Hijack Hijinks: How One Bacteria Takes Over Plant DNA with a Few Special Genes of Its Own

By: Imaan Moin '22 and Ray Stieber '22

A wolf in sheep’s clothing. Stalking quietly, waiting tensely until it’s the perfect time to strike. For many, this imagery may induce panic, discomfort, or even fear. Now imagine the aforementioned wolf to be a scheming bacteria and the unsuspecting sheep to be vulnerable, recently-wounded plant cells. Seems less scary? Think again! These small and seemingly inconsequential bacteria not only attack wounded plant cells, but attack in a way that results in them hijacking the plant’s very own cellular machinery to do their microbial bidding. This mischievous, havoc-wreaking bacteria is known as Agrobacterium tumefaciens and has a quite literally gnarly history of inducing crown galls -- large, amorphous tumors -- in vulnerable plants.

A. tumefaciens cells attaching to the cell wall of a plant cell. (Image sourced from the MicrobeWiki.)

A. tumefaciens is found evenly-concentrated throughout most soils and many plant roots. It is rod-shaped and gram-negative, meaning its bacterial envelope has both an inner and outer cell membrane surrounding a relatively thin layer of peptidoglycan cell wall. As this blog post discusses later on, this outer membrane is integral to the signaling cascade that allows A. tumefaciens to detect the “right time to strike” a plant. Additionally, A. tumefecaiens utilizes transposons to hijack already weakened, wounded plant cells in an effort to take over their autonomy and function. Transposons are sequences of DNA that can be moved around either within a genome or -- in the case of A. tumefaciens -- from genome to genome. These sequences allow A. tumefaciens to send its own DNA into the genomes of plant cells in order to alter the plant cells’ functions. This hijacking causes the chromosomes of the plant cells to take up certain tumor-inducing (Ti) sequences of bacterial DNA, thus causing the plant cells to reproduce the bacterial DNA and proliferate at such a high rate that tumors form on the plant. Although many plants may survive with a crown gall, these tumors usually render crops and other agricultural products undesirable to consumers -- a substantial problem for farmers and agricultural producers at large.

Tumefaciens crown gall on a Spindle Tree (Euonymus europaeus) (Image sourced from the The American Phytopathological Society)

This process of tumorigenesis -- the creation of crown gall tumors in plants -- starts with a signaling cascade that begins when a plant is wounded. According to this explanatory video, wounded plant cells release a signaling molecule that binds to the outer membrane of an A. tumefaciens cell, which then activates the vir (virulence) signaling cascade. This results in the T-DNA -- shorter, specific sequences of tumor-inducing DNA -- to be cut from the overall Ti plasmid -- the circular segment of DNA responsible for tumorigenesis -- and incorporated into the DNA of the wounded plant cell. Then, the T-DNA induces the plant cell to express certain genes that lead to increased growth and cell division.

In addition to the overproduction of plant growth hormones, opines -- carbon and nitrogen-based metabolites -- are produced by plant cells and used by A. tumefaciens cells to sustain their own bacterial metabolism as they spread their T-DNA to other plant cells. These opines lead to greater reproductive success of A. tumefaciens cells, leading to greater competitive success in plant tumors. Particular genes in the Ti plasmid result in processes -- like the infected plant cells’ production of opines -- that directly increase the likelihood that A. tumefaciens will survive, reproduce, and remain competitive within the plant tumor. These genes are known as “fitness genes” and are the focus of a study conducted at the Universite Paris-Saclay led by Marta Torres. To identify specific A. tumefaciens fitness genes in the Ti plasmid, the investigators used a genome-wide screening technique called transposon sequencing (Tn-Sequencing) that allowed them to find genes that lead to the increased reproductive and competitive success of A. tumefaciens -- also known as fitness genes! This method involved using a transposon library to infect plants and testing out various transposon mutants in different growth conditions to discover which genes were necessary for A. tumefaciens reproductive success. The genes that were necessary were labeled as fitness genes and were used to carry out further experiments in the study. The identified fitness genes were found in several locations across the bacteria’s genetic material, with a few located on At and Ti plasmids and the most located on the circular and linear chromosomes.

Here’s one of the figures from the paper which provides a clear visualization of the locations of these genes:

Image Caption: “Fig 1. Genomic location and examples of Agrobacterium tumefaciens fitness genes involved in plant colonization.” (source)

Let’s take a step back and think about how genes and DNA are organized in bacterial cells. Unlike eukaryotes, bacteria can have circular chromosomes alongside or instead of linear chromosomes -- it depends on the bacteria. In A. tumefaciens, there is a circular chromosome, a linear chromosome and a few plasmids: an At plasmid and a Ti plasmid. There are so many different places for genes to be located in A. tumefaciens, so Figure 1 helps visualize the size of each DNA element and the location of the fitness genes found. In the search for genes useful in the colonization of a wide variety of plants, the researchers found 12 possible gene candidates: 9 are on the circular chromosome, and 3 are on the linear chromosome, labeled in black on Figure 1.

