2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This CRIS project has the goal of minimizing unintended and/or potentially negative impacts of plant transformation processes and of the introduction of new genes into the genome. Specifically we seek to understand and reduce or eliminate the effects of transgene insertion and expression on non-targeted plant processes, on the environment, and on the human food supply. Tools to be developed include those that. 1)confine expression of the newly introduced genes to the tissues and developmental stages where and when they are needed;. 2)minimize the disruption of the plant genome by insertion of new sequences;. 3)eliminate extraneous foreign gene sequences not needed to make the desired change; and. 4)allow multiple transgenes to be integrated into the same site in the genome. The approach is a combination of molecular biology and genetic transformation. The characteristics of the resultant plants will be evaluated at the molecular and functional levels. The purpose of the research is to address the perceived lack of safety and predictability that currently limits the usefulness and public acceptance of biotechnology for the improvement of crops. One concern about the commercial release of transgenic crops is the possibility that they will have adverse impacts on the environment and on the food supply. A second concern is that introduction of new genes could have effects on plant metabolism other than those intended. This research will create tools that refine transformation technology and minimize these possibilities. New technologies will be developed to improve the predictability and control of the expression and location of transgenes in the host genome. An expected outcome is the ability to block or increase transgene expression in plant tissues destined for human consumption, thereby increasing public acceptance of transgenic crops for commercial release. 2.List the milestones (indicators of progress) from your Project Plan. FY04: Milestone 1 - Analyze gene expression patterns at the organ level using the rice cDNA microarray. Mine the wheat/barley EST databases for non-seed-expressed promoter candidates. Select 20 candidate promoters from rice, wheat and barley for further characterization. Milestone 2 - Screen recombination systems for ability to function in a eukaryotic environment using an excision based vector test system. Test site-specific recombination in rice using the phiC31 site-specific recombination system. FY05: Milestone 3 - Characterize the expression of the candidate ESTs using northern blots or RT-PCR. For promising candidates, retrieve and sequence 5' flanking regions to use as putative promoters. Milestone 4 - Construct three Agrobacterium transformation vectors, one for hygromycin selection, and two for promoter analyses using GFP and GUS reporters. Fuse candidate promoter regions to the reporter genes in the transformation vectors and initiate their transformation into rice. Milestone 5 - Characterize promising recombination systems. Construct vector test systems to test their ability to facilitate inversion and/or integration events in eukaryotic cells. Optimize integration reactions as needed by the addition of nuclear localization signals or synthetic introns. Screen for functionality in plant cells FY06: Milestone 6 - Document the expression of the first candidates in transgenic rice plants. Identify 10-20 more candidates with novel expression patterns and pursue their characterization. Prepare publications and patent applications. Milestone 7 - Optimize recombination reactions for use in plants. Initiate the integration of a second DNA molecule, the first step in testing gene stacking. 4a.What was the single most significant accomplishment this past year? New site-specific recombination systems that function in plant cells Precise control of the integration of transgenes into plant genomes and the stacking of independent transgenes in a single location require site-specific recombination systems that work accurately and efficiently in plant cells. In FY05, scientists in Albany, CA, screened prokaryote-derived recombination systems for their functionality in Arabidopsis, tobacco and rice. They showed that selected recombination systems mediated recombination in plant cells. These experiments provide reduction to practice for patent claims of new site-specific recombination systems. A non-provisional patent application is being filed with the intention of making these novel molecular tools available in the public domain for use in basic and applied research. 4b.List other significant accomplishments, if any. Promoters with useful expression patterns New well-characterized promoters are needed that confine transgene product accumulation to just the tissues and developmental stages in which it is needed. In FY05, scientists in Albany, CA, used RNA blot analysis to validate potentially useful organ-specific expression patterns for six rice and five barley candidate promoters. The rice candidates under investigation include genes that are putatively organ-specific (e.g. roots, leaves or anthers) and some that are highly expressed in several plant organs, but are undetectable in developing seed tissues ("seed excluded"). Promoter regions for the rice candidates were isolated, sequenced and linked to reporter genes in a plant transformation vector. Thus far, transgenic rice plants containing one of the constructs have been regenerated and construction of a novel Agrobacterium plant transformation vector well suited for promoter testing in monocot crops is also underway for future promoter testing. The promoters identified in this research will be valuable tools for basic and applied biotechnology research. Adaptation of prokaryotic site specific recombination systems for eukaryotic functionality To facilitate their efficient use in higher organisms, site specific recombination systems derived from prokaryotes must be modified for improved functionality in plant and animal cells. In FY2005, the scientists modified each recombinase with addition of nuclear localization and intron sequences and these versions will be tested for improved efficiency in plant cells. Plant vectors containing these versions have been completed and their transformation into rice, tobacco and Arabidopsis is underway. Once recombination efficiency improvements are validated and incorporated into these vectors, they will be made available to biotechnology researchers for use in marker excision, gene stacking and other refinements in transformation technology. 