AWARDEE: Mary-Dell Chilton
FEDERAL FUNDING AGENCIES: Department of Agriculture, Department of Energy, National Institutes of Health, National Science Foundation
The soil beneath our feet teems with life. Beetles scuttle along the surface and burrow down to lay their eggs next to earthworm tunnels, within intricate fungal networks. But at the microscopic level, even more biodiversity emerges — just one teaspoonful of topsoil may contain on the order of a billion bacteria. At any given time, these bacteria are working to decompose organic matter, return nitrogen to the soil, and produce substances that bind soil particles together, improving soil structure. Over millions of years, bacteria have also evolved savvy survival strategies, some of which involve hijacking plants’ systems for their own benefit.
Agrobacterium tumefaciens is one of these bacteria. When it encounters a plant wound — caused perhaps by human pruning, grafting, frost injury, or insect feeding — Agrobacterium can slip into the wound site and cause plant cells to divide uncontrollably, creating a tumorous growth. Agrobacterium can utilize this as a nutrient source while most other bacteria cannot.
Agrobacterium tumefaciens cells (Jing Xu, Indiana University)
Crown galls on a grapevine (Thomas Todaro, Michigan State University)
Agrobacterium isn’t inherently good or bad — it’s just trying to survive — but humans have historically been less than pleased with its effects because the tumors (also known as crown galls) disfigure the plant and interfere with crop growth. The galls can intercept nutrients and water, weakening plants and making them more susceptible to harsh weather conditions and diseases, even killing young plants. In the early 1900s, researchers discovered that Agrobacterium was the culprit behind crown galls on grapevines, which had negative effects on the wine industry. This prompted a search for the mechanism of crown gall formation in the hope of thwarting what humans considered a threat to crops.
Mary-Dell Chilton (Syngenta)
The Seed of an Idea
In the late 1960s, researcher Mary-Dell Chilton was completing a postdoctoral fellowship at the University of Washington (UW) in Seattle. She was intrigued by bacterial transformation, the process by which certain bacteria correct mutations inside their DNA.
She learned a variety of techniques to study bacterial DNA; for example, if she mixed error-free bacterial DNA with bacteria containing mutated DNA and spread the mixture on a surface where the mutant could not grow, a few of the formerly mutant bacteria would grow. This meant the mutant bacteria had somehow been transformed into an error-free version. The original error-free DNA molecules could seemingly enter the mutant bacteria, find the right place to go, and repair the mutant DNA. Importantly, this process of correction (known as “transformation”) would only happen if there was a very good match, or “homology,” between the donor DNA and the DNA of the recipient bacteria. Chilton went beyond this technique and even designed an experiment to measure the percent of mismatch between mutant DNA and error-free DNA for several genes of interest.
Chilton photographing wildflowers with her son Andrew Chilton and late husband Mark Chilton (Syngenta)
After completing the postdoctoral fellowship and caring for her newborn second son for three months, Chilton got a job at UW. She became keenly interested in current publications about Agrobacterium transformation of tobacco plants; some researchers claimed they’d found evidence that Agrobacterium DNA was transferred into plant cells in tobacco crown galls. However, while reviewing these studies, Chilton and her students noticed that important controls had not been performed for the DNA hybridization experiments — the evidence did not support the hypothesis that Agrobacterium was transferring DNA into plant cells.
A Serendipitous Subversion
Chilton, leading a team of researchers funded by the National Institutes of Health’s National Cancer Institute, set out to test properly the claim that Agrobacterium was transferring part of its DNA into plant DNA, a process known as “hybridizing.” Since she’d seen no evidence that this sort of genetic exchange could happen without homology, she anticipated a negative outcome. At first, when Chilton’s team ran the control experiments missing from previous studies, they found no evidence of Agrobacterium DNA transferring to plant DNA.
The key phrase there is that they found no evidence—the methods they were using simply weren’t powerful enough. Indeed, with 20:20 hindsight, there were technical problems with all the experiments looking for Agrobacterium-plant hybridization, but the most fundamental issue was that everyone, Chilton included, was looking for the wrong kind of DNA.
Bacterial cell containing DNA in chromosomal and plasmid form
Quietly, in Jeff Schell and Marc van Montagu’s lab at the University of Ghent in Belgium, an unsung hero of this story made a breakthrough. While investigating Agrobacterium replication, postdoc Ivo Zaenen accidentally stumbled upon giant circular DNA molecules within Agrobacterium. Bacteria often store DNA in tiny circular molecules called plasmids, but these were much larger than expected. What’s more, they were present in virulent Agrobacterium (strains of Agrobacterium that caused crown gall growth) but not avirulent (strains that did not cause crown gall growth). There was soon enough evidence to recognize these large circular molecules were indeed plasmids, but BIG ones, mega plasmids — aptly named tumor-inducing (Ti) plasmids.
