Agrobacterium tumefaciens –
pathogen and useful tool
Agrobacterium tumefaciens is a
plant pathogen that induces
tumors on about 60% of
dicotyledonous angiosperms
and gymnosperms
Cherry
Its tumor-inducing property also
makes it a valuable tool for
introducing genes into plants for
research and agricultural purposes
Casimiro, I., Marchant, A., Bhalerao, R.P., Beeckman, T., Dhooge, S., Swarup, R., Graham, N., Inzé, D., Sandberg, G., Casero, P.J. and Bennett, M.
(2001). Auxin Transport Promotes Arabidopsis Lateral Root Initiation. Plant Cell. 13: 843-852. Herb Pilcher
© 2014 American Society of Plant Biologists
Crown gall disease
The first written record of
crown gall disease, on
grape, dates from 1853
Fridiano Cavara
(1897) found that a
bacterium causes
crown gall in grape
Crown gall induces growths at
wound sites and severely limits
crop yields and growth vigor
Edward L. Barnard, Florida Department of Agriculture and Consumer Services, Bugwood.org; Mike Ellis, Ohio State University; University
of Georgia Plant Pathology Archive, University of Georgia, Bugwood.org; Wikimedia commons
© 2014 American Society of Plant Biologists
is caused
by a
“A1907:
plantCrown
tumor gall
of bacterial
origin”
bacterium
1907 - Erwin Smith and C.O.
Townsend isolated a bacterium
from galls on daisy. When
inoculated onto other plants,
galls were produced
gall
gall
Smith, E.F. and Townsend, C.O. (1907). A plant-tumor of bacterial origin. Science. 25: 671-673.
© 2014 American Society of Plant Biologists
Unusual compounds called opines
are found in many crown galls
The type of opine is determined by
the bacterium, not the plant
Octopineutilizing
strain
Nopalineutilizing
strain
Octopine
Questions raised:
• What are these
compounds?
• Do they cause the
tumors?
• How and why do the
bacteria cause the
plants to make
opines?
Nopaline
1960s – 1970s,
numerous studies
© 2014 American Society of Plant Biologists
Agrobacterium-induced galls do not
require bacterial persistence
Gall tissues without any bacteria can persist
indefinitely in culture, in contrast with most
other pathogen-induced neoplastic growths
that require the presence of the pathogen
Braun made fundamental
discoveries about how
Agrobacterium
transforms plant cells
Armin C. Braun
1911 - 1986
White, P.R. and Braun, A.C. (1941). Crown gall production by bacteria-free tumor tissues. Science. 94: 239-241; Photo from Wood, H.N., and Kelman, A. (1987) Phytopathology 77: 991.
© 2014 American Society of Plant Biologists
Gall tissues can grow indefinitely
without exogenous phytohormones
Auxin
CK
1930s – 1950s,
numerous studies
Normal plant
tissue cannot live
indefinitely in
hormone-free
medium
Normal plant
tissue grows and
survives when
auxin and
cytokinin (CK)
are added to
medium
High levels of auxin
and cytokinin are found
in gall tissues
Crown gall tissue
grows well
without added
hormones
“It is possible for a cell to
acquire the capacity for
autonomous growth as a
result of the permanent
activation of growthsubstance-synthesizing
systems”
-AC Braun 1958
Braun, A.C. (1958) A physiological basis for autonomous growth of the crown-gall tumor cell. Proc Natl Acad Sci U S A. 44: 344–349.
© 2014 American Society of Plant Biologists
A few days after inoculation, tumors
become independent of bacteria
When the tissue was
incubated at room
temperature for one
day before heatkilling the bacteria, no
tumors were formed
Periwinkle (Catharanthus roseus)
stems were inoculated with
Agrobacterium tumefaciens, and
then incubated at room
temperature for various times,
followed by 5 days at 47°C to
kill the bacteria
When the tissue was
incubated at room
temperature for four
days before heat-killing
the bacteria, many
tumors were formed
Viable bacteria are no longer necessary beyond two days post-inoculation. After this
period, tumors become independent of the bacteria, because the bacteria have
altered the host cells, by transferring some factors into them.
Braun, A.C. (1943) Studies on tumor inception in the crown-gall disease. Am. J. Bot. 30: 674-677
© 2014 American Society of Plant Biologists
A large plasmid in gall-inducing
Agrobacterium confers virulence
Virulent
Avirulent
A plasmid
carrying a genetic
marker (antibiotic
resistance) was
shown to be
confer virulence
A very large
plasmid was
identified that is
present in virulent
but absent from
avirulent strains
Heat treatment
removes plasmid
and makes bacteria
non-pathogenic
tumor
heat
No
tumor
Virulent
+
time
tumor
Avirulent
tumor
Zaenen, I., van Larebeke, N., Teuchy, H., van Montagu, M. and Schell, J. (1974). Supercoiled circular DNA in crown-gall inducing Agrobacterium strains. Journal of Molecular Biology. 86: 109-127. Larebeke, N.V., Engler, G.,
Holsters, M., Den Elsacker, S.V., Zaenen, I., Schilperoort, R.A. and Schell, J. (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature. 252: 169-170. Van Larebeke, N., Genetello, C.H.,
Schell, J., Schilperoort, R.A., Hermans, A.K., Hernalsteens, J.P. and Van Montagu, M. (1975). Acquisition of tumour-inducing ability by non-oncogenic agrobacteria as a result of plasmid transfer. Nature. 255: 742-743.
