Download Agrobacterium tumefaciens- mediated Transformation of Plant Cells

Agrobacterium tumefaciensmediated Transformation of
Plant Cells
Andrew Binns, University of Pennsylvania, Philadelphia, Pennsylvania, USA
Angela Campbell, University of Pennsylvania, Philadelphia, Pennsylvania, USA
Introductory article
Article Contents
. Introduction
. Host Plant Characteristics
. The Mechanism of DNA Transfer
. The T-DNA Inside the Plant Cell: Activities Necessary
for Crown Gall Tumour Growth
. Opine Synthesis and Catabolism
. Ti Plasmid Conjugation
Agrobacterium tumefaciens is a Gram-negative soil bacterium that causes plant tumours by
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
Over 90 years ago Smith and Townsend determined that
the Gram-negative soil bacterium Agrobacterium tumefaciens, a member of the eubacterial family Rhizobiaceae, is
the organism responsible for the elicitation of crown gall
tumours in plants. Formation of these tumours (Figure 1)
occurs as a result of bacterial infection, usually at wound
sites, on many dicotyledonous and some monocotyledonous plants. In the 1940s Braun and colleagues demonstrated that the uncontrolled proliferation of the tumour
cells was not dependent on the continuous presence of the
inciting bacteria. During the 1960s Morel and colleagues
demonstrated that various bacteria-free crown gall tumours synthesized unusual amino acid–sugar conjugates
. Summary
(termed ‘opines’) whose chemistry was specified by the
strain of Agrobacterium that incited the tumour. Taken
together, this evidence suggested that a transformation of
the plant cell by Agrobacterium is responsible for the
formation of opine-producing crown gall tumours.
In the mid-1970s several groups discovered that a large
‘tumour-inducing’ (Ti) plasmid in A. tumefaciens is
necessary for this bacterium to incite tumours, and,
moreover, specifies the type of opine produced by that
tumour. Further investigations revealed that a piece of
DNA from this plasmid (the T-DNA) is transferred from
the bacterium into the plant cell where it integrates into the
nuclear DNA and is expressed. The activity of enzymes
encoded on the T-DNA results in the aberrant accumulation 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 Agrobacterium-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
Figure 1 Kalanchoe daigremontia leaves were scratched with a sterile
needle and infected with Agrobacterium tumefaciens carrying a Ti plasmid
(A, B, C) or lacking a Ti plasmid (D). Photograph taken 4 weeks after
infection.
The host range of A. tumefaciens was described originally
using the capacity of the bacterium to induce crown gall
tumours on the subject plant as the criterion. Such tumours
were found in a wide variety dicotyledonous plants, though
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
1
Agrobacterium tumefaciens-mediated Transformation of Plant Cells
occasional reports have appeared describing crown galls
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
for in vitro manipulation of both plant cells and bacteria,
has allowed investigators to screen for phenotypes other
than tumour formation in transformed cells. Studies
utilizing these alternative methods of monitoring for
DNA transfer have demonstrated that the host range of
Agrobacterium is extremely broad, including gymnosperms as well as most dicotyledonous and monocotyledonous plants. The critical feature that determines whether
any one of these highly diverse groups of plants can be
transformed experimentally is the identification of those
cells within the plant that are competent ‘recipients’ in
Agrobacterium-mediated DNA transfer and integration.
Several features of the plant cell are crucial in terms of
this competence. Early studies demonstrated that infection
of wound sites resulted in an extraordinary increase in the
tumour response compared with infection of unwounded
tissues. Two features of wounded plant tissues appear to be
critical. First, cells at the wound site initiate a defence
response that includes the synthesis of large quantities of
phenols, the production and release of sugars involved in
cell wall biosynthesis and the release of protons into the
extracellular space, resulting in acidification of the local
environment. While the phenols are thought to be
produced as antibacterial and antifungal agents, these
molecules, as well as the sugars and acidic pH, are
recognized by invading A. tumefaciens and serve to initiate
the DNA transfer process by this bacterium (see below).
Second, cells at the wound site undergo a few rounds of cell
division, thus helping to repair the wound site. This cell
division appears to be important in increasing the efficiency
with which the DNA transfer and integration takes place.
