Source: OREGON STATE UNIVERSITY submitted to
TRANSFORMATION OF PLANTS BY AGROBACTERIUM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0154817
Grant No.
(N/A)
Project No.
ORE00254
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 1, 2002
Project End Date
Sep 30, 2007
Grant Year
(N/A)
Project Director
Ream, L. W.
Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
MICROBIOLOGY
Non Technical Summary
Crown gall is a problem world wide and causes millions of dollars of damage each year in fruit and nut orchards, vineyards, and nurseries. We have proven that our Agrobacterium oncogene silencing strategy to produce plants resistant to crown gall is effective in a commercially important species (apple). Other than our oncogene silencing strategy, no effective means exists to prevent or cure crown gall. Our goal is to produce crown-gall-resistant rootstocks for grape, walnut, almond, and fruit trees.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2062499104030%
2064010110020%
2121110104015%
2122410104025%
2124010108010%
Goals / Objectives
Crown gall is a problem world-wide and causes millions of dollars of damage each year in fruit and nut orchards, vineyards, and nurseries. We have proven that our Agrobacterium oncogene silencing strategy to produce plants resistant to crown gall is effective in a commercially important species (apple). Other than our oncogene silencing strategy, no effective means exists to prevent or cure crown gall. The same approach should protect other fruit trees, nut trees, grapevines, cane berries, roses, chrysanthemum, and ornamental nursery plants.
Project Methods
Because only rootstocks need protection from crown gall, fruiting wood grafted to gall-resistant rootstock does not need to be genetically engineered. This fact may alleviate consumer concerns regarding genetically modified foods because the fruit from these plants will not contain foreign genes. Because only gene fragments are needed to trigger gene silencing, which is responsible for the gall resistance trait, even the rootstocks do not contain intact foreign genes. Thus, this technology is extremely safe, effective, and inexpensive.

Progress 06/01/02 to 09/30/07

Outputs
OUTPUTS: The results of our research have been disseminated to the scientific community through six publications in peer-reviewed journals, oral presentations at five national conferences, an article in American Nurseryman, chapters in two books, and one textbook. Our work resulted in a patent (Number 6,759,574: Plants having enhanced gall resistance; issued July 6, 2004). The PI trained high school teachers in a NSF-sponsored summer workshop on plant biotechnology. Eight undergraduates and six high school students received training through participation in this project. Approximately 80 undergraduates participated in the Agrobacterium genome annotation project under the guidance of the PI. PARTICIPANTS: Technician Larry Hodges cloned the Agrobacterium rhizogenes GALLS gene; he mutagenized the GALLS ATPase and Nuclear Localization Signal and identified the protein by SDS-PAGE. Deborah Moyer (MS) helped the PI fuse the secretion signal of the GALLS protein to Cre, which is used to detect secretion from Agrobacterium into plant cells. Deborah also fused the GALLS gene to a plant promoter. MS student Eric Coultas helped with the bioinformatic analysis of GALLS. Undergraduate Lindsey Crawford used the Cre-Lox recombination system to introduce the GALLS transgene into a plant transformation vector. Undergraduate Jason Neal-McKinney introduced the wild-type GALLS gene and nuclear localization signal (NLS) deletion and substitution mutations into a broad-host-range vector for transformation into Agrobacterium. Undergraduate Hank McNett worked on the genetic analysis of the alternative translation initiation site in the GALLS gene of Agrobacterium rhizogenes. Undergraduate Chris Brown studied expression of the GALLS proteins. Undergraduate Josh Cuperus built constructions designed to express affinity-tagged GALLS protein and assisted with SDS-PAGE analysis. TARGET AUDIENCES: Target audiences include colleagues in the Agrobacterium and plant biotechnology fields. We reached these colleagues through six publications in peer-reviewed journals and five presentations at national meetings. Our research directly affects the nursery industry and growers involved in fruit, nut, and grape production. We reached this audience through an article in American Nurserymen and through research collaboration with colleagues at nurseries and transgenic plant biotechnology companies. Another target audience is the general public; we reached this audience through and NSF-sponsored training program for high school science teachers. Our final audience is students (high school through PhD); we reached these students through direct involvement in the research project. Undergraduate students were also involved in the Agrobacterium genome annotation project.

