The transfer DNA (abbreviated T-DNA) is the transferred
DNA of the
tumor-inducing (Ti) plasmid of some species of bacteria such as ''
Agrobacterium tumefaciens
''Agrobacterium radiobacter'' (more commonly known as ''Agrobacterium tumefaciens'') is the causal agent of crown gall disease (the formation of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Sympto ...
'' and ''
Agrobacterium rhizogenes
''Rhizobium rhizogenes'' (formerly ''Agrobacterium rhizogenes'') is a Gram-negative soil bacterium that produces hairy root disease in dicotyledonous plants. ''R. rhizogenes'' induces the formation of proliferative multiple-branched adventitious ...
(actually an Ri plasmid)''. The T-DNA is transferred from bacterium into the host plant's
nuclear
Nuclear may refer to:
Physics
Relating to the nucleus of the atom:
* Nuclear engineering
*Nuclear physics
*Nuclear power
*Nuclear reactor
*Nuclear weapon
*Nuclear medicine
*Radiation therapy
*Nuclear warfare
Mathematics
*Nuclear space
*Nuclear ...
DNA
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the ''vir'' genes of the Ti plasmid.
The bacterial T-DNA is about 24,000 base pairs long and contains plant-expressed
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s that code for
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
s synthesizing
opines
Opines are low molecular weight compounds found in plant crown gall tumors or hairy root tumors produced by pathogenic bacteria of the genus ''Agrobacterium'' and ''Rhizobium''. Opine biosynthesis is catalyzed by specific enzymes encoded by gene ...
and
phytohormone
Plant hormone (or phytohormones) are signal molecules, produced within plants, that occur in extremely low concentrations. Plant hormones control all aspects of plant growth and development, from embryogenesis, the regulation of organ size, pat ...
s. By transferring the T-DNA into the plant genome, the bacterium essentially reprograms the plant cells to grow into a tumor and produce a unique food source for the bacteria. The synthesis of the plant hormones
auxin
Auxins (plural of auxin ) are a class of plant hormones (or plant-growth regulators) with some morphogen-like characteristics. Auxins play a cardinal role in coordination of many growth and behavioral processes in plant life cycles and are essenti ...
and
cytokinin
Cytokinins (CK) are a class of plant hormones that promote cell division, or cytokinesis, in plant roots and shoots. They are involved primarily in cell growth and differentiation, but also affect apical dominance, axillary bud growth, and le ...
by enzymes encoded in the T-DNA enables the plant cell to overgrow, thus forming the
crown gall tumor
''Agrobacterium'' is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. '' Agrobacterium tumefaciens'' is the most commonly studied species in this genus. ''Agrobacterium'' i ...
s typically induced by ''Agrobacterium tumefaciens'' infection.
''Agrobacterium rhizogenes'' causes a similar infection known as
hairy root disease. The
opines
Opines are low molecular weight compounds found in plant crown gall tumors or hairy root tumors produced by pathogenic bacteria of the genus ''Agrobacterium'' and ''Rhizobium''. Opine biosynthesis is catalyzed by specific enzymes encoded by gene ...
are
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
derivatives used by the bacterium as a source of carbon and energy. This natural process of
horizontal gene transfer
Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between Unicellular organism, unicellular and/or multicellular organisms other than by the ("vertical") transmission of DNA from parent to offsprin ...
in plants is being utilized as a tool for fundamental and applied research in plant biology through ''Agrobacterium tumefaciens'' mediated foreign gene transformation and insertional mutagenesis.
Plant genomes can be engineered by use of ''
Agrobacterium
''Agrobacterium'' is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. '' Agrobacterium tumefaciens'' is the most commonly studied species in this genus. ''Agrobacterium'' i ...
'' for the delivery of sequences hosted in
T-DNA binary vectors.
