“The natural genetic engineer” Agrobacterium is the causative agent of “Crown Gall Disease” Bacteria isolated from the galls can induce tumours when inoculated into a wound on a different plant. Wounds are important in tumour induction !

Galls can be cultured completely free of bacteria, and show all the characteristics of a animal tumour Rapid, uncontrolled growth, indicating that the plant tissue has been “transformed” by contact with the bacterium. The Tumour Inducing Principle (T.I.P) was postulated to be a toxin, bacteriophage etc…. which converted normal cells into tumourous cells. In 1970’s it was shown (Morel and Petit) that there were 2 sorts of Agrobacterium tumours, caused by 2 sorts of Agrobacteria :-------

Tumours produced by ONE Agrobacterium strain synthesise the carbon compound N--1-carboxyethyl-L-arginine OCTOPINE Tumours produced by the SECOND Agrobacterium strain synthesise N- -1,3, dicarboxylpropyl-L-arginine NOPALINE Agrobacteria which induce tumours can use the carbon compounds produced by the tumours as a carbon and energy source.

Morel proposed that the Agrobacteria were inserting fragments of their own DNA (coding for octopine / nopaline synthesis) into the plant genome. Jeff Schell in Koln discovered (1970) that the TIP was a PLASMID (Ti) because it could be “cured” A cured non-oncogenic strain can be converted into an oncogenic strain by bacterial conjugation. Jeff Schell then proposed the “Genetic Colonisation Concept”

Genetic Colonisation Concept: 1) Ti plasmids are natural gene vectors for plant cells 2) Evolved for the benefit of the bacteria 3) Crown galls are the result of the transformation of healthy cells by Ti plasmids 4) Ti plasmids when introduced into plant cells synthesise opines. 5) Free living Agrobacteria use these opines as a energy + carbon source

Tumour Inducing Plasmids (Ti plasmids) Vir region (50kb) carries genes involved in T-DNA transfer

T-DNA 1) Only the 23kb T DNA is transferred to the plant nucleus. 2) OCS region codes for the synthesis of octopine / nopaline 3) Tms, Tmr code for auxin and cytokinin synthesis 4) T-DNA is flanked by 25bp direct repeat sequences. 5) Repeats are important in transfer.

Early stages in the transfer of the T-DNA from the bacterium > plant T-DNA functions solely as a structural element – DOES NOT encode products for its own transfer. Chromosomal bacterial and plasmid Ti genes are involved in transfer: Bacterium attaches to the plant cell (chromosomal chvA, chvB) chvA = transport protein, chvB = 235kd cyclic glucan. For transformation to occur, the T-DNA has to be “moved” from the Ti plasmid into the plant nucleus – controlled by the Vir operon

Vir gene products (A,B,C,D,E,G) act to transfer the T-DNA How is the vir operon switched on – what controls it ?

How is the vir operon activated and switched on ? A plant molecule called acetosyringone is the major vir activator Acetosyringone 4-acetyl 2,6, dimethoxyphenol is produced by wounded plant cells If Agrobacteria are cultured with acetosyringone, the Vir operon is induced : there must be a recognition system. Acetosyringone is recognised by the products of Vir A and G – these proteins are constitutively synthesised by the Ti plasmid.

VirA is a sensor for Acetosyringone.

Activated VirA phosphorylates VirG Phos VirG is a DNA binding protein which activates the Vir operon to begin the transfer of T-DNA to the plant.

What defines T (Transfer) DNA? RB LB --------------23kb-------------- 1) T DNA is flanked by 25bp border repeats – essential in transfer. 2) Everything between the borders is transferred and integrated. 3) Transfer system only recognises the border sequences. 4) T-DNA transfer is polar, occurs from RB > LB 5) If RB is deleted, transfer of T-DNA does not take place. 6) LB specifies where the T-DNA should terminate.