4c.List any significant activities that support special target populations. None 4d.Progress report. The prospectus for the new 5-year plan was written and approved. In work funded through a Specific Cooperative Agreement with the University of California - Davis, transient and stable assays for site-specific recombination reactions have been established in rice. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. FY05 was the fourth year of this project and the third year at its current funding level. The first year was devoted to staffing, securing laboratory and greenhouse space and purchasing equipment. In the second year, screening of several prokaryotic recombination systems led to identification of a few that can mediate deletions in yeast. With collaborators in Andras Nagy's laboratory at the University of Toronto, the phiC31 system was shown to be capable of mediating precise integration of DNA into the genomes of mouse stem cells, with germline transmission of the introduced transgene. This exemplifies that phiC31 site-specific integration could eventually be used in the precise engineering of farm animals, and suggested that similar achievements could be obtained in plants. We also reported that, in rare instances, certain transgenes expressing the recombinase Cre generate de novo excision of lox-flanked target sequences in each generation. Thus, it is possible to obtain plants where the deleted DNA is not lost, but rather persists in the plant genome as an extra-chromosomal element. In FY04, additional promising new site-specific recombination systems were added to the initial few, making a total of 6 systems to be included in a non- provisional patent filing to be completed in August 2005. Ten rice and ten barley genes that potentially have vegetative-organ-specific or seed-excluded expression patterns were identified. In FY05, two of the new prokaryotic site-specific recombination systems were shown to be functional in plant cells. Novel promoter elements were isolated from rice and fused to reporter genes for rice transformation. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We expect to patent all new discoveries from this project for use in the public domain. Thus far, a patent "DNA recombination in eucaryotic cells by the bacteriophage phiC31 recombination system" has been issued. A non-provisional patent application for the new site-specific recombination systems is near completion. Constraints: Even with these improvements, some members of the food industry and some consumers may continue to oppose genetically engineered crops. As the development time for GM crop plants takes a decade, new technology developed today will take some time before it will be incorporated into commercial products. For example, the marker removal technology was published by ARS in 1991, but it took 15 years for Monsanto to put out a first product using Cre-lox to remove the kanamycin resistance marker from a high lysine corn lines scheduled for the commercial market in 2006. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Invited talks by David Ow: 10/27/04,.at PIPRA (public intellectual property resource for agriculture) workshop on “Optimizing Plant Transformation”, Danforth Center, St. Louis, Oct. 27-28, 2004. “pPIPRA Project - Development of Plant Transformation Vectors with Maximum FTO” 01/20/05, at PIPRA 2005 Annual Membership Meeting, Berkeley, CA, Jan. 20-21, 2005. “Recombinase-mediated plant transformation system”, 03/09/05, at NSF Maize Transformation Workshop, Madison, WI, Mar. 7-10, 2005. “Recombinase-mediated plant transformation”, 03/12/05, at 47th Annual Maize Genetics Conference, Lake Geneva, WI, Mar. 10-13, 2005. “Recombinase-mediated plant transformation”, 04/29/05, at Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, Ottawa, Canada. Invited talk by Roger Thilmony: “Biotechnology risk mitigation research - Creating novel tools for precise genetic engineering of crop plants”, 6/20/2005 at the USDA-AHPIS-Biotechnology Regulatory Services, Riverdale, MD. Poster Abstracts: Thilmony, R., Guttman, M. E. and Blechl, A. E. Promoter Mining For Biotechnology Risk Mitigation In Cereal Grain Crops. At Plant and Animal Genome Conference XIII, January 15 - 19, 2005, in San Diego, CA. http://www.intl-pag.org/13/abstracts/PAG13_P654.html Thomson, J., Ow, D. New recombinases for the genetic manipulation of eukaryotic genomes. At Plant & Animal Genomes XIII Conference, San Diego, CA, Jan. 15-19, 2005. http://www.intl-pag.org/13/abstracts/PAG13_P098.html Review Publications Ow, D.W. 2004. Gene stacking through site-specific integration. In: G.H. Liang, D.Z. Skinner, editors. Genetically Modified Crops, Their Development, Uses and Risks. Haworth Press, Inc. pp. 71-100. Srivastava, V., Ow, D.W. 2004. Marker-free site-specific gene integration in plants. Trends in Biotechnology, Trends in Biotechnology, 22(12):627-629. Ow, D.W. 2005. Transgene management via multiple site-specific recombination systems. In Vitro Cellular and Developmental Biology - Plants 41(3):213-219(7).