Plants vs. Bacteria đź¦
Do you remember the classic game of Plants vs Zombies? Where you, the player, had to place different types of plants and fungi, each with their own unique offensive or defensive capabilities, around a house, in order to stop a horde of zombies from reaching their house.
Now, imagine that, but the house is a plant, there are no plants/fungi to defend the house, and the zombies are actually bacteria. Instead of Plants vs. Zombies, it’s Plants vs. Bacteria instead.
Now let’s turn that into a reality. Introducing Agrobacterium tumefaciens. Agrobacterium tumefaciens is the causal agent of crown gall disease, which is essentially a plant disease. The bacterium causes abnormal growths (galls) on roots, twigs, and branches of shrubs, primarily in the rose family. The bacterium stimulates the rapid growth of plant cells which results in the galls.
Agrobacterium tumefaciens grows optimally at 28°C. The doubling time can range between 2.5 and 4 hours, depending on several factors. Once the temperatures reach above 30°C, Agrobacterium tumefaciens begins to experience a heat shock, which could result in cell division errors.
Method of infection
Agrobacterium tumefaciens infects the plant through its Ti plasmid. The Ti plasmid integrates a segment of its DNA into the chromosomal DNA of its host plant cells. A. tumefaciens has flagella (a hairlike appendage that protrudes from a wide range of microorganisms) that allow it to swim through the soil towards photoassimilates (one of many biological compounds formed by assimilation using light-dependent reactions) that accumulates in the rhizosphere (the soil zone surrounding the plant roots where the biological and chemical features of the soil are influenced by the roots) around roots. Some strains may chemotactically (the movement of an organism or entity in response to a chemical stimulus) move towards chemical exudates from plants, such as sugars, which indicate the presence of a wound in the plant through which bacteria may enter.
A tumour-inducing plasmid (Ti plasmid) is a plasmid found in pathogenic species of Agrobacterium, including A. tumefaciens, A. rhizogenes, A. rubi, etc.
The symptoms are caused by the insertion of a small segment of T-DNA, from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes could be engineered by the use of Agrobacterium for the delivery of sequences hosted in the T-DNA binary vectors.
A minimum of 25 vir genes on the Ti plasmid is needed for tumour induction. The bacteria attachment is a two-step process. After an initial weak and reversible attachment, the bacteria synthesize cellulose fibrils that anchor them to the wounded plant cell to which they were attracted. Four main genes are involved in this process: chvA, chvB, pscA, and att. The products of the first three genes are involved in the actual synthesis of the cellulose fibrils. These fibrils also anchor the bacteria to each other, helping to form a microcolony (a colony of bacteria visible only under a low-power microscope, and growing under insignificant conditions).
Virulence genes: The degree of virulence typically relates to the ability of the pathogen to multiply within the host, and may be affected by other factors. To be virulent, the bacterium contains a Ti plasmid, which contains the T-DNA and all the genes necessary to transfer it to the plant cell.
The main genes: Functional chvA and chvB genes are required for attachment of A. tumefaciens to plant cells, an early step in crown gall tumour formation. The att gene expression is transcriptionally induced by leafy gall extracts, but not by extracts of uninfected plants.
VirC, the most important virulent protein, is necessary for the recombination of illegitimate recombination. It selects the section of the DNA in the host plant that will be replaced and it cuts into this strand of DNA.
Illegitimate recombination is the process by which two unrelated double-stranded segments of DNA are joined.
After the production of cellulose fibrils, a calcium-dependent outer membrane protein called rhicadhesin is produced, which also helps in sticking the bacteria to the cell wall. Homologues of this protein could be found in other rhizobia. Currently, there are numerous reports on the standardization of protocol for Agrobacterium-mediated transformations. The effects of different conditions, such as infection time, have been studied in soybean.
Possible plant compounds that initiate Agrobacterium to infect plants cells:
- Gallic acid — Found in witch hazel, tea leaves, oak bark, etc. 🌳
- Protocatechuic acid — A major metabolite of antioxidant polyphenols found in green tea. 🍵
- Vanillin — The primary component of the extract of the vanilla bean. Synthetic vanillin is used more often than natural vanilla extract as a flavouring agent in foods, beverages, and pharmaceuticals. 🍨
Formation of the T-pilus
To transfer the T-DNA into the plant cell, A. tumefaciens uses a type IV secretion mechanism, involving the production of a T-pilus. When substances are detected, a signal transduction event activates the expression of 11 genes within the VirB operon, which is responsible for the formation of the T-pilus.
Type IV secretion mechanism: A protein complex found in prokaryotes, which is used to transport DNA, proteins, or effector molecules from the cytoplasm to the extracellular space beyond the cell.
An operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter.
The pro-pilin is formed first. This is a polypeptide of 121 amino acids, which requires processing by the removal of 47 residues to form a T-pilus subunit. The subunit is circularized by the formation of a peptide bond between the two ends of the polypeptide.
