Agrobacterium Mediated Gene Transfer In Plants

AAMIR RAINA M Sc, Ph. D (Genetics and Plant Breeding)

Gene Transfer Recombinant DNA technology is based on

the insertion of a DNA fragment (Gene of interest) into a suitable cloning vector and then its introduction into a suitable host to propagate the recombinant DNA.

Gene Carrier Vehicle If a gene is to be introduced into a host

cell,a carrier molecule that can transport the gene into the host cell is required.

Such a molecule is called a cloning vehicle or carrier molecule or a vector.

Gene Carrier Vehicle Following are few gene carrier vehicles. Plasmids Bacteriophages Cosmids Viruses Virus vector for animal cells. Simian Virus

Making Transgenic Plants—Why?1. Study gene function and regulation 2. Making new organismic tools for other

fields of research3. Curing genetic diseases4. Improving agriculture and related raw

materials5. New sources of bioengineered drugs.

Must get DNA:1. into the cells2. integrated into the genome (unless using transient

expression assays)3. expressed (everywhere or controlled)

For (1) and (2), two main approaches for plants:1. Agrobacterium - mediated gene transfer2. Direct gene transfer

For (3), use promoter that will direct expression when and where wanted – may also require other modifications such as removing or replacing introns.

Genetic Engineering of Plants

Agrobacteria soil bacteria, gram-negative, related to Rhizobia species:

It invades many dicot plants when they are injured at the soil level and causes crown gall disease

Agrobacterium tumefaciens-Nature’s smallest genetic engineer

The ability to cause crown gall disease is associated with the presence of Ti Plasmid within bacterial cell

A remarkable feature of Ti Plasmid is that after infection part of molecule is integrated into plant chromosomal DNA. This segment called T-DNA is between 15 and 30 kb in size

It is maintained in a stable form in plant cellc and is passed onto daughter cells as an integral part of chromosome

Agrobacterium tumefaciens-Nature’s smallest genetic engineer

Crown galls caused by A. tumefaciens on nightshade.

More about Galls: http://waynesword.palomar.edu/pljuly99.htmhttp://kaweahoaks.com/html/galls_ofthe_voaks.html

the species of choice for engineering dicot plants; monocots are generally

resistant some dicots more resistant than others (a

genetic basis for this) complex bacterium – genome has been

sequenced; 4 chromosomes; ~ 5500 genes

Agrobacterium tumefaciens

Agrobacterium tumefaciens

Infection occurs at wound sites Involves recognition and chemotaxis of the

bacterium toward wounded cells galls are “real tumors”, can be removed and

will grow indefinitely without hormones

genetic information must be transferred to plant cells

Infection and tumorigenesis

1. Synthesize a unique amino acid, called “opine” octopine and nopaline - derived from arginine agropine - derived from glutamate

2. Opine depends on the strain of A. tumefaciens3. Opines are catabolized by the bacteria, which

can use only the specific opine that it causes the plant to produce.

4. Has obvious advantages for the bacteria, what about the plant?

Tumor characteristics

It was recognized early that virulent strains could be cured of virulence, and that cured strains could regain virulence when exposed to virulent strains; suggested an extra-chromosomal element.

Large plasmids were found in A. tumefaciens and their presence correlated with virulence: called tumor-inducing or Ti plasmids.

Elucidation of the TIP (tumor-inducing principle)

1. An extrachromosomal double stranded circular DNA molecule

2. Tumour inducing3. Large (200-kb in size) and conjugative type4. ~10% of plasmid transferred to plant cell

after infection5. Transferred DNA (called T-DNA) integrates

semi-randomly into nuclear DNA 6. Ti plasmid also encodes:

enzymes involved in opine metabolism proteins involved in mobilizing T-DNA (Vir genes)

Ti Plasmid

auxA auxB cyt ocsLB RB

LB, RB – left and right borders (direct repeat)auxA + auxB – enzymes that produce auxincyt – enzyme that produces cytokininOcs – octopine synthase, produces octopine

T-DNA

These genes have typical eukaryotic expression signals!

auxA auxBTryptophan indoleacetamide indoleacetic acid

(auxin)

cytAMP + isopentenylpyrophosphate isopentyl-AMP

(a cytokinin)

• Increased levels of these hormones stimulate cell division.

• Explains uncontrolled growth of tumor.

1. On the Ti plasmid2. Transfer the T-DNA to plant cell3. Acetosyringone (AS) (a flavonoid) released by

wounded plant cells activates vir genes.4. virA,B,C,D,E,F,G (7 complementation

groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

Vir (virulent) genes

virA - transports AS into bacterium, activates virG post-translationally (by phosphoryl.)

virG - promotes transcription of other vir genes virD2 - endonuclease/integrase that cuts T-

DNA at the borders but only on one strand; attaches to the 5' end of the SS

virE2 - binds SS of T-DNA & can form channels in artificial membranes

virE1 - chaperone for virE2 virD2 & virE2 also have NLSs, gets T-DNA to

the nucleus of plant cell virB - operon of 11 proteins, gets T-DNA

through bacterial membranes

Vir gene functions (cont.)