Mechanism of transformation in nature
The infection process of T-DNA into the host cell and integration into its nucleus involve multiple steps. First, the bacteria multiply in the wound sap before infection and then attach to the plant cell walls. The bacterial virulence genes expression of approximately 10
operon
In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splic ...
s is activated by perception of phenolic compounds such as
acetosyringone
Acetosyringone is a phenolic natural product and a chemical compound related to acetophenone and 2,6-dimethoxyphenol. It was first described in relation to lignan/phenylpropanoid-type phytochemicals, with isolation from a variety of plant source ...
emitted by wounded plant tissue and follows cell-cell contact. Then this process proceeds with the
macromolecular
A macromolecule is a very large molecule important to biophysical processes, such as a protein or nucleic acid. It is composed of thousands of covalently bonded atoms. Many macromolecules are polymers of smaller molecules called monomers. The ...
translocation from ''Agrobacterium'' to cytoplasm of host cell, transmission of T-DNA along with associated proteins (called T-complex) to the host cell nucleus followed by disassembly of the T-complex, stable integration of T-DNA into host plant
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
, and eventual expression of the transferred
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s. The integration of T-DNA into a host genome involves the formation of a single-stranded nick in the DNA at the right border of the Ti plasmid. This nick creates a region of single stranded DNA from the left border of the T-DNA gene over to the right border which was cut. Then, single stranded binding proteins attach to the single stranded DNA. DNA synthesis displaces the single stranded region and then a second nick at the left border region releases the single stranded T-DNA fragment. Further this fragment can be incorporated into a host genome.
''Agrobacterium'' has been known to evolve a control system that uses plant host factors and cellular processes for several pathways of host-plant defense response to invade the host cell nucleus. For the integration of T-DNA into the target host genome, ''Agrobacterium'' carries out multiple interactions with host-plant factors.
To interact with host plant proteins many ''Agrobacterium'' virulence proteins encoded by vir genes. ''Agrobacterium'' ''vir'' gene expression occurs via the VirA-VirG sensor that results in generation of a mobile single-stranded T-DNA copy (T-strand). A processed form of VirB2 is the major component of the T-complex that is required for transformation. VirD2 is the protein that caps the 5′ end of the transferred T-strand by covalent attachment and is transported to the host cell cytoplasm. VirE2 is the single-stranded DNA binding protein that presumably coats the T- strand in the host cytoplasm by cooperative binding. It is then directed into the nucleus via interactions with the host cell proteins such as importin a, bacterial VirE3, and dynein-like proteins. Several other bacterial virulence effectors like VirB5, VirB7 (the minor components of the T-complex), VirD5, VirE2, VirE3, and VirF that may also interact with proteins of host plant cells.
Uses in biotechnology
''Agrobacterium''-mediated T-DNA transfer is widely used as a tool in
biotechnology
Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used b ...
. For more than two decades, ''Agrobacterium tumefaciens'' has been exploited for introducing genes into plants for basic research as well as for commercial production of
transgenic crops
Genetically modified crops (GM crops) are plants used in agriculture, the DNA of which has been modified using genetic engineering methods. Plant genomes can be engineered by physical methods or by use of ''Agrobacterium'' for the delivery of ...
. In
genetic engineering
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
, the tumor-promoting and opine-synthesis genes are removed from the T-DNA and replaced with a gene of interest and/or a selection marker, which is required to establish which plants have been successfully transformed. Examples of selection markers include neomycin phosphotransferase, hygromycin B phosphotransferase (which both phosphorylate antibiotics) and
phosphinothricin acetyltransferase
Phosphinothricin acetyltransferase (, ''PAT'', ''PPT acetyltransferase'', ''Pt-N-acetyltransferase'', ''ac-Pt'') is an enzyme with systematic name ''acetyl-CoA:phosphinothricin N-acetyltransferase''. This enzyme catalyses the following chemical re ...
(which acetylates and deactivates
phosphinothricin
Glufosinate (also known as phosphinothricin and often sold as an ammonium salt) is a naturally occurring broad-spectrum herbicide produced by several species of '' Streptomyces'' soil bacteria. Glufosinate is a non-selective, contact herbicide, ...
