Agrobacterium-mediated gene transfer technique is one of the most effective plants transformation methods. This technique has become very significant for both scientific as well as commercial purposes. The plant transformation is basically done to incorporate our gene of interest in the plant, which will generate a new set of plants with desired traits.
Agrobacterium has served a crucial role in the sector of genetic engineering where vector-mediated gene transfer is concerned. This is because the agrobacterium is nature’s most effective genetic engineer, an expert in interkingdom genetic transfer. And thus, a scientist gives major credit to these bacteria for the success of plant transformation techniques.
What is Agrobacterium?
The agrobacterium is an amazing little bacterium present in soil all around the globe. It is a rod-shaped, motile, gram-negative, soil-borne bacteria which mainly infects dicot plants. These bacteria come under the Rhizobiaceae family. They are present in almost all varieties of soil. The optimal temperature for their growth is about 28oC, with a doubling time ranging from 2.5 to 4 hours. You can easily find these bacteria in the plants like sugar beet, nut trees, grape vines, stone fruits etc.
There are three species of agrobacterium:
- Agrobacterium tumefaciens: Crown gall disease
- Agrobacterium rhizogenes: Hairy root disease
- Agrobacterium radiobacter: Avirulent strain.
Content: Agrobacterium Mediated Gene Transfer
- Crown Galls Disease
- T-DNA Transfer and Integration
- Ti Plasmidon
- Agrobacterium Mediated Gene Transfer Steps
Crown Galls Disease
In 1907, i.e. approx 100 years ago, two scientists named Smith and Townsend found that the causative agent behind the crown gall disease is Agrobacterium tumefaciens. Later, with passing years and advancements in genetics, the importance of agrobacterium was recognized. Gradually, it became a powerful tool for botanists to inculcate the desired gene into plants.
When the plant is cut, scratched or wounded, it releases certain phenolic compounds like acetosyringone and hydroxyacetosyringone. The agrobacterium tumefaciens present in the soil senses these chemical compounds as signals. Thereby, it reaches the wounded site and adheres there. Eventually, it infects that wounded area inducing the formation of a plant tumour called crown gall.
The tumour formation occurs only when the A. tumefaciens releases its Ti plasmid (pTi), i.e. tumour inducing plasmid inside the plant body. Mainly, there is a specified segment of the plasmid known as T- DNA, which is actually transferred from the bacteria to the plant host. After the transfer, this T – DNA containing the virulence factor gets integrated into the plant’s genetic material. In this way, the A. tumefaciens naturally delivers its genetic material into the plant cell chromosome via a naturally evolved genetic engineering process.
T-DNA Transfer and Integration
After the entry into the plant host, it basically does two major things:
- The transferred T DNA contains the genetic codes for proteins required for the biosynthesis of growth hormones like auxins and cytokinins. These hormones will induce the unnatural fastened growth of the cells of that particular region, thereby creating a physical home for the bacteria. Here, it flourishes its colony, remaining protected by the harsh external weather.
- Secondly, it induces the plant cells to manufacture more and more amount of nitrogen-rich compounds called opines and agropines. The opines are the derivatives of amino acids, whereas the agropines are sugar.
These compounds are not part of normal plant production, i.e., are neither produced nor metabolized under normal circumstances. But the bacteria use these compounds as their energy source, and thus, it genetically transforms the plant and generates its own biosynthetic machinery to produce the nutrients required by it.
Ti Plasmid
As we discussed above, the formation of crown gall occurs as a result of the transfer, integration, and expression of the T-DNA of the Ti plasmid.
The Ti plasmid, which has an approximate size of about 200 kb, exists as an independent circular replicating unit. However, the size of the transferred DNA (T-DNA) varies from 12 to 24 kb depending upon the type of strain.
The Ti plasmid comprises three major zones:
1. T-DNA Region
2. Virulence Region
3. Opine Region
- T- DNA Region: This region comprises the genes for the production of three tumour determinant oncogene – auxin(aux), cytokinin(cyt), and opine(ops). These genes remain flacked between the left and the right border.
T DNA borders: It is the sequence which is about 24 kb in size at both sides (left and right) of T DNA. These borders also get transferred to the plant cell during transfer.
