Agrobacterium tumefaciens Unusual disease agent brought into use as plant genetic engineer

A range of interactions exist between bacteria and plants Bacteria associate with plants in ways that benefit both partners - symbiosis. Other bacteria have no beneficial effects - disease. Add picture of nodulated root Rhizobium-legume symbiosis

Cell division sites in the plant are restricted Cells in small regions at the tips of shoots and roots contain cells that expand and divide to increase the size of the plant. These are called apical meristems

Apical meristems Dividing cells lie at the tip of the root below the region where root hairs emerge. They expand and then divide controlled by 2 hormones -auxin and cytokinin Apical meristem producing auxin and cytokinin Cell expansion and division

The view inside a root Root hairs Expanding cells Dividing cells Keiko Fig 2.6 Expanding cells Dividing cells

Some bacteria release the controls on cell division causing a cancerous growth This happens in crown gall disease, producing large masses of disorganised cells. Infection usually follows wounding.

Broad host range The disease affects many different plants including economically important species such as these peach trees Crown gall on Euonymus

There are few limits on the final size! Crown gall on ash.jpg Crown gall on the trunk of an ash tree

All this from one bacterium The production of these millions of extra cells is caused by a bacterium Agrobacterium tumefaciens Agro on plant surface.jpg

Agrobacterium is very common in the soil around roots Agrobacterium is a close relative of Rhizobium species that form nodules on legumes Like Rhizobium, it is common in the rhizosphere, the region around roots rhizosphere.jpg

Another molecular conversation but a different outcome Just like Rhizobium exchanging signals with its legume host, Agrobacterium and its future host exchange signals These activate a mechanism in the bacterium that transfers some bacterial DNA to take control of the plant

DNA - information carrier DNA carries the genetic instructions all organisms (including us) receive from our parents Those instructions determine all inherited features - that make us different ( hair colour, eye colour, blood group etc) and all the features we share DNA directs activities in all cells One enormously long DNA molecule forms each chromosome The information on each chromosome is broken down into many genes Each gene provides the information to make one protein

Agrobacterium controls its plant host by putting a small piece of T-DNA into one of the plant chromosomes The transferred genes form the T-DNA (transferred DNA) region of the Ti plasmid (tumour inducing). T-DNA is less than 10% of the whole plasmid, encodes only 3 or so genes and enters a plant that has at least 25000 genes

But these few genes are enough to take control of the plant Changes the plant’s metabolism to produce food materials (opines) that only the bacterium can use Produces the plant hormones auxin and cytokinin that remove the controls that normally limit cell division and cell expansion. Result - cells showing altered metabolism multiply uncontrollably

Hence the massive disorganised growths

T-DNA, a small region of the Ti plasmid is transferred Functions for DNA transfer and using opines

SUMMARY Gene trans in crowngall.jpg Even if the T-DNA originally change only one cell in the plant, because that cell divides uncontrollably and passes on those bacterial genes to all the new cells, there are soon millions of cells feeding the bacterium at the expense of the plant.

Plant scientists can use Agrobacterium to put other genes into plant chromosomes and so understand what they do

Disarming the Ti plasmid Remove and replace these genes

New genes in the Ti plasmid New genes inserted between borders

“Floral dip” to transfer genes

How my research uses Agrobacterium Mutants have a fault in one gene which stops it directing the cell’s activities in the normal way. Mutants tell us what function a gene normally serves. Make diagram of genetic changes leading to trait

Albino mutants have a faulty gene needed to make leaves green

Plants with a downturned branches have a defective gene needed to make them grow upright cipoupright.gif and cipoweep.gif

Our mutants have defective genes needed to make cellulose We selected mutants unable to make the plant’s major structural material - cellulose. Cellulose makes vegetables crisp, wood strong, paper thin but strong and cotton fibres tough enough to make jeans

Cellulose forms the walls that surround all plant cells Keiko Fig 2.6 When cellulose production stops, the restraint imposed by the wall is removed and the root swells instead of increasing in length

Cellulose structure and appearance in em

The cotton boll Cotton fibres are very long hair cells that form on the cotton seed. They develop thick walls that are almost pure (>95%) cellulose. The fibres are spun into threads to make cotton garments “cotton boll.jpg” Copyright blanked out not for publication

Our mutants have defective genes needed to make cellulose We selected mutants unable to make the plant’s major structural material - cellulose. Cellulose makes vegetables crisp, wood strong, paper thin but strong and cotton fibres tough enough to make jeans

“Which of the 25,000 genes has the fault?” Preliminary work narrows the choice of genes Agrobacterium puts a new copy of each of the suspected genes into the mutant. If the mutant now looks normal, the introduced gene has repaired the fault and the mutant had a faulty copy of the gene delivered by Agrobacterium.

With a new copy of the faulty gene, the mutant looks normal

WITH THE RIGHT GENE, THE CHANGE IS DRAMATIC Make diagram of plasmid with test gene inserted Mutant - swelling not elongating Normal plant Mutant + 1 gene introduced by Agrobacterium

GFP making organelles visible Golgi bodies Endoplasmic reticulum

Some T-DNA insertions inactivate genes in the host plant No effect Inactivates Gene 2 This makes a new insertional mutant

From disease agent to research tool With some changes to the Ti-plasmid, Agrobacterium has been brought from the field to make possible new experiments to understand the function of plant genes

“Floral dip” and seed selection