Progress 03/01/06 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Design and test molecular tools to better control transgene expression and integration. Identify, characterize, and demonstrate the utility of novel gene promoter elements for control of transgene expression in cereal crops. Emphasis is to be placed on promoters that provide developmental or environmental specificity to transgene expression, but are not active in the grains harvested for food or feed. Develop new recombination systems for plants that allow precise integration of DNA into targeted locations and selective removal of unwanted transgenic DNA from the genome. Make promoters and site-specific recombination systems with proven utility available to researchers in the public and private sectors. Approach (from AD-416) Use microarray and computer analyses from in-house and collaborative studies to identify rice, barley and wheat genes that exhibit organ- specific-, pathogen- or abiotic stress-responsive expression patterns. Isolate the corresponding promoters and examine and document their ability to control expression in transgenic cereal plants. Design and build transformation vectors incorporating site-specific recombination systems designed to target predetermined integration sites in cereal genomes and to allow excision of plasmid backbone and marker genes no longer needed after transformants are identified. Optimize codons and protein targeting signals as needed for better functionality in plant cells. Transform plants with recombinase-encoding constructs and target constructs. Demonstrate site-specific excision and/or integration reactions in plant cells. FY10 was the final year of a 5-year project aimed at designing, testing and making available 1) promoters to control transgene expression in cereal crops and 2) site-specific recombination systems for precise excision of DNA in plant cells. Among several novel rice promoters originally identified as exhibiting developmental specificity, four were chosen for detailed characterization in transgenic rice plants. This year marked completion of the analyses of one such promoter, which is active only in roots, primarily in the vascular tissues. In previous years, a rice promoter that is highly active in leaves and other green tissues, and is light-responsive was characterized. This promoter is also responsive to pathogens and environmental stresses. Further characterization of other rice promoters that are active only in the pollen of transgenic rice plants is in progress. None of these promoters is active in rice grains, and thus they would be ideal for genetic engineering of non-seed traits without introducing foreign proteins into the food supply. To test the organ specificity of these and other promoters in temperate cereals, DNA constructions containing promoters with widespread or organ-specific (seed endosperm or green-tissue or root) were transformed into wheat or the model grass Brachypodium distachyon. Examination of reporter gene expression in these transformed plants is ongoing. In parallel, research to develop site-specific recombination systems useful in plants has also progressed. Of seven recombination systems discovered previously to be active in yeast cells, four have been demonstrated in the current project to be functional in plants. Three of these - ParA, Bxb1 and phiC31 - have been fully characterized in transgenic Arabidopsis plants, where they precisely excise DNA between their target sequences from chromosomes. Furthermore, all three recombinases are active in reproductive tissues and can thus be used to excise unwanted DNA, such as transformation selection genes, from plant chromosomes. The resultant loci are faithfully transmitted to progeny plants. To investigate its utility for genetic engineering of cereals, DNA encoding the Bxb1 recombinase was transformed this year into wheat and Brachypodium. In parallel to the work in transgenic plants, a rapid plant cell assay has been developed that generates quantitative estimates of recombinase enzyme activity. This system allows direct comparison of the excision efficiencies of the different recombinases in plant cells. Comparisons include the well-characterized Cre recombinase and wild type versions of the newly characterized recombinases as well as versions of the latter that are modified for enhanced expression in plants. Also under development is a rapid integration activity assay that will allow testing of improvements to genomic targeting in crops. The biotechnology tools developed by the project allow more precise genetic engineering and have been transferred via Material Transfer Agreements to researchers in academia, government and private industry worldwide. Accomplishments 01 Identification of a root-specific rice promoter. Crop biotechnology has the potential to improve the productivity of U.S. agriculture, but tools are needed to precisely control gene expression in grass species like ri and wheat. Few well-characterized organ-specific promoters are available particularly for expression in the non-seed organs of cereal grain crops ARS scientists in Albany, CA, identified a root-specific promoter (named Root3) and characterized it in transgenic rice plants. The promoter reproducibly confers strong expression in the roots, particularly the vascular cells, but exhibits low or undetectable expression in aerial parts of the plant including the leaves, the reproductive tissues or the seeds. The Root3 promoter will enable precise, localized expression of transgenes in the roots of rice and other cereal grain crops and limit t potential for unintended impacts on the grain used for food and feed. 02 A rapid reliable assay for direct comparisons of recombinase efficiencie The adoption of genetically engineered crop plants has met with some consumer concerns about the presence of selectable marker genes (e.g. antibiotic resistance markers) in transgenic plants. To address these concerns and to provide new tools for biotechnology, ARS scientists in Albany, CA, developed a plant cell assay to compare efficiencies of site specific excision mediated by different recombinase enzymes. This assay requires only two days once the DNA constructions are in hand. It is bei used to evaluate modifications of recombinase enzymes for improved activity in crop species. These novel recombination systems will help alleviate public concerns by providing the biotechnology industry convenient options to remove unwanted DNA and precise genomic engineerin of transgenic plants prior to commercialization.