Note: The right border plays a more significant role in T DNA transfer and tumorigenesis. - Virulence Region: Also known as vir region. This region codes for the genes that facilitate the transfer process. There are around nine different vir gene operons, including vir A, vir B, vir G, vir C1, vir D, D2, and D4, and vir E1 and E2.
Virulence Factor Functions Vir A It is a kinase protein present in the bacterial membrane. It serves as a receptor for phenolic compounds like acetosyringone released by the wounded plant cells. Vir G Gets activated by the vir A. plays the role of transcriptional activator for the vir box. Vir D2 Prevents its 5' from getting cleaved by exonuclease. Vir E 69 kDa protein that targets ss T DNA to the plant cell. Also, it functions as ssbp. Vir C1, C2 Promotes the synthesis of high-efficiency ss T-DNA strands. Forms overdrive sequence and stimulates DNA transfer. Vir D Responsible for virulence activity. Vir D1 Shows topoisomerase activity, assists vir D2 to recognize and excise T DNA within 25bp sequence. Vir B Generates conjugational pore between the plant cell and bacterial cell. Vir B 11 Has ATPase activity, provides energy for DNA transfer. Vir H Detoxify components that may hamper the DNA transfer. - Opine Catabolism Region: This region codes for the biosynthesis of proteins responsible for the uptake and metabolism of opines.
Note: Other than these three, the Ori region is there for the initiation of replication.
Agrobacterium Mediated Gene Transfer Steps
1. Signal Induction to Agrobacterium
The process of T DNA transfer begins when the plant releases some phenolic and sugar compounds from its wounded part. These phenolic compounds are acetosyringone and hydroxyacetosyringone. The agrobacterium recognizes these chemicals as a signal. As a result, it stimulates the chain of biochemical events facilitating the transfer mechanism.
2. Attachment of Agrobacterium to Plant
The bacteria produce polysaccharides (mainly cellulose), which aid its attachment to the plant cells. However, some of the virulence (chv) genes also play a part in this attachment procedure.
3. Production of Virulence Protein
After the stimulation of signals, a sequence of events begins for the production of virulence proteins. Initially, vir A is induced by the starting phenolic signals, which in turn activates vir G. This further induces the expression of corresponding proteins such as D1, D2, E2, B etc.
Note: There are certain sugars involved in the induction of these virulence genes. (Example: xylose, galactose, glucose)
4. Production of T DNA Strand
The virulence proteins D1 and D2 recognize the left and right borders of T DNA. Further, they generate a single T DNA (ss DNA) strand. They also protect and export it to the plant cell. During this entire process, vir D2 remains attached to the ss T DNA.
5. Transfer of T DNA out of Agrobacterium
This leads to the formation of ss T DNA – vir D2 complex, which remains associated with vir g. This whole unit is exported from the bacterial cell. The vir B is responsible for making transport apparatus.
6. Transfer of T DNA into a plant
As soon as the complex, i.e., ss T DNA-D2, crosses the plasma membrane, it gets coated with vir E2. Vir E 2 prevents the T DNA from getting degraded due to the activity of nucleases.
Afterwards, the complex ss T DNA – vir D2 – vir E2 enters the nucleus via nuclear pores. Inside the nucleus, the T DNA integrates into the plant’s chromosomal DNA via illegitimate recombination.
Note: Illegitimate recombination doesn’t rely on the similarity in sequences and thus is different from homologous recombination.
Agrobacterium tumefaciens Uses
The agrobacterium is widely used for the production of transgenic plant species. Undoubtedly, it is one of the most successful approaches for indirect gene transfer methods. With the help of this gene transfer technique, we can easily incorporate our desired gene of interest into the plant. The gene – transferring property of agrobacterium is exploited to transfer the gene for a specific trait.
The tumour-inducing gene from the T plasmid is deliberately removed using exonucleases, and the required gene is inserted using ligase. Finally, this recombinant plasmid is again incorporated into the bacteria. Later, the bacteria is allowed to attach to the wounded part of the plant so that it can transfer its genetic material.
Nowadays, plants with pest resistance, herbicide resistance, virus resistance etc., are successfully produced and marketed.
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