But just because these 12 fitness genes have been identified by Tn-Seq doesn’t mean that these are truly fitness genes! In order to “validate” these different potential fitness genes, the researchers made Knock-Out (KO) mutants of each gene -- where the gene is silenced or “knocked-out” -- to see how its disappearance makes an impact on function. If a KO mutant has no impact on the function of interest, then it probably doesn’t play a huge role in that pathway.

Once these twelve genes were confirmed to be relevant fitness genes, it was time to investigate the pathways and roles of the genes. As it turns out, these genes are involved in 3 main processes:

1. Carbon and nitrogen metabolism

2. Synthesis and repair of DNA, RNA, and proteins

3. Bacterial envelope functions

A. tumefaciens exploits tomato plants for nutrients and energy. For the bacteria’s carbon and nitrogen metabolism, these genes allow A. tumefaciens to exploit plants as a resource by allowing for the breakdown of sugars and amino acids. The metabolic pathways of synthesizing and degrading amino acids are also important to A. tumefaciens fitness -- amino acids less abundant in the host need to be synthesized by the bacteria, while the more abundant amino acids can be readily used as nutrients. Meanwhile, other fitness genes are important in synthesizing nucleotides! Some are also involved in the repair and synthesis of proteins, DNA, and RNA. Genes involved in repair pathways could also be involved in “multi-stress responses” of A. tumefaciens during the process of colonizing tumors and when facing stressors like ultraviolet radiation, acidic pH, and oxidative stress. Finally, some of the fitness genes are related to the bacterial cell envelope.

Once the fitness genes were identified, the researchers investigated whether the KO mutants of the genes of interest were relevant to the virulence or abundance of bacterium and tumors, as seen in Figure 5 below.

Image Caption: “Fig 5. Virulence and abundance in tumors of the constructed Agrobacterium tumefaciens knockout (KO) mutants.” (source)

In this experiment, the tomato plants were infected with the mutants, and after 5 weeks of infection the researchers measured tumor weight by mass and the colonization by colony forming units (CFU). Measuring tumor weight allowed researchers to see how virulent or “aggressive” (as labeled in the figure) the bacteria act as a pathogen -- if they are more aggressive, the plant will make a bigger tumor. Meanwhile, the abundance of A. tumefaciens demonstrates how effective it is in reproducing and staying alive in its host. It’s clear that the 12 fitness genes had differing impacts on how virulent the bacteria was by looking at the tomato tumor weight, while the abundance of A. tumefaciens is also variable depending on the gene according to the CFU counts. Important conclusions drawn by the researchers are that “none of the mutants were nonpathogenic” -- in other words, removing a single gene did not entirely prevent the bacteria from harming the plant. However, the mutants did impact the reproduction and aggressiveness of A. tumefaciens. Some mutants prevented tumors from getting as large as the control, but none removed tumors completely. Similarly, some mutants had a decreased abundance of bacteria compared to the wild type control, so these mutated fitness genes are further shown to play a role in how the bacteria hijacks the host plants.

Understanding which genes are important for reproductive fitness of this plant pathogen can help us combat its pathogenic effects in the agricultural industry. The first step was to identify the fitness genes and the roles they play -- now further studies can move towards understanding the specific processes and role each gene plays in its functional pathway. Perhaps future studies can also take another approach to sequencing fitness genes and find genes that were possibly missed due to the technical limits of Tn-Sequencing. Nonetheless, understanding the molecular mechanisms might allow scientists to better protect agricultural crops from this “promiscuous” plant pathogen. But until that research has been conducted, all we can do is keep our eyes peeled for galls on tomato plants and think about the mischievous prokaryote involved!


About the Authors:


Imaan and Ray are more than co-authors - they are co-dependent! Best friends from first year, they are long-term lab buddies and budding TikTok influencers (follow @camplab on TikTok).


Imaan Moin ‘22 (she/her) is a Biochemistry major from Austin, Texas. She is a member of the Camp Lab, a staff writer for the Mount Holyoke News in the Science & Environmental section, and the 2022 Class President, but she spends her free time running the Camp Lab TikTok account, playing the guitar, and making the people around her laugh. After Mount Holyoke, she will be attending graduate school to study science media production in an effort to make science accessible and entertaining for the larger public.


Ray Stieber ‘22 (they/she) is a Biology major and Sociology minor from Sterling Heights, Michigan. They are a member of the Camp Lab, the 2022 Class Board, and an intern at the local Hitchcock Center for the Environment. In her free time, she enjoys romps in the woods and dabbling in various arts and crafts. After Mount Holyoke, they hope to eventually work in the field of experiential education and youth development to help instill a greater sense of confidence and self-efficacy in our next generation of thinkers and change-makers.