Agrobacterium tumefaciens- Introductory article

mediated Transformation of . Introduction

Article Contents

Plant Cells . Host Plant Characteristics

. The Mechanism of DNA Transfer

Andrew Binns, University of Pennsylvania, Philadelphia, Pennsylvania, USA . The T-DNA Inside the Plant Cell: Activities Necessary
for Crown Gall Tumour Growth
Angela Campbell, University of Pennsylvania, Philadelphia, Pennsylvania, USA . Opine Synthesis and Catabolism
. Ti Plasmid Conjugation
Agrobacterium tumefaciens is a Gram-negative soil bacterium that causes plant tumours by . Summary
transferring a portion of DNA from a resident ‘tumour inducing’ (Ti) plasmid into plant cells
where it is integrated into a plant chromosome and expressed. This bacteria’s capacity for
DNA transfer is the basis of most current plant genetic engineering and make it a model
system for the study of pathogenic bacteria that transfer virulence factors to any eukaryotic
cell.

Introduction (termed ‘opines’) whose chemistry was specified by the

Over 90 years ago Smith and Townsend determined that strain of Agrobacterium that incited the tumour. Taken
the Gram-negative soil bacterium Agrobacterium tumefa- together, this evidence suggested that a transformation of
ciens, a member of the eubacterial family Rhizobiaceae, is the plant cell by Agrobacterium is responsible for the
the organism responsible for the elicitation of crown gall formation of opine-producing crown gall tumours.
tumours in plants. Formation of these tumours (Figure 1) In the mid-1970s several groups discovered that a large
occurs as a result of bacterial infection, usually at wound ‘tumour-inducing’ (Ti) plasmid in A. tumefaciens is
sites, on many dicotyledonous and some monocotyledo- necessary for this bacterium to incite tumours, and,
nous plants. In the 1940s Braun and colleagues demon- moreover, specifies the type of opine produced by that
strated that the uncontrolled proliferation of the tumour tumour. Further investigations revealed that a piece of
cells was not dependent on the continuous presence of the DNA from this plasmid (the T-DNA) is transferred from
inciting bacteria. During the 1960s Morel and colleagues the bacterium into the plant cell where it integrates into the
demonstrated that various bacteria-free crown gall tu- nuclear DNA and is expressed. The activity of enzymes
mours synthesized unusual amino acid–sugar conjugates encoded on the T-DNA results in the aberrant accumula-
tion of the plant hormones auxin and cytokinin which, in
turn, leads to uncontrolled cell division and tumour
formation. Moreover, the T-DNA encodes enzymes that
specify the synthesis of opines. Interestingly, these
metabolites provide a dedicated carbon and nitrogen
source for the inciting bacterium and are the likely driving
force in the evolution of this process. The objectives of this
article are to: (1) review the basic steps of the Agrobacter-
ium-mediated transfer of DNA into plant cells; (2) discuss
how the expression of this DNA results in tumour
formation and opine production; (3) examine the role
opine production plays in creating a selective advantage for
the inciting bacteria.

Host Plant Characteristics

The host range of A. tumefaciens was described originally
Figure 1 Kalanchoe daigremontia leaves were scratched with a sterile
needle and infected with Agrobacterium tumefaciens carrying a Ti plasmid using the capacity of the bacterium to induce crown gall
(A, B, C) or lacking a Ti plasmid (D). Photograph taken 4 weeks after tumours on the subject plant as the criterion. Such tumours
infection. were found in a wide variety dicotyledonous plants, though

