ti plasmid presentation-2

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Agrobacterium - mediated Gene Transfer:

Agrobacterium - mediated Gene Transfer Most common method of engineering dicots, but also used for monocots Pioneered by J. Schell (Max-Planck Inst., Cologne) Agrobacteria soil bacteria, gram-negative, related to Rhizobia species: tumefaciens - causes crown galls on many dicots rubi - causes small galls on a few dicots rhizogenes - hairy root disease radiobacter - avirulent

Slide 2:

Crown galls caused by A. tumefaciens on nightshade. More about Galls: http:// waynesword.palomar.edu/pljuly99.htm http://kaweahoaks.com/html/galls_ofthe_voaks.html

Agrobacterium tumefaciens:

Agrobacterium tumefaciens the species of choice for engineering dicot plants; monocots are generally resistant (but you can get around this) some dicots more resistant than others (a genetic basis for this) complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genes

Infection and tumorigenesis:

Infection and tumorigenesis 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

Tumor characteristics :

Tumor characteristics Synthesize a unique amino acid, called “opine” octopine and nopaline - derived from arginine agropine - derived from glutamate Opine depends on the strain of A. tumefaciens Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce. Has obvious advantages for the bacteria, what about the plant?

Elucidation of the TIP (tumor-inducing principle):

Elucidation of the TIP (tumor-inducing principle) 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.

Ti Plasmid :

Ti Plasmid Large (  200-kb) Conjugative ~10% of plasmid transferred to plant cell after infection Transferred DNA (called T-DNA ) integrates semi-randomly into nuclear DNA Ti plasmid also encodes: enzymes involved in opine metabolism proteins involved in mobilizing T-DNA ( Vir genes)

Slide 8:

auxA auxB cyt ocs LB RB LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin Ocs – octopine synthase, produces octopine T-DNA These genes have typical eukaryotic expression signals!

Slide 9:

auxA auxB Tryptophan  indoleacetamide  indoleacetic acid (auxin) cyt AMP + isopentenylpyrophosphate  isopentyl-AMP (a cytokinin) Increased levels of these hormones stimulate cell division. Explains uncontrolled growth of tumor.

Vir (virulent) genes:

Vir (virulent) genes On the Ti plasmid Transfer the T-DNA to plant cell Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes. virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

Vir gene functions (cont.):

Vir gene functions (cont.) virA - transports AS into bacterium, activates virG post-translationally (by phosphoryl.) virG - promotes transcription of other vir genes virD 2 - 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

Slide 12:

VirE2 may get DNA-protein complex across host PM

Slide 13:

Monocots don't produce AS in response to wounding. Important : Put any DNA between the LB and RB of T-DNA it will be transferred to plant cell! Engineering plants with Agrobacterium: Two problems had to be overcome: (1) Ti plasmids large, difficult to manipulate (2) couldn't regenerate plants from tumors

Binary vector system:

Binary vector system 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.

Slide 15:

Binary vector system

Slide 16:

Making a transgenic plant by leaf disc transformation with Agrobacterium.

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Thank you

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