Impacts
We developed two methods to engineer plants resistant to crown gall disease, and we obtained a patent for one of these methods. We used this method to create apple rootstocks resistant to crown gall, and a colleague is using our technology to engineer grape rootstocks resistant to crown gall. We also discovered a novel Agrobacterium virulence protein that transports foreign DNA into the nucleus of plant cells. This protein functions more efficiently than alternative Agrobacterium-encoded nuclear import proteins. This protein allows more efficient production of genetically engineered (transgenic) plants, and it increases the frequency of desirable single-copy transgene insertions.

Publications

  • Hodges, L.D., Lee, L.Y., McNett, H., Gelvin, S.B. and W. Ream. Agrobacterium rhizogenes GALLS gene encodes two secreted proteins required for gene transfer to plants. J. Bacteriol., will be submitted in April, 2008.
  • Ream, W. VirD2 and production of a mobile T-DNA. In: Agrobacterium: From Biology to Biotechnology, Chapter 8, pages 279-313; T. Tzfira & V. Citovsky, eds., Springer (2008).


Progress 01/01/06 to 12/31/06

Outputs
Agrobacterium rhizogenes GALLS Protein Contains Domains for ATP Binding, Nuclear Localization, and Type IV Secretion. Agrobacterium tumefaciens and A. rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system (T4SS), a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells where it binds single-stranded T-DNA and helps target it into the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences (NLS) and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2. VirE1-Mediated Resistance to Crown Gall in Transgenic Arabidopsis thaliana. Crown gall disease, caused by Agrobacterium tumefaciens, remains a serious agricultural problem despite current biocontrol methods. A. tumefaciens transfers single-stranded DNA (T-strands) into plant cells along with several virulence proteins, including a single-stranded DNA-binding protein (VirE2). In plant cells, T-strands are protected from nucleases and targeted to the nucleus by VirE2, which is essential for efficient transmission (transfer and integration) of T-strands. VirE1 is the secretory chaperone for VirE2; it prevents VirE2 from forming aggregates and from binding the T-strands in bacterial cells. Therefore, we hypothesized that sufficient quantities of VirE1 expressed in plant cells might block T-DNA transmission by preventing VirE2 from binding T-strands. Here we show that root explants from Arabidopsis thaliana plants that expressed virE1 formed 3.5-fold fewer tumors than roots from plants without virE1. Also, this resistance was specific for VirE2-mediated Agrobacterium transformation. Plants that have been genetically altered to resist crown gall may prove more effective than biological control.

Impacts
Issue: Crown-Gall-Resistant Plants. Who cares and why? Crown gall disease causes millions of dollars of damage in fruit and nut orchards, vineyards, and nurseries worldwide. Losses in Oregon are ~$400,000 in a typical year. Currently there is no effective means to prevent crown gall (except for our method). What has been done? We expressed a protein in transgenic plants that binds and inactivates an essential Agrobacterium virulence protein that is secreted into infected plant cells. This significantly reduced susceptibility to crown gall. Impact This technology has the potential to prevent crown gall disease in any crop plant amenable to introduction of transgenes. Once gall-resistant transgenic rootstocks are produced, no additional input is required.