From Covey & Grierson

Gauthier, A. et al. (2003) J. Biol. Chem. 278:25273-25276

Type IV Secretion Sys.

• many pathogens, also used in conjugation

• promiscuous • forms T-Pilus

• B7-B10 span OM & IM

• B7-B9 in OM interacts

w/B8 & B10 of IM to form channel

• 3 ATPases

• D4 promotes specific transport

• B2 can form filaments

VirE2 may get DNA-protein complex across host PM

Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

T-DNA is excised from the TI Plasmid and transferred to the nucleus of the plant cell.

Here the T-DNA gets integrated into the plant DNA .

The T-DNA can be passed onto daughter cells as an integral part of plant chromosome

Transfer of tumour inducing Principle

Only small segment of Ti Plasmid is transferred to the host plant cell and gets integrated with the genome

It contains genes for tumour formation(Tum) and nopalina biosynthesis(NOS)

Tum genes encode enzymes that catalyse the synthesis of phytoharmones like IAA and the Cytokinin, Isopentenyl adenosine that causes tumourous growth of cells in crown galls

The T-DNA is bordered by 25 bp repeats, required for the excision and transfer of T-DNA

The T-DNA

Recognition of susceptible wounded plant cell- Plant exudates act as signals by inducing

genes in the Vir genes of the Ti Plasmid - Acetosyringone (AS) ,alpha – hydroxy

acetosyringone- -Binding to wounded cells, controlled by two

chromosomal genes of agrobacterium-chv-A and chv-B

Tumour induction by Agrobacterium

Excision, transfer and integration- The border repeats of the T-DNA play an

important role- Any DNA sequence located between the border

repeats is transferred to the post plant - The T-DNA region is excised from the plasmid

by the enzymes encoded by the Vir region These enzymes specifically recognize the T-

DNA borders The T-DNA enters the plant cell and integrates

into the host genome , mediated by host enzymes

Tumour induction by Agrobacterium

The Ti plasmid has an innate ability to transmit bacterial DNA into plant cell.

This potential is exploited by the genetic engineers to use this as a vector

The gene of a donnor organism can be introduced into the Ti Plasmid at the TDNA region

This plassmid now becomes a recombinant plasmid

By Agrobacterium infection , the donor genes can be transferred and integrated into the genotype of host

This results in the production of a transgenic plant

Ti plasmid as a vector

A mature plant regenerated from transformed cells will contain the cloned gene in every cell and will pass on the cloned gene onto its offsprings

However regeneration of transformed plant can occure only if the Ti plasmid has been disarmed so that transformed cells donot display cancerious properties.

Ti plasmid as a vector

Disarming is possible because cancer genes are not needed for infection

Infectivity is controlled by virulence region of Ti plasmid.

Infactonly parts ofTDNA are involved in infection are two 25bp repeat sequance

Disarmed Ti plasmid

Any DNA placed between these two repeat sequences will be treated as T- DNA and transferred to plant

It is therefore possible to remove all the cancer genes from normal T-DNA and replace them with an entirely new set of genes with out disturbing the infection process.

Disarmed Ti plasmid

Removal of genes responsible of auxin cytokinin and nopaline synthesis.

Disarmed Ti plasmids Deletion of T-DNA REGION. PGV3850 is constructed from pTiC58 It has pBR322 with AmpR Has Border repeats and NOS genes

Disarmed Ti plasmid

Agrobacterium having this PGV 3850 can transfer the modified T-DNA into plant cells.

But the recipient cells will not produce tumour, but could produce nopaline.

This can be used as an efficient vector for introducing foreign genes into plants.

Disarmed Ti plasmid

The main problem is of course that a unique restriction site is an impossibility with a plasmid 200Kb in size.

Novel strategies have to be developed for inserting new DNA into plasmid.

LIMITATIONS

Strategy:1. Move T-DNA onto a separate, small plasmid.2. Remove aux and cyt genes.3. Insert selectable marker (kanamycin resistance) gene

in T-DNA. 4. Vir genes are retained on a separate plasmid.5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids.7. Infect plant with the transformed bacteria.

Binary vector system

Binary vector system

A foreign gene cloned into an appropriate plasmid (pBR322) can be integrated with the disarmed Ti plasmid by a homologous recombination

A compound plasmid called cointegrate is formed.

Construction of Cointegrate

1. Leaf-disc transformation - after selection and regeneration with tissue culture, get plants with the introduced gene in every cell

2. Floral Dip – does not require tissue culture. Reproductive tissue is transformed and the resulting seeds are screened for drug-resistant growth. (Clough and Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743)

2 Common Transformation Protocols

Making a transgenic plant by leaf disc transformation with Agrobacterium.

S.J. Clough, A.F. Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743.

Employing method, genes from entirely unrelated plants can be transferred to other dicots transgenic plants with the following attributes were developed.

Resistance – To antibiotics Pesticides and insect pests Drought and herbicides

Uses

Thank You