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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.
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
Cell expansion
and division
Apical meristem
producing auxin
and cytokinin
The view inside a root
Root hairs
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
There are few limits on the final
size!
• 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
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
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
T-DNA
Functions for
DNA
transfer and
using opines
SUMMARY
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.
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
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
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
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
Mutant swelling not
elongating
Normal plant
Mutant + 1 gene
introduced by
Agrobacterium
GFP making organelles visible
Endoplasmic reticulum
Golgi bodies
Some T-DNA insertions
inactivate genes in the host plant
Gene 1
Gene 2
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