2003 Progress Report: Plant Genes and Agrobacterium T-DNA Integration

EPA Grant Number: R829479C001
Subproject: this is subproject number 001 , 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: Plant Genes and Agrobacterium T-DNA Integration
Investigators: Gelvin, Stanton B.
Institution: Purdue University - Main Campus
EPA Project Officer: Lasat, Mitch
Project Period: July 1, 2002 through June 30, 2004
Project Period Covered by this Report: July 1, 2002 through June 30, 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 objectives of this research project are to: (1) screen T-DNA mutagenized Arabidopsis libraries for resistant to Agrobacterium transformation (rat) mutants; (2) clone T-DNA/plant DNA junctions from various rat mutants; (3) overexpress rat genes in plants to determine whether the resulting plants show enhanced transformation susceptibility; (4) determine the expression patterns of various Arabidopsis histone H2A promoters; and (5) screen T-DNA activation-tagged Arabidopsis libraries for hyper-susceptible to Agrobacterium transformation (hat) mutants.

Progress Summary:

Although we have made progress in each of the objectives stated above, the most significant results lie in three areas: (1) overexpression of rat genes to increase plant susceptibility to transformation; (2) determination of the patterns of expression of various histone H2A promoters; and (3) screening of T-DNA activation-tagged libraries for hyper-susceptible to Agrobacterium transformation (hat) mutants.

Overexpression of Rat Genes Increases Plant Susceptibility to Transformation

We have continued our work to overexpress rat genes in various plants to determine whether the plants become hyper-susceptible to transformation by grobacterium tumefaciens. Most of our work has concentrated on the various Arabidopsis histone genes, but we have begun similar studies on other rat genes. Past work in our laboratory indicated that overexpression of the Arabidopsis histone H2A-1 gene HTA1 in various Arabidopsis ecotypes increases transformation twofold to fivefold. We now have discovered that overexpression of the HTA1 gene in various rat mutants (including many mutants that are not involved in chromatin structure or T-DNA integration) reverses the rat phenotype. This surprising result suggests that overexpression of the HTA1 gene generally sensitizes plant cells to Agrobacterium-mediated transformation. We have performed additional experiments to attempt to discover the mechanism by which this sensitization occurs. Surprisingly, preliminary results indicate that although many rat mutants do not express T-DNA-encoded genes well, the extent of integration of T-DNA into high-molecular weight DNA in these mutants is similar to that of wild-type plants. When rat mutants additionally transgenic for the HTA1 gene are infected by Agrobacterium, however, both T-DNA integration and T-DNA expression are similar to that of wild-type plants. These preliminary results suggest that overexpression of the HTA1 gene either may guide T-DNA to "expressible" loci within the plant genome, or may "reverse" the effects of gene silencing. We currently are conducting experiments to test these various hypotheses.

We have initiated a study to determine which Arabidopsis histone genes, when overexpressed in wild-type Arabidopsis plants, increase transformation-competence. There are 40 core histone genes (H2A, H2B, H3, and H4) encoded by the Arabidopsis genome, but only 22 different proteins are encoded. We have cloned 19 of these 22 cDNAs under CaMV 35S promoter control, and introduced these genes into wild-type Arabidopsis plants (ecotype Ws). We currently are selecting transgenic lines to assay for transformation-competence.

With the help of Dr. Kan Wang (Iowa State University), we have tested whether maize plants transgenic for and expressing the Arabidopsis HTA1 gene are more susceptible to Agrobacterium-mediated transformation. Our initial results suggest that they are. Maize plants expressing "empty vector" DNA transform at the rate of 3-5 percent, whereas plants expressing the HTA1 gene average 8-9 percent transformation (some maize lines show 18 percent transformation frequency).

We have initiated a study to determine whether overexpression of several Arabidopsis genes implicated in T-pilus attachment increases the transformation frequency of Arabidopsis plants. Preliminary data suggest that such overexpression does increase transformation efficiency. We currently are conducting additional experiments to determine the cause for this hyper-susceptibility to transformation.

