Agrobacterium-mediated transfer

The crown gall, and the wounded plant - acetosyringone and hydroxyacetosyringone

The distribution of the vector (requires shockwave)

Agrobacterium tumefaciens, chromosome and plasmid http://www.ppws.vt.edu/~sforza/prokaryote/agrobacterium1.jpg

Rough draft of Ti plasmid (requires shockwave)

The military analogy: Is Agrobacterium like a "mercenary commando"?

Resource pictures: The Pathogenesis of Crown Gall, by Sforza et al.

Octopine-type Ti plasmid T-region - the segment transferred to plants, between Right and Left border Genes for production of: Auxin Cytokinin Opine Outside the T-region - the segment not transferred Opine catabolism genes origin of replication vir genes example: virD encodes a site-specific endonuclease for the Right border sequence, makes a nick.

What is the difference between an element required in cis, and one required in trans?

...a trans element can be moved to a different location, for example from the Ti plasmid to the genome, and it will still function effectively. A gene that makes a diffusible protein product can be moved generally, and that is an example of a "trans" element. An example of a "cis" element is an origin of replication. That cannot be moved, and still serve its purpose. It must be "on" the DNA that needs replicating.

A T-border sequence is a "cis" active element, whereas the vir genes are "trans" active. Vir genes do not need to be on the plasmid carrying the T-DNA region.

From CAMBIA Intellectual Property Resource, a 262 page "white paper" with detailed information on Agrobacterium transformation, intellectual property, and patents: (pdf format)

• Scientific aspects
• Types of tissues to be transformed
• Binary vectors
• Co-integrated vectors
• Monocot transformation
• Dicot transformation

Binary vector systems - widely used

http://www.cambiaip.org/cambiaIP/books/whole.html#agri_page25

Elements:

1. A wide host-range plasmid with

• selectable markers for E. coli, A. tumifaciens, plants
• promoter andgene of interest
• right and left border sequence

2. A "helper" Ti that lacks the T-DNA, but contains the vir genes

Co-integrated or hybrid Ti vector system - oldies but goodies

Elements:

1. A disarmed Ti plasmid

2. An ori-less plasmid that can integrate into the Ti plasmid, containing (typically) cis elements necessary for T-DNA mobilization. (Fig 17.6)

Depends on a single crossover event

Modified Co-integrated vectors - using cre/lox to prepare for directed recombination

Engineering issues:

1. In vitro methodology

2. Transformation of E. coli

3. Transformation or conjugation (with cis/trans function) into A. tumifaciens

4. Transformation of plants

• callus
• immature embryo
• pollen
• shoot apex
• in planta

General Plant Applications:

Monocots

Dicots

• e.g. The Tomato Patents

  The Flavr Savr ... and other delightful fruits at the transgenic tomato site

Microprojectile bombardment - biolistics

Biorad's Helios Gene Gun

The gene is in the plant, so now what?

Detection of foreign DNA expression - Table 17.4

Selectable markers vs. Reporters

e.g.

GUS - beta-D-glucuronidase (from E. coli) plus 5-bromo-4-chloro-3-inolyl B-D-glucuronic acid

• Application - Soybeans
• Application - Arabidopsis flower
• Application - Arabidopsis shoot apex

Luciferase -

• Everything you need to get started
• An example of quantification of expression from the promotor for chlorophyll a/b binding protein

GFP

• Background
• Application - Using GFP to Select for Transgenic Sweet Orange Derived from Electroporated Protoplasts
• Application - microscopy to monitor expression