Transfer DNA binary system

A transfer DNA 'binary system' is a pair of plasmids consisting of a T-DNA binary vector and a vir helper plasmid. The two plasmids are used together to produce genetically modified plants. They are artificial vectors that have been derived from the naturally occurring Ti plasmid found in bacterial species of the genus Agrobacterium, such as A. tumefaciens. The binary vector is a shuttle vector, so-called because it is able to replicate in multiple hosts.
Systems in which T-DNA and vir genes are located on separate replicons are called T-DNA binary systems. T-DNA is located on the binary vector. The replicon containing the vir genes became known as the vir helper plasmid. The vir helper plasmid is considered disarmed if it does not contain oncogenes that could be transferred to a plant

Background of ''Agrobacterium''-mediated transformation

The transfer DNA binary system is derived from the naturally occurring Agrobacterium tumefaciens infection mechanism of plants. Agrobacterium is a parasitic bacterium that naturally occurs in soils and infects plant cells to utilize their biological processes and machinery, integrating its own genetic material into the genome of the plant cell to produce resources that support its survival.

Ti plasmid

Agrobacterium contains a plasmid, a circular piece of DNA, called the "Tumor-inducing plasmid". The Ti plasmid contains the following elements:
The "T-DNA" region: The T-DNA region is the section of the plasmid that becomes integrated into the genome of the host plant cell. Agrobacterium utilizes the plant's transcription and translation machinery to express the genes located within the T-DNA region. It contains the following elements:

Left and right borders : The left and right borders define the boundaries of the T-DNA region. The RB acts as a starting point for the genetic transfer and the LB acts as an endpoint. The borders are recognized and cleaved by the endonucleases encoded by the virD gene.
Auxin and cytokinin genes: These genes encode and force the plant cell to produce auxin and cytokinin, plant hormones that promote cell division and growth. By inducing cell division, the infected cells reproduce more rapidly, increasing the population of cells containing the T-DNA, consequently producing more opines. This rapid cell division leads to the formation of tumors called crown galls.
Opine genes: The opine genes encode and force the plant cell to express enzymes that synthesize opine, a carbon- and nitrogen-rich compound that acts as a food source for Agrobacterium. After synthesis, the opines are secreted into the intercellular space and surrounding environment, allowing them to be taken-up by nearby Agrobacterium. The most common opines produced by plant cells infected with Agrobacterium DNA are nopaline and octopine.

Opine catabolism genes: The opine catabolism genes encode elements of the Agrobacterium opine catabolism pathway. This pathway allows the bacterium to break down and use the opine as an energy source. Only members of the Agrobacterium genus are able to metabolize opine, providing them with a competitive advantage over other soil microbes.
Vir genes cassette: The vir genes, or "virulence genes", encode elements that aid in the transfer of T-DNA from the Ti plasmid into the plant cell genome. There are 6 vir operons involved in the transfer of T-DNA: virA, virB, virG, virC, virD, and virE.
Ori: The ori is the "origin of replication", a site on the plasmid at which the two DNA strands begin to unwind to allow for DNA replication during cell division.
Bacteria are prokaryotic organisms and plants are eukaryotic organisms. Mechanisms of and machinery involved in gene expression differs in prokaryotic and eukaryotic organisms. Agrobacterium has evolved to contain eukaryotic gene elements in the T-DNA region which allows for the genes encoded in the region to be expressed by the plant cells. The remaining Ti plasmid adheres to regular prokaryotic processes.
In genetic engineering of plants, the auxin, cytokinin, and opine genes are replaced with a "gene of interest", the gene to be inserted into the plant. The opine catabolism genes are also removed. The transfer of the gene of interest from the Agrobacterium to the plant cell occurs via the natural infection mechanism of the bacterium.

Infection mechanism

The natural mechanism of Agrobacterium infection of plant cells is mediated via the 6 vir genes located on the Ti plasmid. The process of infection occurs in 2 general steps:

Plant cell recognition and activation of vir gene expression: When a plant cell wall becomes damaged, phenolic compounds such as acetosyringone are released into the surrounding environment. These compounds bind to the cross-membrane receptor kinases located on the membrane of nearby Agrobacterium, signalling to the bacterium that a damaged plant cell is close by. The receptor kinases are encoded by virA. The binding of the phenolic compound to the extracellular domain of the receptor causes the intracellular domain to become phosphorylated. The phosphate group moves from the receptor to a free-floating effector protein encoded by virG. The phosphorylated effector becomes activated and binds to cis-elements within the promoter region of the remaining 4 vir genes, inducing their expression.
Transfer of T-DNA: The virD gene expresses an endonuclease that recognizes the RB sequence. The endonuclease cuts one strand of the RB and covalently binds to the 5' cut end of the T-DNA strand. The break in the DNA triggers the cell's natural DNA repair mechanisms, which begin to synthesize a new T-DNA strand beginning at the 3' cut end of the RB. This new strand pushes the original T-DNA strand away. As this occurs, single-stranded DNA binding proteins, encoded by virE, bind along the length of the original T-DNA to stabilize it and prevent it from being degraded. Another virD endonuclease recognizes and cuts a strand in the LB, releasing the original T-DNA strand from the plasmid. The virB expresses proteins that form a transport channel between the Agrobacterium and plant cells, acting as a physical bridge along which the T-DNA moves from one organism to the other. The virC proteins aid in recruiting virD endonucleases to the borders and directing the released T-DNA to the virB transport channel.

