2004 McCoy Award Recipient
Stanton B. Gelvin
Professor Biological Sciences
Stanton B. Gelvin, professor of biological sciences, has been a Purdue University faculty member since 1981. Born in New York City, he grew up in New Jersey, and received his AB degree in biology from Columbia University in 1970. He went on to attend Yale University, where he received his MPhil degree in 1973 from the Department of Molecular Biophysics and Biochemistry.
After transferring to the University of California, San Diego, he was introduced to the study of plant molecular biology by his doctoral advisor, Dr. Stephen H. Howell. He received his PhD in biology from USCD in 1977, where he isolated and characterized the first non-ribosomal gene from plants, the chloroplast gene encoding the large subunit of the key photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase from the unicellular green alga Chlamydomonas reinhardtii. Gelvin spent another year in Howell’s laboratory as a postdoctoral research fellow investigating unusual repetitive DNA structures in Chlamydomonas chloroplast DNA.
Gelvin subsequently moved to the University of Washington, Seattle, where he worked as a Damon Runyon-Walter Winchell fellow in the laboratories of Drs. Eugene W. Nester and Milton P. Gordon. It was in these laboratories that Gelvin was introduced to the Agrobacterium tumefaciens system, a system that he has continued to investigate for the past 25 years.
Gelvin joined the faculty of Purdue’s Department of Biological Sciences as an assistant professor in 1981 and has investigated a number of aspects of the Agrobacterium-plant interaction. His early work involved an elucidation of transcriptional activating elements of several Agrobacterium T-DNA genes; this work culminated in the development of the “super-promoter,” a strong constitutive promoter that has been used by hundreds of academic and industrial scientists to express genes in transgenic plants. After several years investigating the molecular and genetic mechanisms of Agrobacterium virulence gene induction, Gelvin turned approximately 10 years ago to his current line of investigation, the identification and characterization of plant genes and proteins involved in Agrobacterium-mediated plant genetic transformation. Gelvin was promoted to full professor in 1991; he currently also is an adjunct professor at the Institute of Botany, Academia Sinica, Taipei, Taiwan.
Early in his career at Purdue, Gelvin was selected as a NSF Presidential Young Investigator. He holds five patents and has authored more than 100 publications, including editing a widely used laboratory techniques manual on plant molecular biology. He has been a frequent organizer of the national annual Crown Gall Conferences, and has organized numerous workshops and sessions at international meetings.
Gelvin has served as associate editor for the journals Plant Molecular Biology and Molecular Plant-Microbe Interactions (MPMI), and is a past editor-in-chief of MPMI. He has served on numerous USDA grant review panels and was panel director for the USDA IFAFS panel in 2000. He has been director of the interdisciplinary Purdue Genetics Program since 1999, and was instrumental in initiating and developing the new PULSe interdisciplinary life sciences graduate program.
In his spare time, Gelvin enjoys playing clarinet with the Lafayette Citizens Band and the Lafayette Civic Theater musical productions, gardening with his wife and scientific colleague Dr. Lan-Ying Lee, and playing with his model electric trains.
Agrobacterium tumefaciens, and the related species A. rhizogenes, A. vitis, and A. rubi, cause neoplastic diseases on a wide range of plant species. The molecular mechanism by which Agrobacterium transforms cells involves the transfer of a segment of DNA, the T-(transferred) DNA, from a resident plasmid to the host genome. This horizontal gene transfer between species of different phylogenetic kingdoms is unique in nature, but is an extension of intra-kingdom DNA exchange (conjugation) commonly seen among bacteria. Recently, scientists have learned that this “DNA exchange” really represents “protein exchange” between species; proteins are transferred between organisms using what is known as a Type IV secretion system, with the consequence that DNA linked to these transferred proteins is also exchanged. An identical mechanism of protein transfer is used by many human and animal pathogens, including Bordetella pertusis, Legionella pneumophila, Helicobacter pylori, and various Brucella and Bartonella species, to transfer virulence factors to host mammalian cells. Agrobacterium has become the “model system” to investigate this type of protein transfer from these animal pathogens.
Once inside the host, T-DNA and the associated Virulence (Vir) proteins must traffic through the plant cytoplasm and target the nucleus, where Vir proteins are eventually stripped from the T-DNA and the T-DNA integrates into the host genome, thereby stably genetically transforming the eukaryotic cell. As a result of three decades of intensive investigation by numerous laboratories, scientists now have a reasonably complete understanding of the events that occur within the bacterium to initiate the transformation process. However, until very recently our knowledge of the events that transpire within the host and the contribution of host genes and proteins to this process have remained a mystery. My laboratory has contributed to understanding these latter events.
For the past decade, we have utilized several different approaches to understand the host contribution to Agrobacterium-mediated genetic transformation. The first approach is a “classical” forward genetic screen for plant mutants that are resistant to transformation. Using the model plant species Arabidopsis thaliana, we have identified more than 125 mutants that are resistant to Agrobacterium transformation (rat mutants), and more recently, mutants that are hyper-susceptible to Agrobacterium transformation (hat mutants).
We (along with our international collaborators) have characterized mutants that are defective in each of the steps of transformation. These include mutants defective in bacterial attachment to the plant cell (including plant cell wall synthesis and structural protein mutants), T-DNA and Vir protein transfer (including mutants lacking a putative receptor for the Agrobacterium T-[transfer] pilus), cytoplasmic trafficking (actin mutants), nuclear targeting (importin a and transportin mutants), Vir protein degradation (mutants of the 26S proteosome), T-DNA integration (various histone and chromatin protein mutants), and T-DNA expression (a histone mutant). Interestingly, the vast majority of these mutants have no obvious visible developmental phenotype; the plants appear completely normal. However, Agrobacterium is able to distinguish the loss of minor or partially redundant host proteins.