© 2014 American Society of Plant Biologists
Some DNA from the Ti plasmid is
transferred into the plant cells (1977)
Ti plasmid
Renaturation kinetics of
labeled plasmid DNA
fragments with various
unlabeled DNA samples
Restriction
enzyme
digestion
“Our results suggest that the tumorinducing principle first proposed by
Braun (1947) is indeed DNA, as many
investigators have long suspected.”
The key
finding was
that Ti
plasmid DNA
anneals with
DNA isolated
from the
crown gall,
meaning that
the gall
contains Ti
DNA
Neg. control
(untransformed
plant DNA)
DNA from
crown gall
Pos. controls
(Ti plasmid)
Increasing amounts of
labeled Ti plasmid DNA
Reprinted from Chilton, M.-D., Drummond, M.H., Merlo, D.J., Sciaky, D., Montoya, A.L., Gordon, M.P. and Nester, E.W. (1977). Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis.
Cell. 11: 263-271. with permission from Elsevier. See also Yadav, N.S., Postle, K., Saiki, R.K., Thomashow, M.F. and Chilton, M.D. (1980). T-DNA of a crown gall teratoma is covalently joined to host plant DNA. Nature. 287: 458-461.
© 2014 American Society of Plant Biologists
Structure and function analysis of
the Ti plasmid
T-DNA
Transfer DNA (T-DNA) moves
into the plant cell nucleus. It
is flanked by two direct 25 bp
repeat border sequences,
shown as yellow triangles
pTi
The virulence (vir)
genes are required
for T-DNA
movement into the
plant cell
The organization of Ti plasmids
varies between isolates, but all
carry one or more T-DNA
region and one vir region
© 2014 American Society of Plant Biologists
The T-DNA region: tumor-inducing
genes and opine synthesis genes
Plant cell
Opine synthesis
to “feed”
Agrobacterium
T4SS
T-DNA
pTi
Auxin
synthesis
Cytokinin
synthesis
Autonomous
growth
T4SS = Type IV Secretion System
© 2014 American Society of Plant Biologists
The Ti plasmid can be used to
introduce any gene into plants
T-DNA
pTi
The discovery that T-DNA was inserted into
the plant genome raised the possibility that
“any gene” could be transferred into plants
Gene of interest
Selectable
marker
Tumor-inducing and opine
synthesis genes on T-DNA can be
replaced by a “gene of interest”
and selectable marker
Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983). A binary plant vector strategy based
on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature. 303: 179-180.
© 2014 American Society of Plant Biologists
Applications of Agrobacteriummediated transformation
Basic research – plant transformation
allows in vivo study of plant genes
Lobed-leaf phenotype of
plants overexpressing
KNAT1 gene
Wild type
Expression pattern of an
auxin-inducible promoter
fused to GUS reporter gene
Overexpression
Population
segregating for shorthypocotyl phenotype
conferred by PHYB
overexpression
Wagner, D., Tepperman, J.M. and Quail, P.H. (1991). Overexpression of phytochrome B induces a short hypocotyl phenotype in transgenic Arabidopsis. Plant Cell. 3: 1275-1288; Chuck, G., Lincoln, C.
and Hake, S. (1996). KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell. 8: 1277-1289. Casimiro, I., Marchant, A., Bhalerao, R.P., Beeckman, T., Dhooge,
S., Swarup, R., Graham, N., Inzé, D., Sandberg, G., Casero, P.J. and Bennett, M. (2001). Auxin transport promotes Arabidopsis lateral Root Initiation. Plant Cell. 13: 843-852.
© 2014 American Society of Plant Biologists
Production of genetically-modified
(GM) plants
Bacillus
thuringiensis
expressing Bt
toxin
Transgenic plants
expressing
insecticidal Bt gene
Plant cell
expressing Bt
toxin
Agrobacterium tumefaciens
allows gene transfer into many
crop plants, particularly dicots
like soybean and peanut
Wild-type
peanut plant
Peanut plant
expressing the Bt gene
Photo credits: Herb Pilcher, Scott Bauer
© 2014 American Society of Plant Biologists
Agrobacterium-mediated
transformation has other uses
T-DNA
Insertional
mutagenesis
Mutated, tagged gene
Transient expression studies:
Short-term expression of T-DNA
genes gives results faster than
generating transgenic plants
GFP expression in tobacco epidermal cells
Alonso, J.M. et al., and Ecker, J.R. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 301: 653-657; Reprinted by permission from Macmillan Publishers Ltd Sparkes, I.A., Runions, J.,
Kearns, A. and Hawes, C. (2006). Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protocols. 1: 2019-2025.