Publications

  • Hodges, L.D., Vergunst, A.C., Neal-McKinney, J., den Dulk-Ras, A., Moyer, D.M., Hooykaas, P.J.J. and W. Ream. Agrobacterium rhizogenes GALLS protein contains domains for ATP-binding, nuclear localization, and type IV secretion. J. Bacteriol. 188: 8222-8230 (2006).
  • Humann, J., Andrews, S., and W. Ream. VirE1-Mediated Resistance to Crown Gall in Transgenic Arabidopsis thaliana. Phytopathology 96: 105-110 (2006).
  • Hooven, L.A., Butler, J., Ream, L.W. and P.D. Whanger. Microarray analysis of selenium-depleted and selenium-supplemented mice. Biological Trace Element Res. 109: 173-179 (2006).
  • Leonard, J.A., Shanks, O.C., Hodges, L., Ream, W., Hofreiter, M., Wayne, R.K. and R.C. Fleischer. DNA everywhere: Ancient DNA studies identify extraneous DNA in PCR reagents. J. Archaeological Sci., in press (2006).
  • Ream, W. VirD2 and production of a mobile T-DNA. In: Agrobacterium: From Biology to Biotechnology, T. Tzfira & V. Citovsky, eds., Springer (2006). In press.


Progress 01/01/05 to 12/31/05

Outputs
Crown gall is a problem worldwide and causes millions of dollars of damage each year in fruit and nut orchards, vineyards, and nurseries. This year we proved that expression of a molecular chaperone protein (VirE1) in transgenic plants interferes with the function of an Agrobacterium tumefaciens secreted virulence protein (the DNA-binding protein VirE2) in plants. The VirE2 DNA-binding protein is secreted to infected plant cells along with tumor-inducing bacterial genes. Inside plant cells, VirE2 binds the incoming bacterial oncogenes, thereby protecting them from degradation and targeting them to the nucleus of the plant cell. The VirE1 chaperone protein binds VirE2 and prevents DNA-binding. Normally, VirE1 does not enter plant cells. However, VirE1 produced in transgenic plants prevents VirE2 from binding, protecting, and targeting the incoming bacterial oncogenes, thereby reducing susceptibility to crown gall disease signifcantly. We have also continued our studies to understand the role of a novel Agrobacterium virulence protein in gene transfer to plants. We have proven that this protein is secreted to plant cells, and we have characterized the secretion signal. We have also shown that ATP-binding and nuclear localization signals in this protein are required for its activity.

Impacts
Issue: Crown-Gall-Resistant Plants. Who cares and why? Crown gall disease causes millions of dollars of damage in fruit and nut orchards, vineyards, and nurseries worldwide. Losses in Oregon are ~$400,000 in a typical year. Currently there is no effective means to prevent crown gall (except for our method). What has been done? We expressed a protein in transgenic plants that binds and inactivates an essential Agrobacterium virulence protein that is secreted into infected plant cells. This significantly reduced susceptibility to crown gall. Impact This technology has the potential to prevent crown gall disease in any crop plant amenable to introduction of transgenes. Once gall-resistant transgenic rootstocks are produced, no additional input is required.

Publications

  • Hooven, L.A., Butler, J., Ream, L.W. and P.D. Whanger. Microarray analysis of selenium-depleted and selenium-supplemented mice. Biological Trace Element Res. 108: 1-7 (2005).
  • Shanks, O.C., Hodges, L., Tilley, L., Kornfeld, M., Larson, M.L., and W. Ream. DNA from ancient stone tools and bones excavated at Bugas-Holding, Wyoming. J. Archaeol. Sci. 32: 27-38 (2005).


Progress 01/01/04 to 12/31/04

Outputs
Our work on Agrobacterium tumefaciens aims to achieve two goals: 1) engineer plants resistant to crown gall, and 2) introduce genes into specific locations in plant genomes by homologous recombination. Crown-Gall-Resistant Plants: Crown gall disease causes millions of dollars of damage in fruit and nut orchards, vineyards, and nurseries worldwide. Tumors on roots result from excessive auxin in plant cells genetically transformed by Agrobacterium tumefaciens. High auxin levels result from expression of two oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM and iaaH. We have generated transgenic apple rootstocks resistant to crown gall. Inactivation of iaaM by gene silencing prevented gall formation. Silencing A. tumefaciens oncogenes is a new and effective method to produce plants resistant to crown gall disease. This technology has the potential to prevent crown gall disease in any crop plant amenable to introduction of transgenes. Gene Transfer to Plants by Homologous Recombination: All transgenic crop plants on the market today were made using nonpathogenic strains of Agrobacterium tumefaciens to introduce the transgenes. The economic impact of Agrobacterium-mediated gene transfer technology is several billion dollars annually. We hope to develop a method to introduce genes into plants by homologous recombination. A. tumefaciens-mediated mobilization of genes into plant cells precludes homologous recombination between the incoming DNA and the host genome, except for very rare events. Instead, DNA is normally inherited at unpredictable locations, which often disrupts important plant genes. The inability to move genes into plant chromosomes via homologous recombination is a serious technical limitation for biotechnology and basic plant molecular biology. We will attempt to modify the Agrobacterium DNA transfer system to solve this problem.