The T-DNA then integrates into a random location within the plant cell genome.
The table below is a summary of the vir genes and their function:

Operon	Number of Genes	Type of Protein	Expression Activation	Basal Expression Level	Induced Expression Level	Functional Location	Function
VirA	virA	Cross-membrane sensor kinase		Low		Membrane	Recognizes the phenolic compounds released by nearby damaged plant cells; Autophosphorylates; Transfers phosphate group to virG effector.
VirB	virB1-11		Induced by virA/G		High	Membrane	Form the Type 5 secretion system transfer channel between the Agrobacterium and plant cell.
VirC	virC1, virC2		Induced by virA/G		High	Cytoplasm	Aid in recruiting virD endonucleases to the left and right borders; Direct the T-DNA to the transport channel. VirC1 binds to the overdrive sequence, a region near the RB, to aid in T-DNA processing.
VirD	virD1-5	Helicase ; Nuclease	Induced by virA/G		High	Nucleus	VirD2 recognizes and nicks the left and right borders; Covalently binds to the 5' end of the T-DNA; Contains a nuclear localization signal to direct the T-DNA into the plant nucleus.
VirE	virE1-3	Effector	Induced by virA/G		High	Nucleus	VirE1 prevents virE2 proteins from aggregating with themselves. VirE2 ssDNA binding proteins coat the length of the T-DNA; Stabilize T-DNA to prevent degradation.
VirG	virG	Transcriptional regulator	Induced by positive feedback loop of virA/G	Low	High	Cytoplasm	Becomes activated via phosphorylation by virA; Induces expression of vir genes.

Components of the binary vector system

Binary vector

A binary vector is used in plant genetic engineering to transfer foreign genes into plant cells. The reason for having two separate plasmids is because it is easier to clone and manipulation of genes of interest in E. coli using the T-DNA vector because it is small and easy to work with, while the vir genes remain in Agrobacterium on the helper plasmid to help with plant transformation. The components of the Binary Vector include:

Left and right borders: The binary vector also contains left and right borders, which define the boundaries of the T-DNA region that will be transferred into the plant genome. These border sequences serve as recognition sites for the endonuclease enzymes of Agrobacterium, which nick and cleave the DNA to allow transfer into the plant cell nucleus.
* Inside the T-DNA region, several functional elements are present. First, it contains the gene of interest which encodes the functional protein that researchers aim to introduce into the plant. Secondly fused to the gene of interest is a reporter gene which enable visualization and quantification of successful gene integration in the plant. The reporter genes used could be E. coli lac Z gene, which produces a blue color upon staining, and GFP, which fluoresces under UV light. A promoter is also introduced which drives the expression of the gene of interest within the plant cells. Commonly used promoters include the CaMV 35S promoter and the UBQ10 promoter for constitutive expression.Finally, a terminator sequence signals the end of transcription, ensuring that the gene is expressed properly and consistently in the plant cell
* Inside the T-DNA there is also the plant selectable marker. This marker allows for the selection of plants that have successfully integrated the trans-gene and T-DNA into their nuclear genome. When transformed plants are exposed to a marker such as a herbicide or an antibiotic, only those that have successfully integrated the transgene and the selectable marker gene will survive and grow. Any cells that have not integrated the transgene will be sensitive to the marker and will not survive under selective conditions.
Binary vectors also contain elements necessary for bacterial replication and selection outside of the T-DNA region. A bacterial selectable marker allows for the selection of E. coli cells that have successfully taken up the binary plasmid during cloning and amplification. Examples of bacterial selectable markers include genes for antibiotic resistance such as ampicillin and kanamycin.
The vector also includes an origin of replication for E. coli, which ensures that the plasmid is recognized by the bacterial replication machinery and replicated each time the E. coli cells divide. Additionally, the binary vector contains an origin of replication for Agrobacterium, which is required to ensure that the plasmid can replicate within Agrobacterium cells. After cloning and amplification in E. coli, the plasmid is transferred into Agrobacterium for plant transformation. The origin of replication for Agrobacterium ensures that the plasmid is maintained and as the Agrobacterium cells divide, making it available for T-DNA transfer during the plant infection process.

The combination of these components makes binary vectors versatile and effective tools for plant genetic engineering, allowing researchers to modify and amplify plasmids efficiently in E. coli before introducing them into Agrobacterium for plant transformations.
Representative series of binary vectors are listed below.

Series	Vector	Year	GenBank accession	Size	Autonomous replication in Agrobacterium	Reference
pBIN	pBIN19	1984		11777	Yes
pPVP	pPZP200	1994		6741	Yes
pCB	pCB301	1999		3574	Yes
pCAMBIA	pCAMBIA-1300	2000		8958	Yes
pGreen	pGreen0000	2000		3228	No
pLSU	pLSU-1	2012		4566	Yes
pLX	pLX-B2	2017		3287	Yes