12: The Agrobacterium Ti Plasmid

Question 1

Give an outline of how Agrobacterium tumefaciens causes crown-gall disease

Answer 1

1. Infection of plant wound by agrobacterium
2. Attachment of agrobacterium cells to plant cells via acidic capsular polysaccharide, followed by the production of cellulose fibrils that enmesh large numbers of bacteria at the wound surface
3. Processing of T-DNA from Ti plasmid
4. Transfer of T-DNA to plant cell and integration into plant genome
5. Tumorigenesis and production of opines

Question 2

Why do transformed plant cells proliferate?

Answer 2

Auxin and cytokinin combined causes a hormone imbalance (usually only one secreted at a time) which causes tumour formation (cell proliferation)

Question 3

What is the ecological advantage gained by A. Tumefaciens from gall induction?

Answer 3

The tumours formed produce food (opines) which can only be broken down by the T1 plasmid for agrobacterium

Question 4

What are the regions of the Ti plasmid that play a role in gall induction and/or agrobacterium ecology?

Answer 4

Vir genes and tDNA regions

Question 5

How does A. Tumefaciens perceive the presence of a wounded plant?

Answer 5

Senses stress molecule produced by a wounded plant (acetosyringone) which activates Ti plasmid

Question 6

What does T-DNA encode and why are the genes only expressed in plants?

Answer 6

Codes enzymes for auxin and cytokinin synthesis (tumour formation) and opine synthesis genes (octopine and nopaline) which acts as a carbon and nitrogen source for A. Tumefaciens growth

Question 7

What parts of the T-DNA are required for its transfer to plants?

Answer 7

VirD2 and VirE

Question 8

Give an overview of the molecular mechanism of T-DNA transfer

Answer 8

VirD2 acts as a relaxant (binds to tDNA and transports DNA into plant cell)
VirE is a single stranded binding protein that protects DNA during transport

Both VirD2 and VirE have plant active nuclear localisation sequences which signal plant proteins to phosphorylation it and transport it to the nucleus so the tDNA can integrate the plant DNA
Uses the plant immune system which is activates due to infection to ‘Trojan horse’ tDNA into nucleus

Question 9

What is a disarmed Ti plasmid?

Answer 9

Ti plasmid that has all the Vir genes but does not have the tDNA within it

Question 10

What are the critical components for a vector for use in plant genetic engineering?

Answer 10

The two OriT sites either side of the tDNA

Question 11

What type of secretion system does agrobacterium use

Answer 11

Type IV secretion system