Although “forward genetics” is a powerful tool for investigating gene function, this methodology has limitations. We have, therefore, additionally employed a variety of “reverse genetic” and bioinformatic approaches to understand Agrobacterium-mediated plant transformation. “Forward genetics” starts with a mutant phenotype (the visible characteristics of an organism resulting from the interaction between its genetic makeup and the environment), from which scientists deduce the nature of the genetic lesion. “Reverse genetics” uses the opposite approach: Scientists start with a lesion in a known gene, following which they test the mutant organism for a specific phenotype. We have utilized several different reverse genetic approaches to define plant genes involved in Agrobacterium-mediated transformation. These include PCR-based reverse genetic screens, the use of anti-sense RNA and RNA inhibition (RNAi) technologies, and the use of yeast two-hybrid and in vitro systems to investigate the interaction of Agrobacterium Vir proteins and plant proteins.
We have recently been involved in a multi-laboratory project to develop a plant protein-protein interaction assay system. Taken together, these approaches allowed us to identify and characterize numerous plant proteins that can interact with Vir proteins. Most of these host proteins belong to “families” of highly related proteins. In many instances, mutation of the plant genes encoding these proteins resulted in a loss of transformation competence of the host. Thus, Agrobacterium is able to “pick out” specific members of multi-gene families and use these specific proteins to effect transformation.
Finally, we have used bioinformatic, macroarray, and microarray experimental approaches to identify, on a more global scale, plant genes that respond to Agrobacterium infection. We investigated the nature and kinetics of the response of plant cells to Agrobacterium cells during the first hours of interaction. Several hundred plant genes are either up- or down-regulated during this initial period, and the nature of the plant response depends on the strain of Agrobacterium that the plant cells see. Some plant genes respond to any Agrobacterium strain, whether virulent or avirulent, whereas other plant genes respond only to virulent strains that can transfer T-DNA and Vir proteins. Interestingly, Agrobacterium appears to manipulate plant gene response for its own advantage: The bacterium induces plant genes necessary for transformation and at the same time represses plant defense- and stress-response genes. Thus, Agrobacterium actively suppresses the plant’s defense system while simultaneously making the plant a more suitable host for transformation.
The Molecular Mechanism of Plant Genetic Transformation by Agrobacterium tumefaciens, Nature’s Genetic Engineer
Abstract of Lecture
Agrobacterium tumefaciens is a soil bacterium that causes the neoplastic disease Crown Gall on hundreds of plant species. Agrobacterium is also nature’s genetic engineer. All virulent strains of Agrobacterium harbor a large extra-chromosomal segment of DNA called the Ti- (tumor inducing-) plasmid. During the course of infection, a small region of the Ti-plasmid, termed the T- (transferred-) DNA, is processed from this plasmid and transferred, along with several Virulence (Vir) proteins, from the bacterium to the plant. Once in the plant cell, the T-DNA must traverse the cytoplasm, enter the nucleus, and integrate into the plant genome. Following integration, T-DNA-encoded genes are stably expressed using the host’s transcriptional and translational machinery. Expression of oncogenes from the T-DNA results in over-production of plant growth regulating hormones, the auxins and cytokinins, causing unregulated plant cell growth and, consequently, tumors.
Several genes within the T-DNA also encode enzymes that direct the synthesis of novel low molecular weight compounds, termed opines. Opines can be used as carbon and (sometimes) nitrogen sources for the inciting bacterium to the exclusion of most other soil microorganisms. Thus, Agrobacterium genetically engineers plants to synthesize compounds, the opines, by which it can successfully compete with other organisms in the rhizosphere. Agrobacterium remains the sole known example of natural trans-kingdom (prokaryote to eukaryote) genetic exchange.
Approximately 20 years ago, scientists learned how to “tame” Agrobacterium for use as a genetic engineering organism for plants and, more recently, fungi and human cells. By deleting the oncogenes from the T-DNA, they “disarmed” the bacterium so that it would no longer produce tumors. However, these engineered laboratory strains could still deliver (non-oncogenic) T-DNA to plants, fungi, or mammalian cells. Any DNA segment now incorporated into the T-DNA would likewise be introduced into the host cell. The ability to deliver new genes into plants has formed the basis for modern agricultural biotechnology, resulting in plants resistant to herbicides and pathogens, plants with altered growth, metabolic, and nutritional characteristics, and plants that can be used as “bioreactors” to synthesize valuable pharmaceuticals and antibodies.
Although Agrobacterium-mediated plant transformation has provided a mainstay for plant biotechnology and plant molecular biology studies, many agriculturally important plant species, including corn, soybeans, cotton, fruit trees, trees used for lumber and pulp production, and ornamentals, remain highly recalcitrant to this method of transformation. To some extent, genetic manipulation of the bacterium has resulted in strains with a broader host range. However, major limitations for the use of Agrobacterium still remain. More recently, many scientists have concluded that we may be approaching the limits of our ability to manipulate the bacterium to improve its virulence, and that further success in broadening the host range may lie in an understanding and manipulation of the plant host. Our laboratory has become actively involved in understanding the role of host genes and proteins in the transformation process.
Through a broad range of experimental approaches and international collaborations, we have now identified more than 125 plant genes involved in Agrobacterium-mediated genetic transformation. These approaches have defined numerous host proteins contributing to each of the stages of transformation: bacterial attachment to the host cell, T-DNA and Vir protein transfer, cytoplasmic trafficking of the T-DNA and the associated Vir proteins, nuclear targeting of the T-DNA/Vir protein complex, removal of Vir proteins from the T-DNA, T-DNA integration into the host genome, and T-DNA gene expression. I shall present an overview of what we have learned about the host contributions to the transformation process. This knowledge will likely be applicable to human and animal pathogenesis.