One of the least understood characteristics of plant cells
that defines their competence to be transformed is the cell
wall. Agrobacterium must attach to the plant cell in order
for DNA transfer to occur, and this attachment occurs at
specific but undefined sites on the cell wall. Current
evidence suggests that certain glycoproteins of the wall are
the target for the attachment apparatus of the bacterium,
but the biochemistry of this process represents one of the
important problems of Agrobacterium-mediated transformation that needs further study.
The Mechanism of DNA Transfer
The transfer of the T-DNA between the A. tumefaciens cell
and the plant cell is mediated in trans by virulence gene
products encoded on both the bacterium’s chromosome
and the Ti plasmid. The Ti plasmid is a large (200kilobase), single-copy plasmid (Figure 2) that encodes a
series of important functions which will be described in
2
T-DNA
E
Vir region
D
C
G
Tra region
pTi
B
Occ region
A
Rep
Tra region
Figure 2 Diagram of the Ti plasmid, illustrating the relative locations of
the T-DNA, the border sequences ( 4 ), the virulence (vir) region, the
conjugal transfer (tra) regions and the opine catabolism (occ) region.
detail below. These include expression of the virulence (vir)
gene products that are responsible for processing and
export of the T-DNA as well as genes involved in opine
catabolism and conjugal transfer of the Ti plasmid to other
agrobacteria. In addition, chromosomally encoded virulence genes (chv for chromosomal virulence) participate in
the DNA transfer processes. However, only strains of A.
tumefaciens that have a Ti plasmid are capable of
transforming a plant cell and causing a crown gall tumour.
The T-DNA of the Ti plasmid contains the DNA that
will ultimately be transferred into the plant cell and is
defined by left and right borders, which are 25-base pair
imperfect direct repeats. Any piece of DNA between these
borders will be transferred into the plant cell and randomly
integrated into the plant’s genome. This feature makes A.
tumefaciens quite useful in genetic engineering of plants,
because any gene placed between such borders – even on a
separate plasmid – will be transferred into the plant cell
and integrated into the plant genome. There are no known
limits as to size of the piece of DNA that can be transferred:
by reversing the orientation of the right border investigators have been able to demonstrate that the entire 200
kilobases of the Ti plasmid can be transferred into the plant
cell.
The T-DNA transformation process can be divided,
arbitrarily, into six major steps (Figure 3). First, the bacteria
attach to the plant cell wall in a process mediated by
chromosomally encoded bacterial genes. Second, competent (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 vir gene products are involved in processing the TDNA, in preparation for transfer into the plant cells. This
category contains the virC, virD and virE operons. Fourth,
the T-DNA and associated proteins are transported out of
the bacterium and into the plant cell through the activities
of the VirB proteins which are proposed to form a
membrane-localized pore between the bacterium and the
plant cell. Fifth, the T-DNA and its associated proteins are
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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
T-strand
1. Wounded plant releases
signals recognized by
VirA/VirG two-comp. system
in Agrobacterium
O
VirB pore
OH
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
is integrated into the chromosome. Finally, the genes on
the T-DNA are expressed, producing enzymes involved in
plant hormone synthesis and opine synthesis.
Attachment of the bacterium to the plant cell
wall
As described above, attachment of the bacterium to the
plant cell wall is required for DNA transfer. This is a poorly
understood process though genetic studies have shown
that it occurs even when the bacterium lacks the Ti plasmid.
Rather the products of numerous bacterial chromosomal
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
of DNA transfer. ChvA, ChvB and PscA are important for
synthesis and transport of b-1,2-glucans, which, while
required for attachment, have unknown function, but may
be required in association with plant cell receptors. The
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
Independent of attachment, the first of the Ti encoded steps
in the process of T-DNA transfer is recognition by A.
tumefaciens of the plant-derived molecules released at the
wound site and subsequent activation of vir gene expression. 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
response regulator. VirA is capable of autophosphorylation, and when VirA senses the wounded plant environment this phosphate is transferred to VirG.
Phosphorylated VirG acts as a transcription factor,
inducing expression of all the vir genes, including virA
and virG. This requirement for the plant wound environment prevents unnecessary expression of the other vir gene
products when the bacterium resides in locations not likely
to be targets for transformation.
