Agrobacterium tumefaciens
MICROM 412 Lecture 22: May 23rd, 2025 Alex Meeske -
[email protected] Crown Galls are Plant Tumors
• Tumor growth that
a ects plant health,
but usually does
not kill the plant
• A ects >90 species
of plants, incl.
grapevines, fruit
trees, roses, walnut,
woody plants.
• Caused by a
bacterial pathogen
called
Agrobacterium
tumefaciens
ff
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Galls are associated with Agrobacterium
• Member of the alpha-
Proteobacteria and Rhizobaceae
• Agrobacterium adheres to plant
cells in wound sites
• Agrobacterium infection causes
galls, and Agrobacterium can be
isolated from galls
• NOT an obligate pathogen, it can
grow in the absence of host
plants
Primary Galls are associated with Agrobacterium
• Agrobacterium infection Primary Secondary
causes galls, and Tumor Tumor
Agrobacterium can be
isolated from primary
galls
• Secondary tumors often
form in the plant too, but
these are mostly bacteria-
free
• This indicates that
Agrobacterium makes a
permanent change in the
plant - called
transformation
Once established Galls can persist without Agrobacterium
Tissue from Tissue from
Primary Secondary
Tumor Tumor
• Both primary and
secondary galls can
be grafted onto new
plants
Donor Gall Tissue: Intact Destroyed Intact Destroyed
Agrobacterium in donor tissue? Yes Yes No No
The a ected cells have become anarchists, no longer obeying the
laws of well-ordered society.
-Philip White and Armin Braun “A cancerous neoplasm of plants
produced by bacteria-free crown-gall tissue” Cancer Research 1943
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Plant hormones: Auxins and Cytokinins
Normal Gall
• Crown Galls make high levels of plant
hormones (auxins and cytokinins)
that stimulate plant tissue growth
Indole Acetic Acid
(An Auxin)
Normal Gall
Kinetin
(A cytokinin)
Gall tissue survives even without added hormones
Galls produce Opines - nutrients for Agrobacterium
• Fusions of amino acids with other organic acids (e.g. lysine + pyruvate) - plants do not
make opines unless transformed by Agrobacterium
• Most bacteria cannot grow on them, but Agrobacterium encodes the enzymes necessary
to use them as a carbon and nitrogen source.
• Strains di er in the opines that they can consume, and the opines that they cause the
plant to produce
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The Agrobacterium Ti Plasmid transforms plant tissue
• Growing Agrobacterium at 37°C (high temp) or treating with Ethidium
Bromide (DNA-intercalating agent) makes it unable to cause galls
• Transformation ability can be transmitted between Agrobacterium
species!
A. tumefaciens
Causes galls
(Virulent)
The Agrobacterium Ti Plasmid transforms plant tissue
• Growing Agrobacterium at 37°C (high temp) or treating with Ethidium
Bromide (DNA-intercalating agent) makes it unable to cause galls
• Transformation ability can be transmitted between Agrobacterium
species!
A. radiobacter Does not cause
(Avirulent) galls
The Agrobacterium Ti Plasmid transforms plant tissue
• Growing Agrobacterium at 37°C (high temp) or treating with Ethidium
Bromide (DNA-intercalating agent) makes it unable to cause galls
• Transformation ability can be transmitted between Agrobacterium
species!
A. tumefaciens
A. radiobacter
(Virulent)
(Virulent)
Causes galls
A. radiobacter
(Avirulent)
The Agrobacterium Ti Plasmid transforms plant tissue
• Growing Agrobacterium at 37°C (high temp) or treating with
Ethidium Bromide (DNA-intercalating agent) makes it unable to
cause galls
• The cells treated this way lost a large plasmid
• Transformation ability can be transmitted between Agrobacterium
species!
• The A. radiobacter strain that gained virulence gained a large
plasmid
The plasmid responsible is called the Ti plasmid (Tumor-inducing)
Agrobacterium transfers T-DNA to the plant genome
• At UW, Gene Nester and Mary-Dell Chilton showed that a small
portion of Agrobacterium DNA called T-DNA (transferred DNA) can
be found integrated into the plant DNA.
• The T-DNA is a small portion of the Ti plasmid.
• This was the key insight that enabled genetic engineering of
crops
The Ti Plasmid T-DNA
Oncogenes onc ops Opine Synthases
(Auxin/Cytokinin Synthases)
Transmissibility
genes
Virulence
Ti Plasmid
genes
ONLY the T-DNA is
transferred to the
plant genome.
Opine Catabolism
It is surrounded by 25 genes
bp repeats
Agrobacterium colonization of plants
• Agrobacterium Phenolic compounds
senses phenolic T-DNA and sugars from plant wound
compounds and
Oncogenes
sugars released onc ops Opine Synthases
okinin Synthases) Sensor kinase
from plant wounds
Membrane VirA
Transmissibility
genes
VirG VirG
Virulence
Ti Plasmid Response Regulator
P
genes
VirG vir gene transcription
P
Opine Catabolism
Agrobacterium colonization of plants
• Agrobacterium makes Unipolar Polysaccharide and cellulose
to adhere to plants
Agrobacterum
tumefaciens
T-DNA
PLANT
Ti Plasmid
vir
Agrobacterum
tumefaciens
T-DNA
VirD
PLANT
Ti Plasmid
vir
Agrobacterum
tumefaciens
T-DNA
VirD
VirD
PLANT
single-stranded
Ti Plasmid T-DNA
vir
Agrobacterum
tumefaciens
T-DNA
VirD
VirE VirD
PLANT
Ti Plasmid
vir
single-stranded
T-DNA coated in VirE
Agrobacterum
tumefaciens
T-DNA
VirD
VirB
VirE VirD
Ti Plasmid
vir
Protein-coated T-DNA
is translocated into plant
cytosol through VirB
(type IV secretion system)
Agrobacterum
tumefaciens
VirD has a nuclear
T-DNA
VirD localization signal
(NLS)
VirB
VirE VirD
Ti Plasmid
vir
Protein-coated T-DNA
is translocated into plant
cytosol through VirB
(type IV secretion system)
Using Agrobacterium to make GMO crops
• Uses a “disarmed” Ti plasmid lacking opine synthesis and oncogenesis
functions
• Cargo genes inserted into the T-DNA region can be transferred to plant genome
• T-DNA is anked by 25 bp repeats critical for transfer, cargo is inserted between
these
Cargo T-DNA
T-DNA
25 onc ops 25 25 25
Transmissibility
Ti Plasmid
genes Disarmed
Virulence Virulence
genes
Ti Plasmid
genes
Opine Catabolism
genes
fl
Bt Crops
Engineered to
make insecticidal Bt toxin
Bt toxin from crystal
Bacillus
thuringiensis
Need less forespore
pesticide
Roundup Ready Crops - Herbicide-resistant
Golden Rice
Engineered to
make vitamin A