Products of the VirB genes act at the bacterial surface. On the surface, they play an important role in directing the T-DNA transfer to plant cells.
Genes in the T-DNA
Hormones — To cause gall formation, the T-DNA encodes genes for the production of auxin, via the IAM pathway. This biosynthetic pathway is not used in many plants for the production of auxin, so it means the plant has no molecular means of regulating it and auxin will be produced constitutively. Genes for the production of cytokinins are also expressed, which stimulates cell proliferation and gall formation.
Opines — The T-DNA contains genes for encoding enzymes that cause the plant to create specialized amino acid derivatives that the bacteria can metabolize, called opines. Opines are a class of chemicals that serve as a source of nitrogen for A. tumefaciens, but not for most other organisms. The specific type of opine produced by A. tumefaciens C58 infected plants is nopaline.
Nopaline: A chemical compound derived from the amino acids glutamic acid and arginine.
The first fully sequenced pathovar was first isolated in a cherry tree crown gall and is known as A. fabrum C58. The genome of A. fabrum C58 consists of a circular chromosome, 2 plasmids, and a linear chromosome. The presence of a covalently bonded circular chromosome is common to bacteria, with a few exceptions. However, the presence of both a single circular chromosome and a single linear chromosome is unique to a group in this specific genus.
Genus: A group of closely related species.
Enough about the natural uses, how is it used in research?
The Asilomar Conference established a widespread agreement that recombinant techniques were insufficiently understood and needed to be tightly controlled.
The Asilomar Conference on Recombinant DNA was an influential conference organized by Paul Berg to discuss the potential biohazards and regulation of biotechnology. It was held in February 1975 in Asilomar State Beach, in California. A group of around 140 professionals (biologists, lawyers, physicians, etc) participated in the conference to draw up voluntary guidelines to ensure the safety of recombinant DNA technology.
The DNA transmission capabilities of A. tumefaciens have been explored in biotechnology as a method of inserting foreign genes into plants. Marc Van Montagu and Jozef “Jeff” Schell discovered the gene transfer mechanism between A. tumefaciens and plants, which resulted in the development of methods to modify the bacterium into an efficient delivery system for genetic engineering in plants. The plasmid T-DNA that is transferred to the plant is an ideal apparatus for genetic engineering. This is done by cloning the desired gene sequence into T-DNA binary vectors that will be used to deliver a sequence of interest into eukaryotic cells. It was agreed that “Asilomar-like” protections were needed in plant technologies as well. This process has been performed using the firefly luciferase gene to produce glowing plants. This luminescence has been useful when studying the chloroplast function and when studying how it is used as a reporter gene.
Luciferase gene: Enzymes that produce light when they oxidize their substrate.
Reporter gene: A gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants.
It is also possible to transform Arabidopsis thaliana by dipping flowers into a broth of A. tumefaciens: the seed produced will be transgenic (when one or more DNA sequences from another species have been introduced by artificial means). Under laboratory conditions, the T-DNA has also been transferred to human cells, demonstrating the diversity of insertion applications.
Arabidopsis Thaliana, also known as the mouse-ear cress, is a small flowering plant that is normally found along the shoulders of roads and in disturbed land. The plant is native to both Eurasia and Africa, and is naturalized in Canada and the USA, among other countries. It’s a popular organism used in plant biology and genetics. A. thaliana has a comparatively small genome, with around 135 megabase pairs. It was the first plant to have its genome sequenced, and is often used to understand the molecular biology of many plant traits. 🌻
The mechanism by which A. tumefaciens inserts materials into the host cell is by a type IV secretion system which is very similar to mechanisms used by pathogens to insert materials (usually proteins) into human cells by type III secretion. It also employs a type of signalling conserved in many Gram-negative bacteria called quorum sensing. This also makes A. tumefaciens an important topic of medical research.
Natural genetic transformation
There’s also the potential use of natural genetic transformation. Natural genetic transformation in bacteria is a sexual process involving the transfer of DNA from one cell to another through the intervening medium, and the integration of the donor sequence into the recipient genome by homologous recombination. A. tumefaciens can undergo a natural transformation in soil without any specific physical or chemical treatment.
What’s The Big Deal?
In short, Agrobacterium tumefaciens’s role in the production of transgenic plants is using the genes from A. tumefaciens, and inserting them into plant DNA to give the plant different desired traits. These plants are better known as genetically modified organisms (GMO’s), or in this case, genetically modified crops. Genetically modified crops are pest-resistant, drought-resistant, disease-resistant, resistant to herbicides, and many more. 👩‍🔬
👋 Hey, I’m Aneeva! I’m a 16-year-old genomics + gene editing enthusiast, working to understand and research how we could use these 2 technologies together to combat climate-driven issues. If you want to follow along on my journey, subscribe to my personal newsletter! You can also find me on LinkedIn and Twitter.