© 2014 American Society of Plant Biologists
Summary – the development of a
vector for plant transformation
Introduce gene of interest into
T-DNA region, and introduce into
Agrobacterium carrying vir genes
Agrobacterium
tumefaciens
Inoculate plant with
engineered Agrobacterium
T-DNA
Regenerate plant
from transformed cells
Arabidopsis floral dip
transformation method
Photo by Peggy Greb, USDA
© 2014 American Society of Plant Biologists
Inside the black box – how
Agrobacterium transfers DNA
1. Chemoattraction and
activation of virulence
2. T-DNA excision
4. Nuclear import and
integration of T-DNA
3. Movement of
T-DNA out of the
bacterium
Agrobacterium
Plant cell
5. Expression
of T-DNA and
pathogenicity
© 2014 American Society of Plant Biologists
Many of the genes needed for T-DNA
transfer are found on the Ti plasmid
T-DNA
xin
LB
inin opine
cytok
au
RB
traR
Conjugal
Transfer
tra
A
B
Ti plasmid
G
214233bp
vir
C
occ
D
Virulence
genes
traI
The Ti plasmid carries
genes required for
T-DNA transfer, Ti
plasmid conjugation
and opine metabolism
E
F
replication
Opine
catabolism
© 2014 American Society of Plant Biologists
Perception of host signals induces
expression of vir genes
Plant-derived small
molecules such as
acetosyringone induce
Agrobacterium vir genes
Acetosyringone is
likely perceived by the
VirA protein encoded
on the Ti plasmid
VirA and VirG induce other
vir genes in response to
plant signals
Stachel, S.E., Messens, E., Van Montagu, M. and Zambryski, P. (1985). Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature. 318:
624-629; Stachel, S.E., Nester, E.W. and Zambryski, P.C. (1986). A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc. Natl. Acad. Sci. USA. 83: 379-383.
© 2014 American Society of Plant Biologists
T-DNA transfers through a multisubunit type IV secretion system
Tilted outer
membrane side
Plant cell
Side view
Outer membrane
VirB9
and B7
VirB10
Inner membrane
Agrobacterium
Tilted inner
membrane side
Reprinted by permission from Macmillan Publishers Ltd. Fronzes, R., Christie, P.J. and Waksman, G. (2009). The structural biology of type IV secretion systems. Nat. Rev. Micro. 7: 703-714.
© 2014 American Society of Plant Biologists
Conjugation spreads the Ti plasmid
throughout the population
Agrobacterium
with Ti plasmid
Opine
Cell division
Conjugation –
horizontal gene
transfer
Agrobacterium
without Ti plasmid
Replication of the large Ti plasmid is metabolically costly. When opines are
present, the Ti plasmid is amplified in the population by conjugation. Thus, a small
number of individuals carrying Ti can serve as a reservoir for the larger population
© 2014 American Society of Plant Biologists
SUMMARY (Animated)
.
Plant cell
Agrobacterium
Transfer
T-DNA processing
D2
vir
genes
vir genes
LB
RB
E3
E2
E2
D2
F
T4SS
E2 E3
F
D2
D5
Integration
of T-DNA
VirB/D4
T-DNA
nucleus
Ti
Plasmid
induction
Expression of
T-DNA: auxin,
VirG
p
VirG p
VirA
Phenolics
cytokinin, opine
Signaling
in rhizosphere
© 2014 American Society of Plant Biologists
Agrobacterium rhizogenes: inducer
of roots
Infection by A. rhizogenes leads to
production of a large root mass, rather
than a tumor. Therefore, the large
plasmid carried by A. rhizogenes is
called a Root-inducing (Ri) plasmid
The oncogenic genes
on A. rhizogenes TDNA are not as well
understood as those
on the Ti plasmid
T-DNA
pRi
Mulberry infected
with A. rhizogenes
Image credits: William M. Brown Jr., Bugwood.org; Reprinted from Dhakulkar, S., Ganapathi, T.R., Bhargava, S. and Bapat, V.A. (2005). Induction of
hairy roots in Gmelina arborea Roxb. and production of verbascoside in hairy roots. Plant Sci. 169: 812-818 with permission from Elsevier.
© 2014 American Society of Plant Biologists
Conclusions
Agrobacterium is an amazing
organism, with a unique ability to
transfer DNA into diverse host
genomes, which has been exploited
to facilitate research and breeding
Agrobacterium research and its
application went far beyond what
Smith and Townsend could foresee
when they found crown gall was
caused by the bacterium in 1907
Edward L. Barnard, Florida Department of Agriculture and Consumer Services, Bugwood.org; Mike Ellis, Ohio State University;
© 2014 American Society of Plant Biologists