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 Agrobacterium tumefaciens-mediated Transformation of Plant Cells

occasional reports have appeared describing crown galls T-DNA

on some monocots. However, the development of plant
selectable markers (e.g. antibiotic or herbicide resistance)
that can be engineered into the T-DNA, and the capacity E
for in vitro manipulation of both plant cells and bacteria, D
has allowed investigators to screen for phenotypes other C Tra region
Vir region
than tumour formation in transformed cells. Studies G pTi
utilizing these alternative methods of monitoring for
B
DNA transfer have demonstrated that the host range of
Agrobacterium is extremely broad, including gymnos- A Occ region
perms as well as most dicotyledonous and monocotyledo-
nous plants. The critical feature that determines whether
Rep Tra region
any one of these highly diverse groups of plants can be
transformed experimentally is the identification of those Figure 2 Diagram of the Ti plasmid, illustrating the relative locations of
cells within the plant that are competent ‘recipients’ in the T-DNA, the border sequences ( 4 ), the virulence (vir) region, the
Agrobacterium-mediated DNA transfer and integration. conjugal transfer (tra) regions and the opine catabolism (occ) region.
Several features of the plant cell are crucial in terms of
this competence. Early studies demonstrated that infection detail below. These include expression of the virulence (vir)
of wound sites resulted in an extraordinary increase in the gene products that are responsible for processing and
tumour response compared with infection of unwounded export of the T-DNA as well as genes involved in opine
tissues. Two features of wounded plant tissues appear to be catabolism and conjugal transfer of the Ti plasmid to other
critical. First, cells at the wound site initiate a defence agrobacteria. In addition, chromosomally encoded viru-
response that includes the synthesis of large quantities of lence genes (chv for chromosomal virulence) participate in
phenols, the production and release of sugars involved in the DNA transfer processes. However, only strains of A.
cell wall biosynthesis and the release of protons into the tumefaciens that have a Ti plasmid are capable of
extracellular space, resulting in acidification of the local transforming a plant cell and causing a crown gall tumour.
environment. While the phenols are thought to be The T-DNA of the Ti plasmid contains the DNA that
produced as antibacterial and antifungal agents, these will ultimately be transferred into the plant cell and is
molecules, as well as the sugars and acidic pH, are defined by left and right borders, which are 25-base pair
recognized by invading A. tumefaciens and serve to initiate imperfect direct repeats. Any piece of DNA between these
the DNA transfer process by this bacterium (see below). borders will be transferred into the plant cell and randomly
Second, cells at the wound site undergo a few rounds of cell integrated into the plant’s genome. This feature makes A.
division, thus helping to repair the wound site. This cell tumefaciens quite useful in genetic engineering of plants,
division appears to be important in increasing the efficiency because any gene placed between such borders – even on a
with which the DNA transfer and integration takes place. separate plasmid – will be transferred into the plant cell
One of the least understood characteristics of plant cells and integrated into the plant genome. There are no known
that defines their competence to be transformed is the cell limits as to size of the piece of DNA that can be transferred:
wall. Agrobacterium must attach to the plant cell in order by reversing the orientation of the right border investiga-
for DNA transfer to occur, and this attachment occurs at tors have been able to demonstrate that the entire 200
specific but undefined sites on the cell wall. Current kilobases of the Ti plasmid can be transferred into the plant
evidence suggests that certain glycoproteins of the wall are cell.
the target for the attachment apparatus of the bacterium, The T-DNA transformation process can be divided,
but the biochemistry of this process represents one of the arbitrarily, into six major steps (Figure 3). First, the bacteria
important problems of Agrobacterium-mediated transfor- attach to the plant cell wall in a process mediated by
mation that needs further study. chromosomally encoded bacterial genes. Second, compe-
tent (for transformation) plant cells are recognized
through the activities of virA and virG, resulting in the
expression of the other virulence genes. Third, several of
The Mechanism of DNA Transfer the vir gene products are involved in processing the T-
DNA, in preparation for transfer into the plant cells. This
The transfer of the T-DNA between the A. tumefaciens cell category contains the virC, virD and virE operons. Fourth,
and the plant cell is mediated in trans by virulence gene the T-DNA and associated proteins are transported out of
products encoded on both the bacterium’s chromosome the bacterium and into the plant cell through the activities
and the Ti plasmid. The Ti plasmid is a large (200- of the VirB proteins which are proposed to form a
kilobase), single-copy plasmid (Figure 2) that encodes a membrane-localized pore between the bacterium and the
series of important functions which will be described in plant cell. Fifth, the T-DNA and its associated proteins are

