BLGY1211 Agrobacterium transformation
Agrobacterium tumefaciens – a natural genetic engineer
Gram-negative soil bacterium causing Crown Gall disease on most dicots
The tumorous growth results from the expression of genes transferred from the
bacterium to the plant chromosome
In this case the natural horizontal gene transfer – the only known example of DNA
transport between kingdoms
Engineering a biological niche
The transferred segment of DNA (T-DNA) encodes genes with 2 main functions;
synthesis of auxin and cytokinin and production of unusual amino acid derivatives
Major opines are octopine and nopaline
By engineering the plant to produce a food source that only Agrobacterium can use,
it creates a biological niche for itself
Requirements for Plant Transformation
Requirements for Plant Transformation
3 genetic components of Agrobacterium required for the plant transformation
process; T- DNA and Virulence (vir) region, both carried on the Tumour inducing
plasmid, and chromosome virulence genes
T-DNA
T-DNA is delimited by flanking 25bp direct repeats called borders
Any DNA between the borders can be transferred to a plant cell
Unlike transposons, the T-DNA does not encode the products that mediate its
transfer
Vir-region
Encodes the products that mediate T-DNA transfer
A region of the Ti-plasmid approx.. 35kb in length – lies to the left of the T-DNA
Six vir-loci found in all Ti plasmids A, B, C, D, E and G
Some loci encode more than one gene product
Vir genes are tightly regulated – expression only occurs in the presence of
susceptible wounded plant cells
The Inducer
Mass < 1,000 daltons
Organic molecule
First molecules to be identifies were purified from culture media of tobacco cells
Purified by FPLC fractions assayed for vir-induction Acetosyringone
Chromosomal virulence genes
11 chromosomal genes so far identified as necessary for tumour formation
4 of the best characterized are involved in the attachment of the bacteria to the
plant cell – chvA, chvB, pscA and att
Agrobacterium tumefaciens – a natural genetic engineer
Gram-negative soil bacterium causing Crown Gall disease on most dicots
The tumorous growth results from the expression of genes transferred from the
bacterium to the plant chromosome
In this case the natural horizontal gene transfer – the only known example of DNA
transport between kingdoms
Engineering a biological niche
The transferred segment of DNA (T-DNA) encodes genes with 2 main functions;
synthesis of auxin and cytokinin and production of unusual amino acid derivatives
Major opines are octopine and nopaline
By engineering the plant to produce a food source that only Agrobacterium can use,
it creates a biological niche for itself
Requirements for Plant Transformation
Requirements for Plant Transformation
3 genetic components of Agrobacterium required for the plant transformation
process; T- DNA and Virulence (vir) region, both carried on the Tumour inducing
plasmid, and chromosome virulence genes
T-DNA
T-DNA is delimited by flanking 25bp direct repeats called borders
Any DNA between the borders can be transferred to a plant cell
Unlike transposons, the T-DNA does not encode the products that mediate its
transfer
Vir-region
Encodes the products that mediate T-DNA transfer
A region of the Ti-plasmid approx.. 35kb in length – lies to the left of the T-DNA
Six vir-loci found in all Ti plasmids A, B, C, D, E and G
Some loci encode more than one gene product
Vir genes are tightly regulated – expression only occurs in the presence of
susceptible wounded plant cells
The Inducer
Mass < 1,000 daltons
Organic molecule
First molecules to be identifies were purified from culture media of tobacco cells
Purified by FPLC fractions assayed for vir-induction Acetosyringone
Chromosomal virulence genes
11 chromosomal genes so far identified as necessary for tumour formation
4 of the best characterized are involved in the attachment of the bacteria to the
plant cell – chvA, chvB, pscA and att
