You can create stably transformed plants expressing your gene of interest; be it for the subcellular localization of your protein or simply for the in planta protein expression and purification. Whatever it is, you can do wonders with plant transformation.
Sound difficult? It isn’t. Just like there are millions of microbes that interact with us, there are bacteria and viruses that naturally infect plants. And these can be exploited to help you out.
For example, Agrobacterium-mediated plant transformation makes things easier these days. This transformation is based on the ability of Agrobacterium to transfer a part of its DNA (called T-DNA or transferred DNA) to the host plant genome. Today, I am going to tell you about binary vectors and the biology behind Agrobacterium-mediated plant transformation.
The Story Behind Agrobacterium-mediated Transformation
In short, transformation is mediated by the plant’s bacterial virulence system. Once it is induced, the T-DNA will be transferred to the nucleus of the host plant cell and will be integrated into the plant genome.
There are 3 major genetic components in the Agrobacterium that are required for transformation of plants:
- The T-DNA, which acts like a mobile genetic element, is integrated into the plant genome. However, it cannot act alone like classical transposable elements.
- Depending on the phenotype it causes in plants, the virulence (vir) genes in either the tumor-inducing (Ti) or root-inducing (Ri) plasmid are required for transfer of T-DNA.
- Three chromosomal virulence loci, chvA, chvB, and pscA that are important for the transfer. In wild-type Agrobacterium, the T-DNA and Ti/Ri plasmids are one genetic element.
How can I Transform Plants?
First, create your transformation plasmid by inserting your gene of interest into a specific region containing multiple cloning sites within the T-DNA. Second, transform the plant cells via an injured region. Agrobacterium will recognize signals coming from the exposed plant tissues and will turn on vir gene expression from either the Ti /Ri plasmid. vir genes regulate the processing and transfer of the T-DNA containing your gene. The transferred T-DNA will then integrate into the host genome and express your gene of interest.
But Wait, There are Problems
The Ti/Ri plasmids are very large and low in copy number in Agrobacterium. They are therefore difficult to isolate. They also do not replicate well in E. coli. Furthermore, it is even more difficult to manipulate the plasmids in vitro. For example, the wild type T-DNA region is generally very large and does not contain restriction digestion sites. Inserting your gene becomes a nightmare! How do you clone your gene then?
The Solution: Binary Vectors
The good news is that some genetic sequences within the T-DNA are not essential for transfer, so wild type genes can be deleted and replaced with selectable markers and your gene of interest. More importantly, the vir and T-DNA regions of Ti plasmids can be separated as two replicons and work in trans as long as you place them in the same Agrobacterium. Tada!
This system, in which T-DNA and vir genes are separated into two different replicons, is called the binary system. You call the vector containing the T-DNA region the binary vector and the other one the vir helper (or helper plasmid).
Now, you simply clone your gene of interest into the T-DNA regions. After you verify your construct in E.coli, you can easily mobilize the T-DNA binary vector into the Agrobacterium strain of your choice containing the appropriate vir helper. That’s all!! You are ready to transform your plants.
You can manipulate binary vectors quite a lot and it’s a great help in plant transformation. Here are some basics that you should know about binary vectors.
Copy & Paste: Insertion Site for the Gene of Interest
Inserting your gene of interest in the T-DNA is the most important part. Fortunately, most of the options available for cloning in E. coli can also be used in Argobacterium. For example, you can insert your gene using restriction endonucleases. In many vectors, multiple cloning sites are already flanked by promoters and poly-A tail addition signals. Some vectors contain expression cassette tags to produce fluorescent proteins. These days you can also get binary vectors that have Gateway sites, which make gene insertion easier. I personally have had a good experience using LIC (ligase independent cloning) for cloning. Below are some more tips on how to clone and maintain the plasmids that contain the T-DNA sequence in E.coli:
Compatible Start: Origin(s) of Replication
Since you have to maintain your plasmid clones in E.coli before moving to Agrobacterium to make the whole cloning procedure easier, you need an origin of replication (ori) that allows the maintenance of the plasmid in a wide host range. Sometimes, one origin of replication that functions in Agrobacterium (e.g. those from Ri plasmids) will not work in E.coli. In that case, you have to make sure you have another functional origin of replication added to the vector.
Transfer it all: T-DNA Borders
To define and transfer the T-DNA region, you need left and right border repeat sequences. These 25bp long repeat sequences are highly conserved in all Ti and Ri plasmids. The polarity of the T-DNA transfer is from the right border (RB) to the left border (LB). After entering the plant, the T-strand is frequently chewed back, mainly because of exonucleases. But fortunately, the 5` end being linked to a protein responsible for T-DNA processing and transfer, stays protected and therefore loss of nucleotides at this end is minimal (only a few nucleotides). However, the end remains unprotected and you can lose several hundred nucleotides. So the best place to insert your gene is near the right border.
Choose the Right Clone & Selection Markers
As you have to clone your gene of interest first in E.coli and later in Agrobacterium, you need to think carefully about your selection markers. For selection in E. coli, use an antibiotic resistance gene. Many available vectors have a kanamycin resistant gene as the bacterial selection marker.
Plant-Specific Selectable Marker Gene
The easiest way to select your successfully transformed plants is to make them resistant to a specific antibiotic or herbicide. Commonly used antibiotics and herbicides include kanamycin, hygromycin and phosphinothricin/gluphosinate. Remember that some plant species can only tolerate low levels of kanamycin; so use the minimum concentration of the antibiotic that will kill the non-transformed tissues completely but keep your desired plant growing. These selectable markers are generally placed near the LB (left border).
Last but not Least
It is very important to carefully match your choice of binary vector with the respective vir helper Agrobacterium. Some binary vectors, such as pGreen based vectors are modified to give you a relatively small plasmid for efficient plasmid proliferation, flexible transformation and extensive multiple cloning site. But, these vectors cannot replicate in Agrobacterium if these strains do not contain another plasmid, pSoup, which provides replication functions in trans for pGreen.
Many Agrobacterium strains express genes for specific resistance. Therefore, you cannot simply use a binary vector with the same selection marker. For example, pBIN19 based T-DNA binary vectors often utilize kanamycin resistance for bacterial selection; you cannot easily use it in A. tumefaciens EHA101 as it is also kanamycin resistant. So, remember to check the strains and the binary vectors for their resistance to antibiotics before pairing them. You can find a list of different available binary vectors and strains online.
Binary vectors are a real blessing for us. You will see.