Impacts
(N/A)
Publications
- Thilmony, R.L., Guttman, M.E., Thomson, J.G., Blechl, A.E. 2009. The LP2 leucine-rich repeat receptor kinase promoter directs organ-specific, light- responsive expression in transgenic rice. Plant Biotechnology Journal. 7:867-882.
- Thomson, J.G., Chan, R., Thilmony, R.L., Ow, D.W. 2010. The phiC31 Recombinase Demonstrates Heritable Passage of Site-specific Genomic Excision in Arabidopsis. BioMed Central (BMC)Biotechnology. Available: doi:10.1186/1472-6750-10-17.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Design and test molecular tools to better control transgene expression and integration. Identify, characterize, and demonstrate the utility of novel gene promoter elements for control of transgene expression in cereal crops. Emphasis is to be placed on promoters that provide developmental or environmental specificity to transgene expression, but are not active in the grains harvested for food or feed. Develop new recombination systems for plants that allow precise integration of DNA into targeted locations and selective removal of unwanted transgenic DNA from the genome. Make promoters and site-specific recombination systems with proven utility available to researchers in the public and private sectors. Approach (from AD-416) Use microarray and computer analyses from in-house and collaborative studies to identify rice, barley and wheat genes that exhibit organ- specific-, pathogen- or abiotic stress-responsive expression patterns. Isolate the corresponding promoters and examine and document their ability to control expression in transgenic cereal plants. Design and build transformation vectors incorporating site-specific recombination systems designed to target predetermined integration sites in cereal genomes and to allow excision of plasmid backbone and marker genes no longer needed after transformants are identified. Optimize codons and protein targeting signals as needed for better functionality in plant cells. Transform plants with recombinase-encoding constructs and target constructs. Demonstrate site-specific excision and/or integration reactions in plant cells. (Replaces 5325-21000-008-00D, 2/06.) Significant Activities that Support Special Target Populations Research characterizing several novel gene promoters active only in specific plant organs continued. A rice promoter that is light-responsive in leaves and other green tissues was characterized in multiple rice transgenic plants. This promoter is also responsive to pathogen and environmental stresses. Further characterization of other rice promoters that are active only in the roots or the anthers/pollen of transgenic rice plants is in progress. Transgenic wheat plants containing the rice leaf-specific promoter and a wheat senesence-associated promoter were generated and are being characterized. Characterization of promoters expressed in green tissues of wheat has proved to be difficult because the promoters are silenced in the majority of transformants generated by particle bombardment. Resolution of this issue will be necessary for efficient future characterization of promoter function in wheat. Research utilizing the model grass Brachypodium distachyon as an alternative species for promoter analysis was pursued. Several organ-specific promoters with endosperm, green tissue or root specificities have been transformed into Brachypodium and examination of reporter gene expression is underway. Research has also continued to develop site-specific recombination systems useful in plants. The original strategy for detection of excision events in transient assays did not provide reliable quantitative estimates of recombinase enzyme activity. Several different modifications of the transient assay and the constructs have been made and are being tested so that efficiencies of the different recombinases can be directly compared to one another in plant cells. The functionality of three recombinases in transgenic plants has been demonstrated. ParA, Bxb1 and phiC31 recombinases were expressed in the model plant Arabidopsis, where they precisely excise DNA between their target sequences from plant chromosomes. The efficiency of excision of these recombinases in Arabidopsis approaches that of the control recombinase Cre. Transmission of the excision events in the absence of a co-transmitted recombinase gene has been confirmed. This indicates that these recombinases are active in reproductive tissues and can be used to excise unwanted DNA from plant chromosomes. Currently the Bxb1 recombinase is being investigated for activity in the monocots Brachypodium and wheat. Technology Transfer Number of New/Active MTAs(providing only): 8 Number of Invention Disclosures submitted: 1
Impacts
(N/A)
Publications