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 Agrobacterium tumefaciens-mediated Transformation of Plant Cells

Plant cell
T-DNA 6. T-DNA is stably
integrated into the
plant’s genome

5. T-DNA is targeted
for the plant nucleus
by NLSs found in
VirD2 and VirE2

1. Wounded plant releases T-strand

signals recognized by
VirA/VirG two-comp. system
in Agrobacterium

O
OH VirB pore

VirA

2. vir gene expression

induced by VirG-P

VirG
vir genes
VirD2 VirE2

Ti plasmid
4. T-strand, covered by
VirE2 to form T-complex,
exits through the VirB pore

3. T-strand formed
by displacement;
capped by VirD2
Agrobacterium

Figure 3 Steps in Agrobacterium-mediated DNA transfer into a plant cell.

transported to the nucleus of the plant cell, where the DNA understood process though genetic studies have shown
is integrated into the chromosome. Finally, the genes on that it occurs even when the bacterium lacks the Ti plasmid.
the T-DNA are expressed, producing enzymes involved in Rather the products of numerous bacterial chromosomal
plant hormone synthesis and opine synthesis. virulence genes (e.g. chvA, chvB, pscA and various att
genes) are necessary. Mutations in these genes result in
bacteria incapable of attaching to plant cells and incapable
Attachment of the bacterium to the plant cell of DNA transfer. ChvA, ChvB and PscA are important for
wall synthesis and transport of b-1,2-glucans, which, while
required for attachment, have unknown function, but may
As described above, attachment of the bacterium to the be required in association with plant cell receptors. The
plant cell wall is required for DNA transfer. This is a poorly biochemical roles of the att gene products are unknown.

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 Agrobacterium tumefaciens-mediated Transformation of Plant Cells