, a potent inhibitor of
glutamine synthetase
Glutamine synthetase (GS) () is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine:
Glutamate + Adenosine triphosphate, ATP + NH3 → Glutamine + Ad ...
) or a
herbicide
Herbicides (, ), also commonly known as weedkillers, are substances used to control undesired plants, also known as weeds.EPA. February 201Pesticides Industry. Sales and Usage 2006 and 2007: Market Estimates. Summary in press releasMain page fo ...
formulations such as Basta or Bialophos.
Another selection system that can be employed is usage of metabolic markers such as phospho-mannose isomerase. ''Agrobacterium'' is then used as a vector to transfer the engineered T-DNA into the plant cells where it integrates into the plant genome. This method can be used to generate
transgenic plant
Genetically modified plants have been engineered for scientific research, to create new colours in plants, deliver vaccines, and to create enhanced crops. Plant genomes can be engineered by physical methods or by use of '' Agrobacterium'' for the ...
s carrying a foreign gene. ''Agrobacterium tumefaciens'' is capable of transferring foreign DNA to both
monocotyledons
Monocotyledons (), commonly referred to as monocots, (Lilianae ''sensu'' Chase & Reveal) are grass and grass-like flowering plants (angiosperms), the seeds of which typically contain only one embryonic leaf, or cotyledon. They constitute one of t ...
and
dicotyledonous
The dicotyledons, also known as dicots (or, more rarely, dicotyls), are one of the two groups into which all the flowering plants (angiosperms) were formerly divided. The name refers to one of the typical characteristics of the group: namely, t ...
plants efficiently while taking care of critically important factors like the genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of ''Agrobacterium'', selection marker genes and selective agents, and various conditions of tissue culture.
The same procedure of T-DNA transfer can be used to disrupt genes via
insertional mutagenesis In molecular biology, insertional mutagenesis is the creation of mutations of DNA by addition of one or more base pairs. Such insertional mutations can occur naturally, mediated by viruses or transposons, or can be artificially created for researc ...
.
Not only does the inserted T-DNA sequence create a mutation but its insertion also 'tags' the affected gene, thus allowing for its isolation as T-DNA flanking sequences. A reporter gene can be linked to the right end of the T-DNA to be transformed along with a plasmid replicon and a selectable antibiotic (such as
hygromycin Hygromycin may refer to either of two chemically dissimilar antibiotics produced by the bacterium '' Streptomyces hygroscopicus'':
* Hygromycin A
* Hygromycin B, an aminoglycoside that kills bacteria, fungi and higher eukaryotic
Eukaryotes ...
)-resistance gene and can explicit approximately 30% of average efficiency having successful T-DNA inserts induced
gene fusions in ''Arabidopsis thaliana''.
Reverse genetics
Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proce ...
involves testing the presumed function of a gene that is known by disrupting it and then looking for the effect of that induced mutation on the organismal phenotype. T-DNA tagging mutagenesis involves screening of populations by T-DNA insertional mutations. Collections of known T-DNA mutations provide resources to study the functions of individual genes, as developed for the model plant ''
Arabidopsis thaliana
''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small flowering plant native to Eurasia and Africa. ''A. thaliana'' is considered a weed; it is found along the shoulders of roads and in disturbed land.
A winter a ...
'' .
Examples of T-DNA insertion mutations in ''Arabidopsis thaliana'' include those associated many classes of phenotypes including seedling-lethals, size variants, pigment variants, embryo-defectives, reduced-fertility, and morphologically or physiologically aberrant plants
See also
*
Transfer DNA binary system A transfer DNA (T-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 (thus ''binary'') to produce genetically modified plants. They are artificial vectors th ...
References
Further reading
*
{{DEFAULTSORT:T-Dna
Biotechnology
Bacterial plant pathogens and diseases
DNA mobile genetic elements