- Thomson, J.G., Yuan-Yeu, Y., Blanvillain, R., Chiniquy, D., Thilmony, R.L., Ow, D.W. 2009. ParA resolvase catalyzes site-specific excision of DNA from the Arabidopsis genome. Transgenic Research. 18:237-248.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Design and test molecular tools to better control transgene expression and integration. Identify, characterize, and demonstrate the utility of novel gene promoter elements for control of transgene expression in cereal crops. Emphasis is to be placed on promoters that provide developmental or environmental specificity to transgene expression, but are not active in the grains harvested for food or feed. Develop new recombination systems for plants that allow precise integration of DNA into targeted locations and selective removal of unwanted transgenic DNA from the genome. Make promoters and site-specific recombination systems with proven utility available to researchers in the public and private sectors. Approach (from AD-416) Use microarray and computer analyses from in-house and collaborative studies to identify rice, barley and wheat genes that exhibit organ- specific-, pathogen- or abiotic stress-responsive expression patterns. Isolate the corresponding promoters and examine and document their ability to control expression in transgenic cereal plants. Design and build transformation vectors incorporating site-specific recombination systems designed to target predetermined integration sites in cereal genomes and to allow excision of plasmid backbone and marker genes no longer needed after transformants are identified. Optimize codons and protein targeting signals as needed for better functionality in plant cells. Transform plants with recombinase-encoding constructs and target constructs. Demonstrate site-specific excision and/or integration reactions in plant cells. (Replaces 5325-21000-008-00D, 2/06.) Significant Activities that Support Special Target Populations FY08 is the second year of this research project. Research characterizing several novel gene promoters active only in specific plant organs has continued. The activity of a rice promoter that responds to light and is active in green tissues has been thoroughly tested in multiple rice transgenic plants. In progress is further characterization of promoters from rice that appear to be active only in roots or only in pollen. Several novel organ-specific candidate promoter sequences have been isolated from barley genomic DNA. Selection of wheat and barley candidate promoters that respond to pathogens and/or environmental stresses has been initiated. The expression patterns for several pathogen- and water stress-responsive genes are being evaluated to identify appropriate candidates for promoter isolation and characterization. Difficulties encountered in detecting the expression of the GUS reporter in some transformed wheat tissues has required the investigation of alternative approaches that may be used to document promoter function. Resolution of this issue will be necessary for efficient future characterization of promoter function in this species. Construction of the DNAs that allow expression of 6 new recombinases in plants has been completed as has construction of the test vectors for chromosomal integration and excision by these recombinases. The activity of the 6 novel recombinases was assayed and compared to that of previously characterized recombinases Cre and phiC31. The overall strategy allowed the successful detection of excision activity in plant cells, but it did not provide reliable quantitative estimates of recombinase enzyme activity. Attempts to modify the assay to allow for recombinase activity quantification have been unsuccessful. Based on these results, an alternative strategy to examine recombinase function in transgenic plants is being utilized. Using this strategy, ParA and Bxb1 recombinases were expressed in the model plant Arabidopsis, where they were shown to precisely excise DNA between their target sequences from plant chromosomes. The activity of ParA and Bxb1 enzymes in Arabidopsis is comparable to that observed for the control recombinases Cre and phiC31. Transmission of the recombinase- mediated excision chromosomes to progeny plants in the absence of the recombinase enzyme parA has been confirmed. The data indicate that the ParA is active in the reproductive tissue and can be used to excise unwanted DNA from plant chromosomes. This research addresses National Program 302 Component 3, Plant Biotechnology Risk Assessment and Problem Statement 3A, Improving and Assessing Genetic Engineering Technology. Technology Transfer Number of New/Active MTAs(providing only): 4 Number of Web Sites managed: 1 Number of Other Technology Transfer: 2
Impacts
(N/A)
Publications
- Thilmony, R.L., Thomson, J.G. 2008. A simple and inexpensive method for sending binary vector plasmid dna by mail. Plant Biotechnology. 25:403- 406.