Prior studies hadn’t found detectable Agrobacterium DNA in plants because they needed this last clue: Agrobacterium keeps some of its DNA in these circular molecules. When Chilton’s team looked for specific pieces of the Ti plasmid in the DNA of transformed plant cells, rather than DNA from the entire bacterium or the entire plasmid, they found conclusively that part of the plasmid was indeed in the gall cells. That was the component of the Agrobacterium DNA causing the tumorous crown galls to grow on plants. More and more researchers confirmed the finding using various methods, shifting the entire research community’s conception of how DNA transfer can occur.
Mary-Dell Chilton (second from top right) with fellow researchers Don Merlo, Martin Drummond, Eugene Nester, Daniela Sciaky, Alice Montoya, and Milton Gordon (Esther M. Zimmer Lederberg Memorial Website)
Chilton recalls with delight proving herself wrong:
“I had a tape with the radioactivity measurements from our experiment and was performing the calculations at my kitchen table after the kids had gone to bed. I said, ‘My God, the DNA is there!’ Before that experiment, I was sure that you could not get bacterial genes to recombine with plant genes—there is no homology between the two. I just absolutely did not believe it. It went against everything I had ever learned. But in the process of trying to prove the idea wrong, I proved it was indeed right. It is important to remember that even if the evidence is incorrect, that does not make the idea wrong!”
Chilton examining tobacco plants at Washington University in St. Louis (Syngenta)
Tiny Bacterium, Huge Breakthrough
Chilton didn’t stop there. After Washington University in St. Louis hired her as an associate professor of biology, she started exploring ways to take advantage of Agrobacterium’s natural ability to add DNA to plant cells. With grants from the Department of Energy and National Science Foundation, along with private funding, Chilton’s team built on the techniques she’d used before to create the world’s first transgenic tobacco plant. She recalls the irony of giving cancer to tobacco plants, but that wasn’t the ultimate goal; later, she figured out a way to disarm the Agrobacterium Ti plasmid so it did not cause tumorous galls to grow but maintained its ability to transfer genes to plants. Later, the National Institute of Food and Agriculture funded research that improved transformation efficiency and expanded to other types of field crops based on the findings of Chilton’s team.
Patent for the regeneration of plants containing genetically-engineered T-DNA (Syngenta)
Chilton speaks often about the many researchers with whom she worked directly and indirectly, as well as collaboration with international researchers, who were essential in the journey toward these breakthroughs. One such researcher, Professor Andrew Binns of University of Pennsylvania, was the member of Chilton’s team who coaxed transformed tobacco cells to grow, produce shoots and roots, and finally set fertile seeds. Participants in this collaborative effort forged a bond of trust and fellowship that has strengthened over the decades.
The company CIBA-Geigy (now Syngenta) recruited Chilton in 1983 to launch its Agricultural Biotechnology Research Unit and conduct a research program that would produce genetically modified seed. By the mid-1990s, the first transgenic crops were cultivated and made commercially available.
Reaping the Rewards of Research
Chilton’s outside-the-box thinking and willingness to follow the evidence — even when she was proving her own beliefs wrong — didn’t just facilitate the understanding of this bacterial gene transfer mechanism that existed for millions of years; she repurposed the natural gene transfer ability of the bacterium into a technique now ubiquitous across biotechnology, now known as Agrobacterium-mediated transformation (AMT).
Chilton receiving the USDA Hall of Heroes Plaque in 2015 (Bob Nichols, USDA)
AMT’s environmental and economic impacts on agriculture have been massive. The incredibly powerful technique can introduce a DNA sequence that causes a plant to produce a protein that kills one type of pest when the pest tries to eat the plant, but does not harm any other non-targeted insects and animals. Genetically modified cotton with pest-resistant traits, called Bt cotton, has contributed to a significant decrease in insecticides applied (66% between 1994 and 2019), which has in turn decreased costs for farmers and lessened environmental impacts, including bioaccumulation, water contamination, and deaths of non-pest insects. Bt cotton and corn have also increased yields (by mitigating losses to pests) and profit margins for U.S. farmers compared to conventional seeds.
The AMT technique is so useful, in fact, that it is still used to deliver components of CRISPR/Cas9 — the new and very powerful gene editing tool — into plants, and researchers are now using Agrobacterium T-DNA sequences to study epigenetics, how environmental factors impact gene expression.
The magnitude of development in this field sparked by Chilton’s work is remarkable. By following her fascination with science, Chilton took the seed of a federally funded experiment on a soil bacterium and cultivated an entirely new field of biotechnology research, which continues to grow, with the promise of more economic benefits and scientific advances to come.
By Gwendolyn Bogard