Determination of the Expression Patterns of Arabidopsis Histone H2A Genes

We have made translational fusions of the gusA gene to the first approximately 10 amino acids of 9 of the 13 histone H2A genes (HTA1,2,3,4,5,6,8,10, and 13). We have derived multiple T2 generation transgenic lines from each construction and determined the expression patterns of each of the histone promoters. The data indicate that there are substantial differences in the patterns of expression of the various promoters. In addition, we have quantified, using real-time polymerase chain reaction, the expression levels of the various histone H2A genes in Arabidopsis plants. There is a 3-to-4 order of magnitude range of expression levels of the various genes. Interestingly, HTA1 is expressed at very low levels compared to many other histone H2A genes.

Screening of Arabidopsis T-DNA Activation Tagged Libraries for Hat Mutants

We have begun screening an Arabidopsis T-DNA activation tagged library for hat mutants. We have screened individually approximately 2,000 lines using a tumorigenic strain of Agrobacterium at very dilute concentrations (approximately 105-106 cells/mL). At these low concentrations, only 5-10 percent of the root segments of wild-type plants show tumors. We have identified at least three lines that, through three rounds of screening, consistently show threefold to fivefold increases in transformation. We currently are characterizing these hat mutants further.

Future Activities:

Future activities will include: (1) the assay of transgenic plants overexpressing various histone genes for increased transformation-competence; and (2) further identification and characterization of Arabidopsis hat mutants.


Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other subproject views: All 56 publications 8 publications in selected types All 8 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 Gelvin SB. Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiology and Molecular Biology Reviews 2003;67(1):16-37. R829479 (2006)
R829479C001 (2003)
R829479C001 (Final)
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  • Journal Article Gelvin SB. Improving plant genetic engineering by manipulating the host. Trends in Biotechnology 2003;21(3):95-98. R829479 (2006)
    R829479C001 (2003)
    R829479C001 (Final)
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  • Journal Article Tian L, Wang JL, Fong MP, Chen M, Cao HB, Gelvin SB, Chen ZJ. Genetic control of developmental changes induced by disruption of Arabidopsis histone deacetylase 1 (AtHD1) expression. Genetics 2003;165(1):399-409. R829479 (2006)
    R829479C001 (2003)
    R829479C001 (Final)
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  • Journal Article van Attikum H, Bundock P, Overmeer RM, Lee L-Y, Gelvin SB, Hooykaas PJJ. The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T-DNA. Nucleic Acids Research 2003;31(14):4247-4255. R829479 (2006)
    R829479C001 (2003)
    R829479C001 (Final)
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  • Journal Article Zhu Y, Nam J, Humara JM, Mysore KS, Lee L-Y, Cao HB, Valentine L, Li JL, Kaiser AD, Kopecky AL, Hwang H-H, Bhattacharjee S, Rao PK, Tzfira T, Rajagopal J, Yi HC, Veena, Yadav BS, Crane YM, Lin K, Larcher Y, Gelvin MJK, Knue M, Ramos C, Zhao X, Davis SJ, Kim S-I, Ranjith-Kumar CT, Choi Y-J, Hallan VK, Chattopadhyay S, Sui X, Ziemienowicz A, Matthysse AG, Citovsky V, Hohn B, Gelvin SB. Identification of Arabidopsis rat mutants. Plant Physiology 2003;132(2):494-505. R829479 (2006)
    R829479C001 (2003)
    R829479C001 (Final)
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  • Supplemental Keywords:

    Agrobacterium tumefaciens, plant genetic transformation, rat mutants, hat mutants, histone., Scientific Discipline, Sustainable Industry/Business, Genetics, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, agrobacterium, bioengineering, T-DNA, transgenic plants, plant genes, Arabidopsis, biotechnology, remediation, plant biotechnology, cloning

    Relevant Websites:

    http://www.bio.purdue.edu/about/faculty/gelvin/index.html Exit
    http://www.bio.purdue.edu/about/faculty/gelvin/gelvinweb/gelvin.html Exit

    Progress and Final Reports:

    Original Abstract
  • Final Report

  • 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