Induction of vir gene expression bound to the VirD2–T-strand. The capacity for both
VirE2 and the VirD2–T-strand to move out of the bacterial
Independent of attachment, the first of the Ti encoded steps cell suggests that the DNA transfer process may actually be
in the process of T-DNA transfer is recognition by A. a protein transfer process, and that one of the transferred
tumefaciens of the plant-derived molecules released at the proteins is covalently attached to DNA, resulting in the
wound site and subsequent activation of vir gene expres- transfer of this macromolecule as well.
sion. A two-component regulatory system, composed of
the constitutively expressed vir gene products VirA and
VirG, is necessary for these events to occur. VirA is a
membrane-bound sensor kinase, whereas VirG is the Macromolecular transport into the plant cell
response regulator. VirA is capable of autophosphoryla- The 11 VirB proteins, as well as VirD4, are postulated to
tion, and when VirA senses the wounded plant environ- form a membrane-bound complex that is responsible for
ment this phosphate is transferred to VirG. the transfer of VirD2–T-strand, VirE2 and other sub-
Phosphorylated VirG acts as a transcription factor, strates (see below) across the bacterial membranes and into
inducing expression of all the vir genes, including virA the plant cell. While a good deal is known about the
and virG. This requirement for the plant wound environ- individual VirB proteins, information about the interac-
ment prevents unnecessary expression of the other vir gene tions that occur between them that are necessary for VirB
products when the bacterium resides in locations not likely complex formation and activity is much less complete and
to be targets for transformation. the subject of many current research projects. Genetic and
While in most cases so far described, the phenols and an microscopic evidence indicates that all of the VirB proteins
acidic pH are required for vir gene induction, the sugars are are required to form a pilus (composed mainly of VirB2),
not required, although their presence will make the VirA/ but has not yet revealed the nature of the association of this
VirG system approximately 100 times more sensitive to the pilus, or other parts of the VirB complex, with the plant
phenols. The recognition of the wound environment by cell.
VirA may not be direct. For example, wound-released Several pieces of evidence indicate that the T-DNA
sugars are sensed by ChvE, a chromosomal virulence transfer process is homologous to conjugal plasmid DNA
factor, which then interacts with the periplasmic region of transfer between bacteria. First, the DNA processing steps
VirA. Moreover, the actual phenol binding site has not are quite similar. In both cases a single-strand, site-specific
been demonstrated. While genetic evidence suggests that nick defines the origin of transfer, the 5’ nick site is
phenols are recognized by and bind to VirA, there is no covalently attached to the nicking protein and in both cases
physical evidence of this. Similarly, while there is physical the transfer is polar, with the 5’-capped single-stranded
evidence that other, chromosomally encoded proteins bind DNA–protein complex the first to enter a recipient cell.
the inducing phenols, there is no genetic evidence that Second, there is extensive homology between the proteins
proves the relevance of this binding to the induction responsible for conjugation and T-DNA transfer. These
process. include the processing proteins such as VirD2 and TraI (of
plasmid RP4) as well as the VirB proteins and those
Production of transferred macromolecules proteins proposed to build the membrane-bound transfer
apparatus of IncN and IncP conjugal plasmids. Perhaps
As a result of vir gene expression, the T-DNA is processed the most convincing evidence that T-DNA transfer and
in preparation for transfer into the plant cell. The left and conjugal DNA transfer are homologous comes from two
right borders of the T-DNA are recognized by VirD1/D2, remarkable findings. First, the promiscuous, broad host
which acts as an endonuclease and makes a single-strand range plasmid RSF1010 (IncQ) can be transferred from
nick of the T-DNA. VirD2 forms a covalent bond to the 5’ Agrobacterium to plant cells, in a process that requires the
phosphate at the nick and a single-stranded intermediate – mobilization of genes on the plasmid as well as most of the
‘VirD2–T-strand’ – is thought to form by strand displace- Vir proteins of the Ti plasmid. The second finding that has
ment resulting from repair DNA synthesis starting at the profound implications regarding study of the VirB
nick site. VirC1 may enhance VirD2–T-strand formation complex was the observation that conjugal transfer of
under conditions of limiting VirD1/D2 by helping these RSF1010 between Agrobacterium is dependent on the VirB
proteins bind to the border region. Another crucial element system. Moreover, point mutations in the virB genes that
in the T-DNA transfer and integration process is VirE2, a affect T-DNA transfer to plants affect interbacterial
single-stranded DNA-binding protein. This protein can conjugal transfer of RSF1010 in a quantitatively similar
coat the length of the T-strand and is thought to protect it fashion. Thus, analysis taking advantage of the conjugal
from attack by exonucleases. Intriguingly, there is strong activity of the VirB complex is directly relevant to the
evidence that both VirE2 and VirD2–T-strand can move activity of this complex in plant transformation.
out of the bacterium independently and then interact once Recent studies have shown that characterization of the
inside the plant cell to form a ‘T-complex’ in which VirE2 is VirB complex and its activities have importance that

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 Agrobacterium tumefaciens-mediated Transformation of Plant Cells