Impacts
Issue: Crown-Gall-Resistant Plants. Who cares and why? Crown gall disease causes millions of dollars of damage in fruit and nut orchards, vineyards, and nurseries worldwide. Losses in Oregon are ~$400,000 in a typical year. Currently there is no effective means to prevent crown gall (except for our method). What has been done? We used gene-silencing technology to generate transgenic apple rootstocks resistant to crown gall. Impact This technology has the potential to prevent crown gall disease in any crop plant amenable to introduction of transgenes. Gall-resistant transgenic apple rootstocks are already available. Once gall-resistant transgenic rootstocks are produced, no additional input is required. Issue: Gene Transfer to Plants. Who cares and why? The inability to move genes into plant chromosomes via homologous recombination is a serious technical limitation for biotechnology. Using current technology, DNA normally inserts at unpredictable locations, often disrupting important plant genes. Hundreds of lines must be tested to find a useful line. Transgenes introduced by homologous recombination would drastically reduce this number. What has been done? We discovered a new pathway for gene transfer to plants. We will use it to introduce genes by homologous recombination. Impact Agrobacterium-mediated gene transfer technology generates billions of dollars annually. Introduction of transgenes at specific chromosomal locations by homologous recombination would significantly reduce the cost of producing transgenic plants.

Publications

  • Hodges, L.D., Cuperus, J. and W. Ream. Agrobacterium rhizogenes GALLS Protein Substitutes for A. tumefaciens Single-Stranded DNA-Binding Protein VirE2. J. Bacteriol. 186: 3065-3077 (2004).
  • Viss, W., Pitrak, J., Humann, J.L., Cook, M., Driver, J. and W. Ream. Crown-gall-resistant transgenic apple trees that silence Agrobacterium tumefaciens oncogenes. Molecular Breeding 12: 283-295 (2003).
  • Lee, H., Humann, J., Pitrak, J., Cuperus, J., Parks, T.D., Whistler, C., Mok. M. and W. Ream. Translational Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes. Plant Physiol. 133: 966-977 (2003).


Progress 01/01/03 to 12/31/03

Outputs
Crown gall disease is an economically significant problem in fruit and nut orchards, vineyards, and nurseries worldwide. Tumors on stems and leaves result from excessive production of the phytohormones auxin and cytokinin in plant cells genetically transformed by Agrobacterium tumefaciens. High phytohormone levels result from expression of three oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM, iaaH, and ipt. The iaaM and iaaH oncogenes direct auxin synthesis, and the ipt oncogene causes cytokinin production. In contrast to other tissues, roots do not respond to high cytokinin levels, and auxin overproduction is sufficient to cause tumor growth on roots. Inactivation of iaaM abolished gall formation on apple tree roots. Transgenes designed to express double-stranded RNA from iaaM and ipt sequences prevented crown gall disease on roots of transgenic apple trees. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5' end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease.

Impacts
Crown gall disease causes millions of dollars of damage in fruit and nut orchards, vineyards, and nurseries worldwide. We have generated transgenic apple rootstocks resistant to crown gall. This technology has the potential to prevent crown gall disease in any crop plant amenable to introduction of transgenes.