While in most cases so far described, the phenols and an
acidic pH are required for vir gene induction, the sugars are
not required, although their presence will make the VirA/
VirG system approximately 100 times more sensitive to the
phenols. The recognition of the wound environment by
VirA may not be direct. For example, wound-released
sugars are sensed by ChvE, a chromosomal virulence
factor, which then interacts with the periplasmic region of
VirA. Moreover, the actual phenol binding site has not
been demonstrated. While genetic evidence suggests that
phenols are recognized by and bind to VirA, there is no
physical evidence of this. Similarly, while there is physical
evidence that other, chromosomally encoded proteins bind
the inducing phenols, there is no genetic evidence that
proves the relevance of this binding to the induction
process.
Production of transferred macromolecules
As a result of vir gene expression, the T-DNA is processed
in preparation for transfer into the plant cell. The left and
right borders of the T-DNA are recognized by VirD1/D2,
which acts as an endonuclease and makes a single-strand
nick of the T-DNA. VirD2 forms a covalent bond to the 5’
phosphate at the nick and a single-stranded intermediate –
‘VirD2–T-strand’ – is thought to form by strand displacement resulting from repair DNA synthesis starting at the
nick site. VirC1 may enhance VirD2–T-strand formation
under conditions of limiting VirD1/D2 by helping these
proteins bind to the border region. Another crucial element
in the T-DNA transfer and integration process is VirE2, a
single-stranded DNA-binding protein. This protein can
coat the length of the T-strand and is thought to protect it
from attack by exonucleases. Intriguingly, there is strong
evidence that both VirE2 and VirD2–T-strand can move
out of the bacterium independently and then interact once
inside the plant cell to form a ‘T-complex’ in which VirE2 is
4
bound to the VirD2–T-strand. The capacity for both
VirE2 and the VirD2–T-strand to move out of the bacterial
cell suggests that the DNA transfer process may actually be
a protein transfer process, and that one of the transferred
proteins is covalently attached to DNA, resulting in the
transfer of this macromolecule as well.
Macromolecular transport into the plant cell
The 11 VirB proteins, as well as VirD4, are postulated to
form a membrane-bound complex that is responsible for
the transfer of VirD2–T-strand, VirE2 and other substrates (see below) across the bacterial membranes and into
the plant cell. While a good deal is known about the
individual VirB proteins, information about the interactions that occur between them that are necessary for VirB
complex formation and activity is much less complete and
the subject of many current research projects. Genetic and
microscopic evidence indicates that all of the VirB proteins
are required to form a pilus (composed mainly of VirB2),
but has not yet revealed the nature of the association of this
pilus, or other parts of the VirB complex, with the plant
cell.
Several pieces of evidence indicate that the T-DNA
transfer process is homologous to conjugal plasmid DNA
transfer between bacteria. First, the DNA processing steps
are quite similar. In both cases a single-strand, site-specific
nick defines the origin of transfer, the 5’ nick site is
covalently attached to the nicking protein and in both cases
the transfer is polar, with the 5’-capped single-stranded
DNA–protein complex the first to enter a recipient cell.
Second, there is extensive homology between the proteins
responsible for conjugation and T-DNA transfer. These
include the processing proteins such as VirD2 and TraI (of
plasmid RP4) as well as the VirB proteins and those
proteins proposed to build the membrane-bound transfer
apparatus of IncN and IncP conjugal plasmids. Perhaps
the most convincing evidence that T-DNA transfer and
conjugal DNA transfer are homologous comes from two
remarkable findings. First, the promiscuous, broad host
range plasmid RSF1010 (IncQ) can be transferred from
Agrobacterium to plant cells, in a process that requires the
mobilization of genes on the plasmid as well as most of the
Vir proteins of the Ti plasmid. The second finding that has
profound implications regarding study of the VirB
complex was the observation that conjugal transfer of
RSF1010 between Agrobacterium is dependent on the VirB
system. Moreover, point mutations in the virB genes that
affect T-DNA transfer to plants affect interbacterial
conjugal transfer of RSF1010 in a quantitatively similar
fashion. Thus, analysis taking advantage of the conjugal
activity of the VirB complex is directly relevant to the
activity of this complex in plant transformation.
