development of an effective system for gene stacking in crop improvement. The genetic improvement of crops is one of the most effective ways to increase their productivity in agriculture. Until now, it has been difficult to genetically engineer improvements in complex traits like yield or disease resistance that require the action of multiple genes. An ARS scientist in Albany, California, developed a novel technology called Gene Assembly in Agrobacterium by Nucleic Acid Transfer using Recombinase technology (GAANTRY), that allows the efficient assembly and introduction of multiple genes into plants. The system can be used to join multiple genes together in a simple, reliable and highly effective process, and then be used to generate transformed plants that frequently produce all the introduced traits as desired. This technological breakthrough enables the use of crop biotechnology to effectively improve complex traits in a wide array of crop plants.
Review Publications
Sade, N., del Mar Rubio-Wilhelmi, M., Ke, X., Brotman, Y., Wright, M., Khan, I., De Souza, W., Bassil, E., Tobias, C.M., Thilmony, R.L., Vogel, J.P., Blumwald, E. 2018. Salt tolerance of two perennial grass Brachypodium sylvaticum accessions. Plant Molecular Biology. 96(3):305-314. https://doi.org/10.1007/s11103-017-0696-3.
Scott, R.A., Thilmony, R.L., Harden, L.A., Zhou, Y., Brandl, M. 2017. Escherichia coli O157:H7 converts plant-derived choline to glycine betaine for osmoprotection during pre- and post-harvest colonization of injured lettuce leaves. Frontiers in Microbiology. 8:2436. https://doi.org/10.3389/fmicb.2017.02436.
Wang, Y., Xu, L., Thilmony, R.L., You, F., Gu, Y.Q., Coleman-Derr, D.A. 2016. PIECE 2.0: an update for the plant gene structure comparison and evolution database. Nucleic Acids Research. 45(D1):1015-1020. https://doi.org/10.1093/nar/gkw935.