extends beyond DNA transfer systems to include virulence cally, most T-DNAs encode two enzymes whose activities
activities in a variety of pathogenic bacteria. For example, convert tryptophan into the active auxin, IAA. In addition,
the Ptl (pertussis toxin liberation) proteins of Bordetella a third T-DNA gene encodes an enzyme that converts
pertussis, used to transport the pertussis toxin into host dimethylallyl pyrophosphate and adenosine monopho-
cells, are homologous to the VirB proteins. Similarly, sphate into isopentyl adenosine monophosphate. Endo-
proteins encoded by genes of the Helicobacter pylori cagII genous plant enzymes can then convert this metabolite into
pathogenicity island, and some of the Legionella pneumo- other molecules that have cytokinin activity. Thus, A.
niae pathogenicity proteins are also homologous to the tumefaciens T-DNA activity in the plant results in the
VirB proteins. In many cases, insights and discoveries synthesis and accumulation of the two plant hormones that
concerning the VirB proteins and their activities have been can stimulate continuous cell division. Other T-DNA
directly applicable to these virulence systems. Moreover, genes appear to affect hormone responsiveness of the
the homology between conjugal transfer and these protein transformed cells, but their mode of action is not known.
transfer systems also supports the hypothesis that the While most strains of A. tumefaciens induce crown gall
DNA transfer observed in T-DNA transformation of plant tumour growth by the transformed cells, a related
cells, as well as bacterial conjugation, is actually based on bacterium, Agrobacterium rhizogenes, causes the so-called
mechanisms evolved for protein transport. hairy-root disease, in which the transformed cells form
numerous roots. These bacteria also carry a large plasmid,
in this case called the Ri (root-inducing) plasmid, which has
a DNA transfer mechanism that is interchangeable with
The T-DNA Inside the Plant Cell: that of the Ti plasmid. The only real difference between
these plasmids is in their T-DNA gene products that affect
Activities Necessary for Crown Gall plant cell growth. While the Ri plasmid T-DNA encodes
Tumour Growth the same auxin biosynthesis enzymes as does the Ti
plasmid, the Ri T-DNA also carries genes (the rol genes)
Inside the plant cell, the T-complex is targeted to the plant that encode a series of proteins which condition the
nucleus by the nuclear-localization signals found in VirD2 transformed cells to respond more than usual to the root-
and VirE2, which interact with the endogenous nuclear inducing activity of auxin. This includes, but is not limited
localization machinery. Once in the nucleus, the DNA to, a greater sensitivity to this plant hormone. A finding of
integrates stably into the plant genome in an as yet poorly considerable interest to those scientists involved in the
characterized process that is likely to include the activities evolutionary implications of T-DNA transfer is that some
of both vir gene products and host enzymes. The Nicotiana species appear to have acquired certain rol genes,
integration site appears to be random and more than one which are integrated into the genome of these species and
T-DNA can integrate into a single genome. Once this are expressed. The activity these plant rol genes may have is
integration takes place, the genes on the T-DNA are stably unknown.
maintained in the chromosome and transcribed and
translated. The T-DNA genes, though derived from a
bacterium, are considered eukaryotic because they have
eukaryotic expression signals such as a TATA box and Opine Synthesis and Catabolism
polyadenylation signals that are utilized by plant-specific
regulatory mechanisms. Additionally, expression of the T- The T-DNA in both A. tumefaciens and A. rhizogenes
DNA genes is influenced by the site of integration in the transformed plant cells encodes enzymes that synthesize
plant’s genome. novel amino acid–sugar conjugates called ‘opines’
One intriguing question has been: how does the T-DNA (Figure 4). Whereas these opines cannot be metabolized
activity result in plant tumour formation? In the 1950s by the plant, their catabolism is encoded by the Ti and Ri
Skoog and Miller demonstrated that nontransformed plasmids. The opine catabolic enzymes encoded by a
plant tissues generally require both an auxin, such as particular Ti plasmid will metabolize only those opines
indole-3-acetic acid (IAA) and a cytokinin, such as N6- whose synthesis is specified by that Ti plasmid’s T-DNA.
isopentenyl adenosine, in order to proliferate continuously For example, the Ti plasmid that contains the T-DNA
in vitro. Intriguingly, Braun showed that cultured crown encoding octopine synthesis by the plant also encodes
gall tumours did not require these hormonal supplements octopine metabolism genes, whereas nopaline synthesis
for continuous growth. Once the T-DNA was shown to be and metabolism genes are encoded by the nopaline Ti
responsible for tumorous growth of transformed plant plasmid. Thus, A. tumefaciens and A. rhizogenes geneti-
cells, the genes required for this phenotype were elucidated. cally engineer the plant so as to create a growing region –
These studies demonstrated that the T-DNA from field the crown gall tumour or hairy root – that produces the
isolates of A. tumefaciens normally contains genes encod- opines for use by the bacteria as a specific nutrient source.
ing for the biosynthesis of auxin and cytokinin. Specifi- The ability to use opines specifically as a nutrient source is

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 Agrobacterium tumefaciens-mediated Transformation of Plant Cells