Publications

  • Hodges, L.D., Cuperus, J. and W. Ream. Agrobacterium rhizogenes GALLS Protein Substitutes for A. tumefaciens Single-Stranded DNA-Binding Protein VirE2. J. Bacteriol. Accepted pending revision (2003).
  • Viss, W., Pitrak, J., Humann, J.L., Cook, M., Driver, J. and W. Ream. Crown-gall-resistant transgenic apple trees that silence Agrobacterium tumefaciens oncogenes. Molecular Breeding 12: 283-295 (2003).
  • Lee, H., Humann, J., Pitrak, J., Cuperus, J., Parks, T.D., Whistler, C., Mok. M. and W. Ream. Translational Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes. Plant Physiol. 133: 966-977 (2003).


Progress 06/01/02 to 12/31/02

Outputs
Crown gall disease is a significant problem in fruit and nut orchards, vineyards, and nurseries. Tumors on roots result from excessive production of the phytohormones auxin and cytokinin in plant cells genetically transformed by Agrobacterium tumefaciens. High phytohormone levels result from expression of three oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM, iaaH, and ipt. Inactivation of iaaM abolished gall formation on apple tree roots. A transgene designed to express double-stranded RNA from a portion of the iaaM oncogene prevented crown gall disease on roots of transgenic apple trees. The goal of our research was to provide a new, more effective method to produce fruit and nut trees, grapevines, and other crop plants resistant to crown gall disease. Using a gene silencing strategy we produced six transgenic apple lines that were completely resistant to crown gall. Agrobacterium tumefaciens is the most widely used means to insert foreign genes into plants because it provides proteins that protect transferred DNA from rearrangements. VirE2 is the most abundant A. tumefaciens protein that accompanies DNA into plants, where it binds single-stranded DNA transferred from Agrobacterium. Because VirE2 plays a crucial role in transfer of foreign genes into plant cells, we examined the role of VirE1 in export of VirE2 protein and T-DNA from A. tumefaciens. Export of VirE2 to plant cells required VirE1. In the absence of VirE1, VirE2 remained stable in A. tumefaciens but was unable to exit the bacterial cells. Because VirE1 binds VirE2, we reasoned that VirE1 produced in plant cells might interfere with VirE2 function and interfere with DNA transfer and tumor formation. Our work showed this was the case.

Impacts
Crown gall disease is a significant problem in fruit and nut orchards, vineyards, and nurseries. Using a gene silencing strategy we produced six transgenic apple lines that were completely resistant to crown gall. Our oncogene silencing technology can be used to prevent crown gall disease in any crop. Our work has shown that production of the molecular chaperone VirE1 in plants interferes with development of crown gall disease. This offers a second strategy to engineer gall-resistant plants. Combining the oncogene-silencing and chaperone interference strategies in a single plant line should lead to plant lines with more durable crown gall resistance.

Publications

  • No publications reported this period


Progress 01/01/01 to 12/31/01

Outputs
Agrobacterium tumefaciens is the most widely used means to insert foreign genes into plants because it provides proteins that protect transferred DNA from rearrangements. VirE2 is the most abundant A. tumefaciens protein that accompanies DNA into plants, where it binds single-stranded DNA transferred from Agrobacterium. Because VirE2 plays a crucial role in transfer of foreign genes into plant cells, we created a genetic map of virE2 and identified domains in VirE2 protein that interact with its secretory chaperone, VirE1. We introduced the virE1 gene into plants, which exhibited resistance to crown gall tumorigenesis in lines that expressed high levels of VirE1. Crown gall tumors result from expression of three A. tumefaciens oncogenes in plant cells. This leads to overproduction of plant growth hormones auxin and cytokinin, which causes unorganized plant cell growth. We constructed transgenes designed to trigger post-transcriptional gene silencing of these oncogenes. These transgenes blocked production of excess auxin that normally occurs as a result of A. tumefaciens infection. This interfered with crown gall tumor development. These transgenes have been introduced into apple where they protected the roots from crown gall. Patent Application No. US99/26100 entitled "Plants Having Enhanced Gall Resistance" was filed on November 4, 1999, and is still pending.

