2003 Progress Report: Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants

EPA Grant Number: R829479C004
Subproject: this is subproject number 004 , 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: Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
Investigators: Hogenhout, Saskia A. , German, Thomas L.
Institution: The Ohio State University - Main Campus , University of Wisconsin - Madison
EPA Project Officer: Lasat, Mitch
Project Period: October 2, 2002 through April 3, 2004
Project Period Covered by this Report: October 2, 2002 through April 3, 2003
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation

Objective:

The objective of this research project is to develop a reverse genetics system that can be used to express, in a regulated manner, one or more foreign genes from the monocot infecting negative strand RNA rhabdovirus, maize fine streak virus (MFSV).

Progress Summary:

The first step was to determine whether all open reading frames (ORFs) detected on the MFSV genome are transcribed into mRNAs. The MFSV putative gene order is N-P-ORF3-ORF4-M-G-L. All ORFs contain gene junction regions consisting of a 13-nt stutter sequence for poly(A)tailing, an intergenic noncoding region of 4 nucleotides, and transcription start site of 3 nucleotides. The start sites of several MFSV transcripts have been confirmed by 5' RACE and subsequent sequencing of the polymerase chain reaction (PCR) products. The intergenic regions are highly conserved within the MFSV genome and among plant and animal rhabdoviruses. Northern blot analysis of transcripts of the MFSV genome showed that all seven mRNAs can be detected in total RNA isolated from MFSV-infected plants, thereby confirming the sequence data. The 3' and 5' leader and trailer sequences have been revealed and appear to be most similar to those of Rice yellow stunt rhabdovirus (RYSV). A publication reporting the results of MFSV sequencing and Northern blot analysis is in preparation.

The second step was to develop a set of vectors with which to express viral nucleic acids or proteins in yeast cells. The rationale for this approach is to express the minimum set of viral nucleic acids. We will express the 5' and 3' regions of the virus known to be required for replication and the intergenic regions known to be required for imitation and termination of transcription (mRNA production) on a single RNA molecule (a "replicon"). A reporter gene will be inserted in the replicon such that transcription can be monitored from either a physical reporter or a selectable marker. To copy this molecule in its entirety (replication) and produce mRNA from it, we will express it and the three viral proteins (N, P, and L) known to be the replicase/transcriptase from individual plasmids. Each plasmid will have a separate selectable marker so we can be sure that they are all in the same yeast cell. The replicon RNA will be expressed from a plasmid with the appropriate DNA sequence downstream from a T-7 promoter recognizable by bacteriophage T-7 RNA dependent RNA polymerase (T-RdRp) that also will be expressed by means of a yeast cell promoter containing plasmid.

To meet this goal, we have:

• Become proficient at manipulation of plasmids and expression of proteins in yeast cells.

• Cloned and expressed T-7RdRp modified by adding a nuclear localization signal.

• Constructed yeast plasmids designed to express a GUS reporter gene driven by a wild-type T-7 promoter or a modified promoter lacking the terminal GGG sequence so that the transcription product will not have an additional 5' G that is not viral encoded (which may be critical for replication by the authentic viral polymerase complex).

• Transformed yeast cells with the above constructs and showed that the modified T-7RdRp can recognize the wild-type promoter in such a way that produces a strong signal from the GUS reporter.

• Confirmed that the truncated promoter works to a lesser extent than the wild type so that if this approach is required, we can use it.

• Constructed yeast expression plasmids with different selectable markers (leu, tryp, ura) and convenient cloning sites for addition of viral proteins.

• Constructed a plasmid for expression of the replicon that will have the above-described T-7 promoter and that contains a ribozyme (HDV) that will cleave the viral replicon RNA at the 3' end to produce an exactly authentic viral termini (again, may be essential for identification by viral replication complex).

Future Activities:

The next phase of work will be to construct the cDNA for the replicon (based on known sequence) and insert it into the above-described vector between the T-7 promoter and the HDV ribozyme. Then we will clone and insert the viral N, P, and L proteins into the yeast expression vectors. We will transform yeast cells with these constructs individually and assay for expression of either RNA or individual viral proteins as appropriate. After this, we will do complex transformations to insert all required vectors into yeast and look for assembly of replicons (replicon RNA in complex with viral N protein) based on their biophysical properties (sucrose gradient profile, RNase protection, etc). After that, we will screen for reporter or selectable marker activity from cells transfected with all required plasmids.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 10 publications 2 publications in selected types All 2 journal articles
Other center views: All 211 publications 48 publications in selected types All 44 journal articles
Type Citation Sub Project Document Sources
Journal Article Hogenhout SA, Redinbaugh MG, Ammar E-D. Plant and animal rhabdovirus host range: a bug's view. Trends in Microbiology 2003;11(6):264-271. R829479 (2006)
R829479C004 (2003)
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  • Journal Article Redinbaugh MG, Seifers DL, Meulia T, Abt JJ, Anderson RJ, Styer WE, Ackerman J, Salomon R, Houghton W, Creamer R, Gordon DT, Hogenhout SA. Maize fine streak virus, a new leafhopper-transmitted rhabdovirus. Phytopathology 2002;92(11):1167-1174. R829479 (2006)
    R829479C004 (2003)
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  • Supplemental Keywords:

    yeast expression vectors, viral vector, gene, insect, plants, maize fine streak virus, MFSV, rhabdovirus, open reading frame, ORF., RFA, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Sustainable Environment, Genetics, Technology, Technology for Sustainable Environment, New/Innovative technologies, Agricultural Engineering, negative sense viral vectors, bioengineering, yeast expression vectors, plant genes, environmental engineering, biotechnology, plant biotechnology, RNA rhabdovirus, maize fine streak virus

    Relevant Websites:

    http://www.cpbr.org Exit

    Progress and Final Reports:

    Original Abstract
  • Final

  • 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