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2006 Progress Report: Engineered Plant Virus Proteins for Biotechnology
EPA Grant Number: R829479C030Subproject: this is subproject number 030 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Engineered Plant Virus Proteins for Biotechnology
Investigators: Scholthof, Herman B , Scholthof, Karen-Beth G
Institution: Texas A & M University,Texas Agricultural Experiment Station
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 2004 through September 30, 2007 (Extended to December 31, 2007)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001)
Research Category: Hazardous Waste/Remediation , Targeted Research
Description:
Objective:The overall aim of the project is to determine conditions that improve the performance and stability of virus vectors for optimum expression of foreign proteins in plants. The objectives are:
- Test the protective effect of the coat protein of satellite panicum mosaic virus on the stability of plant virus-derived gene expression vectors.
- Test the effect of RNA silencing suppressors on the performance of plant virus-derived gene expression vectors.
- Determine the effect of host species background on the performance and stability of plant virus-derived gene expression vectors.
Although the project was not initiated until the beginning of April 2006, substantial progress has been made towards the stated objectives. Accomplishments will be listed per objective:
Objective 1
To start addressing this objective, a p otato virus X (PVX) gene vector was constructed to express the coat protein of satellite panicum mosaic virus (Scp). To test the stated hypothesis that Scp has a stabilizing effect on virus vector integrity, a passage experiment was conducted. Nicotiana benthamiana plants were inoculated with infectious RNA transcripts of the PVX-Scp vector. After symptoms were established, infected leaves of the transcript-inoculated plants were pulverized in virus inoculum buffer and used as inoculum for second -passage plants, and so on, until passage five. As a control, a PVX vector was designed that carries the same Scp nucleotide sequence but in the opposite orientation, and thus not conducive to translation of Scp.
Leaves of each passage plant were collected and analyzed for expression of Scp with immunoblotting techniques (i.e., western blots) using our Scp-specific rabbit antiserum. Preliminary results show detectable levels of Scp until passage three. Experiments are currently being repeated with shorter time periods between inoculation and harvest for subsequent passage. In addition, primers have been designed for reverse transcriptase (RT) PCR to amplify the Scp sequence from the vectors after each passage. This will allow a comparison between Scp expressing vectors and the control (with the inverted Scp sequence).
Objective 2
We have generated, or collected from colleagues, Agrobacterium tumefaciens binary T-DNA vectors that, when introduced into plant leaves, express different plant virus suppressors of gene silencing. In the first set of experiments, we conducted trials with a construct expressing the protein P19 (and a defined mutant) of t omato bushy stunt virus (TBSV). P19 is an example of a well-characterized suppressor of gene silencing. Four days after agroinfiltration, leaves were collected and tested for P19 accumulation with western blots using our recently raised P19-specific rabbit antiserum. Results showed that P19 could be readily detected. Experiments will now be conducted to determine the effect of P19 expression (and that of other agroinfiltrated suppressors) on the integrity of virus vectors using the new quantification technique developed in our laboratory under Objective 3.
In a related set of experiments we aimed to develop a quick assay for the effect of P19 mutants on foreign gene expression prior to their subcloning in Agrobacterium vectors. Towards this purpose we agroinfiltrated N. benthamiana with Agrobacterium, expressing the foreign gene for green fluorescent protein (GFP), and a few days later these same leaves were inoculated with TBSV expressing either wild-type P19 or not expressing P19. The hypothesis was that the presence of P19 would boost the GFP expression by preventing the latter from being targeted by gene silencing. Preliminary results are in agreement with this hypothesis. Future experiments aim to test a variety of P19 mutants for their ability to suppress gene silencing and then test them as described above for their effect on virus vector integrity.
Objective 3
To test virus vector integrity others have used RT-PCR to monitor the maintenance of foreign genes by virus vectors. However, this technique does not directly assay for maintenance of foreign gene expression. For this purpose, we developed a new quantification technique. This novel method is based on the property that on cowpea leaves TBSV produces readily quantifiable local lesions. Furthermore, upon UV illumination, TBSV expressing the foreign gene for GFP gives bright green fluorescence in those lesions. However, in case the foreign gene is (partially) deleted or mutated, green fluorescence no longer occurs, yet the lesions still form. Thus, when 100 percent of the lesions give green fluorescence this means that 100 percent of the virus vector inoculum contained the intact foreign gene. However, if for instance 50 percent of all lesions give green fluorescence, this means that 50 percent of the inoculum contained a mutation or deletion in the foreign gene.
The hypothesis for this objective is that virus gene vectors (such as TBSV expressing GFP) are more stable in some hosts then in others. The above technique allows us to quantify and compare. For this purpose, we inoculate the test host with transcripts of TBSV-GFP and after 4-5 days we measure the green fluorescence (to verify that infection occurred) and then collect the infected leaves and use these as the source of inoculum for cowpea. Then. we determine the percent of lesions that give green fluorescence.
We have tested several hosts for susceptibility to inoculation with TBSV-GFP and results showed that several Nicotiana species —tomato, spinach, pumpkin, and beets —yield green fluorescence. Subsequent transfer to cowpea was performed in preliminary tests with TBSV-GFP- infected N. benthamiana. Results of these tests showed that 40-50 percent of the lesions on cowpea gave green fluorescence. This means that in these tests 40-50 percent of the TBSV-GFP vector molecules that had been amplified in N. benthamiana had lost the ability to express GFP. We are now going to conduct the same experiments with the other hosts to determine whether a higher percentage can be obtained in certain plant species.
Future Activities:These are summarized with the progress summaries provided above for each objective separately.
Supplemental Keywords:TREATMENT/CONTROL, Sustainable Industry/Business, Scientific Discipline, Technology, New/Innovative technologies, Agricultural Engineering, Ecology and Ecosystems, Geochemistry, Genetics, plant genes, biotechnology, bioengineering, plant biotechnology, cloning, transgenic plants, plant virus proteins, environmentally friendly turfgrass, bioenergy
Progress and Final Reports:
Original Abstract
Main Center Abstract and Reports:
R829479 The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829479C001 Plant Genes and Agrobacterium T-DNA Integration
R829479C002 Designing Promoters for Precision Targeting of Gene Expression
R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
R829479C005 Development of Novel Plastics From Agricultural Oils
R829479C006 Conversion of Paper Sludge to Ethanol
R829479C007 Enhanced Production of Biodegradable Plastics in Plants
R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
R829479C025 Chemical Induction of Disease Resistance in Trees
R829479C026 Development of Herbicide-Tolerant Hardwoods
R829479C027 Environmentally Superior Soybean Genome Development
R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
R829479C030 Engineered Plant Virus Proteins for Biotechnology