Impacts
We have proven that our strategy to produce plants resistant to crown gall is effective in a commercially important species (apple). The same approach should protect other fruit trees, nut trees, grapevines, cane berries, roses, chrysanthemum, and ornamental nursery plants. Crown gall is a problem world wide and causes millions of dollars of damage each year in fruit and nut orchards, vineyard, and nurseries. Other than our strategy, no effective means exists to prevent or cure crown gall. Because only rootstocks need protection from crown gall, fruiting wood grafted to gall-resistant rootstock does not need to be genetically engineered. This fact may alleviate consumer concerns regarding genetically modified foods because the fruit from these plants will not contain foreign genes. Because only gene fragments are needed to trigger gene silencing, which is responsible for the gall resistance trait, even the rootstocks do not contain intact foreign genes. Thus, this technology is extremely safe, effective, and inexpensive.

Publications

  • Viss, W., Pitrak, J., Humann, J.L., Cook, M., Driver, J. and W. Ream. Crown-gall-resistant transgenic apple trees that silence Agrobacterium tumefaciens oncogenes. Submitted to Molecular Breeding (2002).
  • Hamilton, C.M., Lee, H., Li, P.L., Cook, D.M., Piper, K.R., Beck von Bodman, S., Lanka, E., Ream, W. and S.K. Farrand. TraG and its homologs from pTiC58 and RP4 confer relaxosome specificity to the Ti plasmid conjugal transfer system. J. Bacteriol. 182: 1541-1548 (2000).
  • Sundberg, C.D. and W. Ream. Agrobacterium tumefaciens chaperone-like protein, VirE1, interacts with VirE2 at domains required for single-stranded DNA binding and cooperativity. J. Bacteriol. 181: 6850-6855 (1999). Mysore, K.S., Bassuner, B., Deng, X., Darbinian, N.S., Motchoulski, A., Ream, W. and S.B. Gelvin. Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration. Mol. Plant-Microbe Interactions 11: 668-683 (1998).
  • Dombek, P.D. and W. Ream. Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2. J. Bacteriol. 179: 1165-1173 (1997).
  • Sundberg,C., Meek, L., Carroll,K., Das, A., and W. Ream. VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells. J. Bacteriol. 178: 1207-1212 (1996).
  • Haas, J.H., Moore, L.W., Ream, W., and S. Manulis. Universal PCR primers for detection of pathogenic Agrobacterium species. Appl. Environ. Microbiol. 61: 2879-2884 (1995).


Progress 01/01/94 to 12/30/94

Outputs
ORE00254 We developed a very sensitive method to detect pathogenic strains of Agrobacterium; this technique uses polymerase chain reaction (PCR) to identify a highly conserved portion of a virulence gene (VirD2). A publication describing this work is in press in Applied and Environmental Microbiology (listed below). We have submitted relevant documents to Bill Hostetler (OSU's Technology Transfer Director); we hope to patent the PDR primers and market them as a diagnostic tool to test soil and plant material for pathogenic Agrobacterium strains. We initiated work to create transgenic plants (tobacco) resistant to crown gall disease, which is caused by Agrobacterium tumefaciens. Bacterial genes, expressed by infected plant cells, result in over production of two plant growth hormones (auxin and cytokinin), which causes galls to form. We believe cosuppression techniques pioneered by Bill Dougherty will permit us to block expression of these genes in engineered plants. We have chosen tobacco as a model, and we have created a number of transgenic plants that will soon be large enough to test. If successful in tobacco, we will introduce these cosuppression transgenic genes into plants where crown gall is a costly problem: grapes and fruit trees.

Impacts
(N/A)

Publications

  • Haas, J. N., L. W. Moore, W. Ream, and S. Manalis. Universal PCR Primers for Detection of Pathogenic Strains of Agrobacterium. Applied and Environmental Microbiology, in press. 1995.