- Ow, D.W. 2008. Chance Events Shape a Career. In: C. Neal Stewart, Jr., Plant Biotechnology and Genetics: Principles, Techniques and Applications. Adobe E-Book, "lifebox." Wiley and Sons, New York, pp. 238-240.
- Stewart, C., Ow, D.W. 2008. The Future of Plant Biotechnology. In: C. Neal Stewart, Jr., Plant Biotechnology and Genetics: Principles, Techniques and Applications. Adobe E-Book. Wiley and Sons, New York, pp. 357-369.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Design and test molecular tools to better control transgene expression and integration. Identify, characterize, and demonstrate the utility of novel gene promoter elements for control of transgene expression in cereal crops. Emphasis is to be placed on promoters that provide developmental or environmental specificity to transgene expression, but are not active in the grains harvested for food or feed. Develop new recombination systems for plants that allow precise integration of DNA into targeted locations and selective removal of unwanted transgenic DNA from the genome. Make promoters and site-specific recombination systems with proven utility available to researchers in the public and private sectors. Approach (from AD-416) Use microarray and computer analyses from in-house and collaborative studies to identify rice, barley and wheat genes that exhibit organ- specific-, pathogen- or abiotic stress-responsive expression patterns. Isolate the corresponding promoters and examine and document their ability to control expression in transgenic cereal plants. Design and build transformation vectors incorporating site-specific recombination systems designed to target predetermined integration sites in cereal genomes and to allow excision of plasmid backbone and marker genes no longer needed after transformants are identified. Optimize codons and protein targeting signals as needed for better functionality in plant cells. Transform plants with recombinase-encoding constructs and target constructs. Demonstrate site-specific excision and/or integration reactions in plant cells. (Replaces 5325-21000-008-00D, 2/06.) Significant Activities that Support Special Target Populations In this, the first year of the project, reporter gene expression for three new rice promoters was characterized in rice, substantially meeting the objectives of Milestone 1 of the project plan. Expression of the GUS reporter in some transformed wheat tissues is inhibited by a cellular factor and this slowed characterization of rice and wheat promoters in wheat. Test vectors for demonstrating the functionality of new recombinase systems in plants were constructed, substantially meeting the objectives of Milestone 3. These included plasmids to detect excision from and integration into plant chromosomes and for expression of six novel recombinases in plant cells. Recombinase-mediated excision has been demonstrated in cells of the plant Arabidopsis for three of these systems. Work is in progress to improve efficiencies. Accomplishments Site-Specific Excision of DNA The adoption of genetically engineered crop plants has been limited by consumer concerns about the presence of marker genes in transgenic plants. To address these concerns and to provide new tools for biotechnology, scientists in Albany, CA have tested a number of prokaryotic recombinase systems for their ability to excise marker genes from plant chromosomes. They showed that the parA and Bxb1 recombinases can excise marker genes from the Arabidopsis genome. These systems will facilitate precise genome engineering of plants, thereby helping both the biotechnology industry to achieve more predictable results and alleviating one of the public concerns over genetically engineered crops. Technology resulting from this project has already been distributed to seven different national and international research groups. This research addresses National Program 302 Component 3, �Plant Biotechnology Risk Assessment� and Problem Statement 3A, �Improving and Assessing Genetic Engineering Technology�. New Rice Promoters with Organ-specific Expression New well-characterized promoters are needed that confine transgene product accumulation to just the tissues and developmental stages in which it is needed. In particular, promoters are needed for strong expression in the non-seed organs of cereal plants. Scientists in Albany, CA, discovered two new rice promoters, one of which can be used to express genes only in leaves and other green tissues, and the other of which can be used for root-specific expression. The novel promoters have specificity in rice plants that will potentially be useful for changing traits in the leaves or roots of genetically engineered cereals without affecting the composition of the grain used for food and feed. This research addresses National Program 302 Component 3, �Plant Biotechnology Risk Assessment� and Problem Statement 3A, �Improving and Assessing Genetic Engineering Technology�. Technology Transfer Number of New CRADAS and MTAS: 4 Number of Non-Peer Reviewed Presentations and Proceedings: 2 Number of Newspaper Articles,Presentations for NonScience Audiences: 1
Impacts
(N/A)
Publications
- Thomson, J.G., Ow, D.W. 2006. Site-Specific Recombination Systems for the Genetic Manipulation of Eukaryotic Genomes. Genesis 44(10):465-476.
