Agrobacterium tumefaciens –

pathogen and useful tool

Agrobacterium tumefaciens is a
plant pathogen that induces
tumors on about 60% of
dicotyledonous angiosperms
and gymnosperms

Its tumor-inducing property also

makes it a valuable tool for
Cherry 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

 “A1907:
plantCrown
tumor gall is caused
of bacterial by a
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 Questions raised:
• What are these
compounds?
• Do they cause the
Octopine- tumors?
utilizing Octopine • How and why do the
strain bacteria cause the
plants to make
opines?

Nopaline- Nopaline
utilizing
strain 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 High levels of auxin
and cytokinin are found
in gall tissues

“It is possible for a cell to

acquire the capacity for
Normal plant
Normal plant Crown gall tissue autonomous growth as a
tissue cannot live
tissue grows and grows well result of the permanent
indefinitely in
survives when without added activation of growth-
hormone-free
auxin and hormones substance-synthesizing
medium
cytokinin (CK) systems”
are added to -AC Braun 1958
medium
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 heat-
killing the bacteria, no
tumors were formed
Periwinkle (Catharanthus roseus)
stems were inoculated with
When the tissue was
Agrobacterium tumefaciens, and
incubated at room
then incubated at room
temperature for four
temperature for various times,
days before heat-killing
followed by 5 days at 47°C to
the bacteria, many
kill the bacteria
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 A very large
plasmid was Heat treatment tumor
identified that is removes plasmid
heat
and makes bacteria
Avirulent present in virulent
non-pathogenic
but absent from
avirulent strains No
tumor

A plasmid Virulent
carrying a genetic + time
tumor
marker (antibiotic
resistance) was Avirulent
shown to be
confer virulence 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)
Renaturation kinetics of
Restriction labeled plasmid DNA
enzyme
Ti plasmid digestion fragments with various
unlabeled DNA samples
The key
Neg. control
finding was (untransformed
that Ti plant DNA)
plasmid DNA
DNA from
anneals with crown gall
DNA isolated
from the
crown gall, Pos. controls
meaning that (Ti plasmid)
the gall
“Our results suggest that the tumor- contains Ti
DNA Increasing amounts of
inducing principle first proposed by labeled Ti plasmid DNA
Braun (1947) is indeed DNA, as many
investigators have long suspected.”
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
Transfer DNA (T-DNA) moves
T-DNA 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
T4SS to “feed”
T-DNA Agrobacterium

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
The discovery that T-DNA was inserted into
the plant genome raised the possibility that
“any gene” could be transferred into plants
T-DNA

pTi 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 Agrobacterium-
mediated transformation
Basic research – plant transformation
allows in vivo study of plant genes

Expression pattern of an
Lobed-leaf phenotype of
auxin-inducible promoter
plants overexpressing
fused to GUS reporter gene
KNAT1 gene

Wild type Overexpression

Population
segregating for short-
hypocotyl 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
Transgenic plants
Bacillus expressing
thuringiensis insecticidal Bt gene
expressing Bt
toxin

Plant cell
expressing Bt
toxin

Agrobacterium tumefaciens
allows gene transfer into many
crop plants, particularly dicots
Wild-type Peanut plant
like soybean and peanut
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
Inoculate plant with
Introduce gene of interest into
engineered Agrobacterium
T-DNA region, and introduce into
Agrobacterium carrying vir genes
Agrobacterium
tumefaciens 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 5. Expression
bacterium of T-DNA and
Plant cell pathogenicity
Agrobacterium

© 2014 American Society of Plant Biologists

Many of the genes needed for T-DNA
transfer are found on the Ti plasmid

T-DNA cytok
inin opine RB
Conjugal
xin Transfer
au traR
LB
tra
The Ti plasmid carries
A genes required for
traI
B Ti plasmid
T-DNA transfer, Ti
G 214233bp plasmid conjugation
C
vir and opine metabolism
D occ
Virulence E
Opine
F
genes catabolism
replication

© 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 multi-
subunit 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
Opine
with Ti plasmid
Agrobacterium
Conjugation – without Ti plasmid
horizontal gene
Cell division transfer

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 E3
E2
D2
E2 D5
T-DNA processing F
E2 E3 T4SS
F VirB/D4 Integration
D2 LB RB
D2 of T-DNA
T-DNA
vir nucleus
genes Ti
Plasmid
vir genes
induction Expression of
VirG
p
T-DNA: auxin,
VirA
cytokinin, opine
VirG p Phenolics

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

T-DNA
on A. rhizogenes T-
DNA are not as well
pRi
understood as those
on the Ti plasmid

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