Progress 01/01/93 to 12/30/93

Outputs
1) We pursued several different issues relevant to transformation of plants by Agrobacterium. First, we helped develop a method to detect DNA helicase activity in crude protein extracts. We tested the method on proteins prepared from E. coli and then searched for novel helicase activities amongst proteins isolated from A. tumefaciens. This work resulted in one publication (listed below). 2) We developed a very sensitive method to detect virulent strains of Agrobacterium; this technique uses polymerase chain reaction (PCR) to identify a highly conserved virulence gene (virD2). This work resulted in a published abstract (listed below), and a full paper is in preparation. 3) We participated in a collaborative project to identify a tobacco protein that binds specifically to the nuclear localization signal that we identified within the Agrobacterium VirD2 virulence protein. A manuscript describing this work will be submitted shortly (listed below).

Impacts
(N/A)

Publications

  • KEIM-MILLER, C. A.; REAM, W.; MOSBAUGH, D. 1993. DNA helicase activity detected in situ following polyacrylamide gel electrophoresis. Methods in Molecular and Cellular Biology, 3:259-269.
  • LU, S.; CANFIELD, M.; HAAS, J. H.; MANULIS, S.; REAM, W.; MOORE, L. W. 1993. Use of sensitive nonradioactive methods to detect Agrobacterium tumefaciens in crown gall tumors of naturally infected woody plants. Sixth International Congress. LIN, Z. W.; GUPTA, V.; SHURVINTON, C. E.; REAM, W.; GELVIN, S. B. (199X). Identification of a tobacco nuclear localization sequence-binding protein that interacts with the Agrobacterium tumefaciens VirD2 NLS. (To be submitted).


Progress 01/01/92 to 12/30/92

Outputs
Agrobacterium tumefaciens is an economically important plant pathogen which causes significant damage in fruit tree nurseries and vineyards. We study the stable transfer of disease-causing genes from A. tumefaciens to plant cells during crown gall tumor induction. Specific bacterial genes transfer into plant cells where these genes enter the nucleus and integrate into plant nuclear DNA. Transfer of genes between the bacterial and plant kingdoms requires DNA to cross the plant nuclear membrane. Two bacterial proteins attach to the transferred DNA and accompany it into plant cells. We discovered that one of these proteins helps pilot bacterial DNA across the nuclear membrane and into the nucleus PNAS 89: 11837-11841 (1992). We have designed strategies which may allow us to disrupt this process. Our work will continue to focus on nuclear transport in plants. A. tumefaciens provides an efficient means to introduce foreign genes into plants. We can replace the tumor-inducing genes with beneficial genes. Studies in our laboratory identified essential components of the gene transfer apparatus, and we discovered ways to stimulate this process more than 100 fold. We also devised a method to increase by 10 fold the number of genes A. tumefaciens can transfer simultaneously into plants (J. Bact. 174: 2288-2297 (1992). Thus, our work has improved our ability to introduce novel genes into plants.

Impacts
(N/A)

Publications


    Progress 01/01/91 to 12/30/91

    Outputs
    Our research efforts this past year focused primarily on two Agrobacterium tumefaciens proteins which play a crucial role in transfer of bacterial DNA into plant cells and subsequent development of crown gall tumors. We created a number of mutations in the protein (VirD2) which attaches covalently at the origin of transfer in the bacterial DNA; VirD2 protein appears to pilot the Agrobacterium genes into host cells and across the nuclear membrane. We identified amino acid sequences in VirD2 likely to mediate nuclear transport and, through mutagenesis experiments, demonstrated their importance for virulence. We also replaced these native nuclear localization signals with a heterologous sequence from tobacco etch virus; the TEV sequences substituted for the native ones. These studies have addressed nuclear transport, a fundamental process which is largely uncharacterized in plants. Our work has increased our understanding of an important plant pathogen and suggested ways to prevent crown gall disease. A single-strand DNA-binding protein (VirE2) is also essential for virulence of Agrobacterium. We have isolated mutations which do not affect the ability of this protein to bind DNA but do dramatically reduce its ability to promote crown gall tumorigenesis. We will characterize this mutations.

    Impacts
    (N/A)

    Publications