- Ow, D.W. 2007. GM Maize from Site-Specific Recombination Technology, What Next? Current Opinion in Biotechnology 18(2):115-20.
- Ow, D.W. 2007. Site-Specific Recombination for Plant Genetic Engineering: Strategy for Agro-Mediated Gene Stacking. In: Litz, R.E., Scorza, R., eds. ISHS Acta Horticulturae 738, International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species. The Netherlands, Drukkerij Jansen BV, pp. 117-127.
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Progress 10/01/05 to 09/30/06
Outputs
Progress 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? Why does it matter? This CRIS project is part of National Program 302, Plant Biological and Molecular Processes. It 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 or to the environmental situations in which 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. 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 decrease transgene expression in plant
tissues destined for human consumption, thereby increasing public acceptance of transgenic crops for commercial release. 2. List by year the currently approved milestones (indicators of research progress) Year 0 (FY 06): None, first four months of project. Year 1 (FY07): Milestone 1 - Characterize and document reporter gene expression in transgenic rice containing candidate native organ-specific promoters. Milestone 2 - Initiate research to identify several candidate crop promoters that are responsive to biotic and/or abiotic stress. Characterize and validate the stress responsive expression of selected candidates using northern blots or quantitative RT-PCR. Milestone 3 - Complete recombinase test constructs for excision in planta (pTC1), recombinase test constructs for integration in planta (pTC2, pTC3), recombinase test constructs for chromosomal integration in planta (pTC4) and the recombinase expression vectors (pRE-X) for the 6 recently discovered recombinase genes plus Cre and
phiC31 as controls. Year 2 (FY08): Milestone 4 - Retrieve and sequence 5' flanking regions of validated stress responsive genes to use as putative promoters. Fuse candidate promoter regions to GFP and GUS reporter genes in Agrobacterium vectors and initiate plant transformation. Milestone 5 Complete the transient excision assay using pTC1 in dicot and monocot protoplasts. Repeat with new pRE-X constructs if needed. Complete the transient integration assay using pTC2 and pTC3 in dicot and monocot protoplasts. Repeat with new pRE-X constructs if needed. Milestone 6 - Production of the transgenic plants for the genomic excision assay using pTC1, and the genomic integration assays using pTC2 and pTC3 as the chromosomal targets. Year 3 (FY09): Milestone 7 - Create transgenic wheat and/or barley plants containing candidate organ-specific promoters. Milesone 8 Complete the the genomic excision assay using pTC1, and the genomic integration assays using pTC2 and pTC3 as the chromosomal
targets. Repeat with new pRE-X constructs if needed. Milestone 9 - Complete the crosses for the genomic excision assay. Year 4 (FY10): Milestone 10 - Create transgenic wheat, barley and/or Brachypodium distachion plants containing pathogen-induced or abiotic stress- responsive promoters. Milesone 11 Complete progeny analyses for genomic excision assay. Repeat with new pRE-X constructs if needed. Milestone 12 Conduct recombinase mediated genomic integration with best pRE-X constructs. Year 5 (FY11): Milesone 13 - Characterize and document reporter gene expression in transgenic plants containing heterologous candidate organ-specific and stress responsive promoters. Milestone 14 - Analysis of pRE-X recombinase mediated genomic integration events. Additional recombinase optimization experiments if needed. 4a List the single most significant research accomplishment during FY 2006. None, first four months of the new project. Please see report for in- house project 5325-21000-008-00D. 4d
Progress report. The OSQR project plan for the new 5-year plan was written and approved. The research for this project plan began in February, 2006. 5. Describe the major accomplishments to date and their predicted or actual impact. None. This report covers the first few months of this project. For previous project, see report for CRIS # 5325-21000-008-00D. Research in this project will contribute to Component 3 of NP 302 Plant Biotechnology Risk Assessment, specifically Problem Statement 3A, Improving and Assessing Genetic Engineering Technology. 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 by academic and biotechnology company scientists. 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 line 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). None yet for this project; see annual report for Project 5325-21000-008- 00D.
Impacts
(N/A)
Publications
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