Agrobacterium tumefaciens is an instrumental part of modern plant science, as it has allowed researchers to genetically transform plants long before the discovery of CRISPR Cas9. A. tumefaciens is a bacterium that is naturally found in the soil living off plant nutrients, but can also edit the genomes of plants for its own gain. In nature, the bacterium causes crown gall tumors, large knobby lumps that protrude from the plant.

To get a better picture of how a bacterium can genetically modify a plant, it’s important to understand the genes of A. tumefaciens itself. Unlike humans, who have 46 unique linear chromosomes (often depicted as X-shaped), bacteria have a large circular chromosome that makes up the bulk of their genetic information. A. tumefaciens has important pathogenic genes on this main chromosome that help the bacterium infiltrate plant systems. In addition to their circular nucleoid though, bacteria can also have plasmids, which are smaller loops of DNA. In bacteria, plasmids are often swapped with other bacteria and the environment to maximize genetic diversity. In A. tumefaciens, one plasmid is responsible for hacking plant genomes, the tumor inducing (Ti) plasmid. This plasmid contains virulence (Vir) genes, including Vir A, which codes for a receptor protein on the bacterial cell surface. When plant tissues are injured, their cells release chemoattractants including Acetosyringone, which binds to the surface receptor. This activates a protein encoded by the Vir G gene, which goes on to activate transcription factors for other Vir genes. 

To infect the plant cell, the bacterium makes an extending chute called a pilus that connects the two cells. To insert its DNA into the plant, it first makes a single-stranded DNA copy of the section it wants to insert, which is called the T-DNA region. The T-DNA travels through the pilus and into the plant cell, where it is inserted in the host genome. The T-DNA encodes for the production of opines, nitrogen-rich compounds that feed the Agrobacterium, and cytokinins and auxins, plant hormones that stimulate cell division resulting in the crown gall tumors. So, an edited cell will make food for the bacteria, and the rapid division ensures there will be plenty to go around.

That is how A. tumefaciens controls plants in the natural world. In modern biotechnology, however, humans are able to edit the T-DNA region to their own liking, so instead of causing a tumor, the infected plants can be edited to carry desirable traits. Since E. coli is easier to manipulate than Agrobacterium, researchers produce the plasmid containing the edited T-DNA within E. coli and replicate it. However, it’s easier for E. coli to maintain smaller plasmids, and the Ti plasmid is rather large. As a result, researchers split the large plasmid into two smaller plasmids, one with the T-DNA containing the gene of interest, and one with the Vir genes. Each plasmid is replicated in E. coli first, then introduced to A. tumefaciens, which can then go onto infect plant cells. To ensure that the transformation process is successful, both plasmids have selective marker genes, usually coding for different types of antibiotic resistance. By culturing the modified A. tumefaciens on both antibiotics, scientists can confirm that both plasmids are present. A marker can also be placed on the T-DNA region, which is expressed once the plant is modified. If this marker codes for resistance to a particular herbicide, resulting plants that survive exposure to the herbicide are confirmed for genetic modification. 

Since the discovery of CRISPR is relatively new, I knew that we had been genetically engineering stuff via other methods, but it wasn’t until now that I really understood how. If the Flavr Savr tomato was on the market before we developed CRISPR/Cas9, how did we genetically engineer it? Now we know!

My project at the Danforth Center this summer involves Agrobacterium-mediated transformation, so I’ve done a bit of investigation to fully understand this mechanism. It’s kind of shocking how little I had heard of Agrobacterium prior to this project, but now I feel comfortable explaining what it is and how it can genetically modify plants. My project is on a species of flower commonly known as columbine, so although it won’t be directly addressing food insecurity or climate change or something like that, this research will be improving the methods we use to transform these plants, which is really exciting. Not to mention I’ll be gaining experience with techniques I will use at my lab at Berkeley transforming chickpea, including CRISPR! 

Also, even though plants edited by humans via Agrobacterium are considered to be genetically modified, the process is, indeed, natural. Researchers have even found a portion of the T-DNA region in sweet potatoes!

I really really enjoyed writing this week’s article. It feels a little weird because I know I will be learning this in class at some point, but it still fits with my theme of investigating progress in biotechnology. Unlike most of my articles, where I have an idea of the change being made (like introducing a gene for flood tolerance), I’m really understanding the cellular mechanisms at work. It’s kind of funny when you do something you like and you’re reminded of why you like it. I haven’t been learning too much about cellular mechanisms in school, much to my dismay, but I almost forgot how much I enjoy it. In preparation for writing this article, and also just understanding Agrobacterium more in general, I had a discussion/argument with my flatmate about various questions I had about its infection system for like two hours. She’s a rising senior in Genetics and Plant Biology at Berkeley, so I was able to ask her a lot of questions, but I spent a really long time trying to get her to explain topics that evaded my understanding, like why we needed to separate the Ti plasmid into two smaller plasmids. If it works in the wild Agrobacterium, why is it too big now? I kept getting “because it’s easier when it’s smaller,” or “because it’s more efficient,” but not why, until it was finally revealed that E. coli prefers the smaller fragments, and it’s replicated there before moving to the Agro. That discussion, despite the fact that we were yelling at each other, was really fun, and I feel like my understanding of the whole thing was really solidified. I hope I get to talk to people about cellular mechanisms all the time once I finally start at Berkeley.

There’s stuff I didn’t fit in about Agrobacterium in this post, and I’m sure I’ll learn plenty more, but I’m happy to share with you what I know now. Stay tuned to learn with me!

Main background of A. tumefaciens: https://link.springer.com/article/10.1007/s12033-023-00788-x

Additional background and also a picture of a massive crown gall on a tree at UC Davis: Ronald, Pamela C., and Raoul W. Adamchak. Tomorrow’s Table : Organic Farming, Genetics, and the Future of Food. Oxford University Press, 2018.

T-DNA delivery naturally and engineered: https://www.youtube.com/watch?v=CBlliZRELm4 (this is a really good video)