Octopine expression. The opine that induces conjugation is the same

opine synthesized by the T-DNA of that Ti or Ri plasmid.
H2N
This conjugation process is regulated by an autoinducer
C NH (CH2)3 CH COOH
HN
similar to those involved in a variety of different ‘quorum
NH sensing’ (cell density sensing) mechanisms in prokaryotes.
CH3 CH COOH The production of Agrobacterium autoinducer (AAI), an
acyl homoserine lactone, is induced by the opines, and
Nopaline AAI, in turn, induces expression of the Ti (or Ri) localized
tra genes that are required for conjugal DNA transfer
H2N
between agrobacteria. This opine dependency of conjuga-
C NH (CH2)3 CH COOH
HN
tion results in the transfer of the inciting Ti or Ri plasmid to
NH plasmid-free agrobacteria in the soils and ensures that the
HOOC (CH2)2 CH COOH recipient bacterium will attain a Ti plasmid that will direct
metabolism of the opines specified by the inciting
bacterium. Thus, the opine-mediated conjugal transfer of
Mannopine the Ti or Ri plasmid results in a greater proportion of the
Agrobacterium population carrying the selective advan-
CH2OH (CHOH)4 CH2
tage of being able to utilize opines produced by the
NH transformed plant cells.
O
HOOC CH (CH2)2 C
NH2

Agropine Summary
CHOH (CHOH)3
NH
Agrobacterium tumefaciens and Agrobacterium rhizogenes
are nature’s most successful plant genetic engineers. These
O bacteria have evolved the capacity to deliver DNA into
(CH2)2 CONH2
plant cells. The expression of this T-DNA not only results
O
in the proliferation of the cell carrying it, but causes such
Figure 4 The chemical composition of some well-characterized opines. transformed cells to produce nutrients that serve as
dedicated carbon and nitrogen sources to the inciting
bacterium. Plant and agricultural scientists have been able
believed to give A. tumefaciens its selective advantage in the to harness the DNA transfer activity of Agrobacterium so
crown gall environment because, with only a few excep- that it is now possible to genetically engineer a wide variety
tions, other soil bacteria cannot metabolize these mole- of plants. In addition to this remarkable technical advance,
cules. Moreover, recent studies have shown that opines can the study of Agrobacterium continues to provide novel
move out of the tumour, via roots, into the surrounding insights into the general mechanisms whereby both plant
soil, thus serving as a nutrient source to promote, and animal pathogens transfer macromolecules into host
selectively, the growth of Agrobacterium within the rhizo- cells, resulting in disease states in the host organisms.
sphere.
Further Reading
Ti Plasmid Conjugation Binns AN and Howitz VR (1994) The genetic and chemical basis of host
recognition by Agrobacterium tumefaciens. Current Topics in Micro-
biology and Immunology 192: 119–138.
While strains that contain the Ti plasmid can metabolize Christie PJ (1997) Agrobacterium tumefaciens T-complex transport
opines, the majority of strains isolated in nature do not apparatus: a paradigm for a new family of multifunctional transpor-
contain a Ti plasmid. This leads to the question, how does ters in Eubacteria. Journal of Bacteriology 179: 3085–3094.
the genetic engineering of a plant cell by A. tumefaciens to Kado CI (1998) Agrobacterium-mediated horizontal gene transfer.
form an opine-producing tumour result in a selective Genetic Engineering 20: 1–24.
advantage if most of the A. tumefaciens in the surrounding Spaink H, Kondorosi A and Hooykaas PJJ (eds) (1998) The
Rhizobiaceae. Dordrecht: Kluwer Press.
environment cannot metabolize these compounds? The
Winans SC, Burns DL and Christie PJ (1996) Adaptation of a conjugal
answer lies in the ability of strains that contain the Ti (or transfer system for the export of pathogenic macromolecules. Trends
Ri) plasmid to conjugate it into strains lacking such a in Microbiology 4: 64–68.
plasmid. Intriguingly, Ti and Ri plasmid conjugation is Zupan J and Zambryski P (1997) The Agrobacterium DNA transfer
induced by opines and occurs independently of vir gene complex. Critical Reviews in Plant Science 16: 279–295.

6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net