Recent studies have shown that characterization of the
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
activities in a variety of pathogenic bacteria. For example,
the Ptl (pertussis toxin liberation) proteins of Bordetella
pertussis, used to transport the pertussis toxin into host
cells, are homologous to the VirB proteins. Similarly,
proteins encoded by genes of the Helicobacter pylori cagII
pathogenicity island, and some of the Legionella pneumoniae pathogenicity proteins are also homologous to the
VirB proteins. In many cases, insights and discoveries
concerning the VirB proteins and their activities have been
directly applicable to these virulence systems. Moreover,
the homology between conjugal transfer and these protein
transfer systems also supports the hypothesis that the
DNA transfer observed in T-DNA transformation of plant
cells, as well as bacterial conjugation, is actually based on
mechanisms evolved for protein transport.
The T-DNA Inside the Plant Cell:
Activities Necessary for Crown Gall
Tumour Growth
Inside the plant cell, the T-complex is targeted to the plant
nucleus by the nuclear-localization signals found in VirD2
and VirE2, which interact with the endogenous nuclear
localization machinery. Once in the nucleus, the DNA
integrates stably into the plant genome in an as yet poorly
characterized process that is likely to include the activities
of both vir gene products and host enzymes. The
integration site appears to be random and more than one
T-DNA can integrate into a single genome. Once this
integration takes place, the genes on the T-DNA are stably
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
polyadenylation signals that are utilized by plant-specific
regulatory mechanisms. Additionally, expression of the TDNA genes is influenced by the site of integration in the
plant’s genome.
One intriguing question has been: how does the T-DNA
activity result in plant tumour formation? In the 1950s
Skoog and Miller demonstrated that nontransformed
plant tissues generally require both an auxin, such as
indole-3-acetic acid (IAA) and a cytokinin, such as N6isopentenyl adenosine, in order to proliferate continuously
in vitro. Intriguingly, Braun showed that cultured crown
gall tumours did not require these hormonal supplements
for continuous growth. Once the T-DNA was shown to be
responsible for tumorous growth of transformed plant
cells, the genes required for this phenotype were elucidated.
These studies demonstrated that the T-DNA from field
isolates of A. tumefaciens normally contains genes encoding for the biosynthesis of auxin and cytokinin. Specifi-
cally, most T-DNAs encode two enzymes whose activities
convert tryptophan into the active auxin, IAA. In addition,
a third T-DNA gene encodes an enzyme that converts
dimethylallyl pyrophosphate and adenosine monophosphate into isopentyl adenosine monophosphate. Endogenous plant enzymes can then convert this metabolite into
other molecules that have cytokinin activity. Thus, A.
tumefaciens T-DNA activity in the plant results in the
synthesis and accumulation of the two plant hormones that
can stimulate continuous cell division. Other T-DNA
genes appear to affect hormone responsiveness of the
transformed cells, but their mode of action is not known.
While most strains of A. tumefaciens induce crown gall
tumour growth by the transformed cells, a related
bacterium, Agrobacterium rhizogenes, causes the so-called
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
that of the Ti plasmid. The only real difference between
these plasmids is in their T-DNA gene products that affect
plant cell growth. While the Ri plasmid T-DNA encodes
the same auxin biosynthesis enzymes as does the Ti
plasmid, the Ri T-DNA also carries genes (the rol genes)
that encode a series of proteins which condition the
transformed cells to respond more than usual to the rootinducing activity of auxin. This includes, but is not limited
to, a greater sensitivity to this plant hormone. A finding of
considerable interest to those scientists involved in the
evolutionary implications of T-DNA transfer is that some
Nicotiana species appear to have acquired certain rol genes,
which are integrated into the genome of these species and
are expressed. The activity these plant rol genes may have is
unknown.
Opine Synthesis and Catabolism
The T-DNA in both A. tumefaciens and A. rhizogenes
transformed plant cells encodes enzymes that synthesize
novel amino acid–sugar conjugates called ‘opines’
(Figure 4). Whereas these opines cannot be metabolized
by the plant, their catabolism is encoded by the Ti and Ri
plasmids. The opine catabolic enzymes encoded by a
particular Ti plasmid will metabolize only those opines
whose synthesis is specified by that Ti plasmid’s T-DNA.
For example, the Ti plasmid that contains the T-DNA
encoding octopine synthesis by the plant also encodes
octopine metabolism genes, whereas nopaline synthesis
and metabolism genes are encoded by the nopaline Ti
plasmid. Thus, A. tumefaciens and A. rhizogenes genetically engineer the plant so as to create a growing region –
the crown gall tumour or hairy root – that produces the
opines for use by the bacteria as a specific nutrient source.
The ability to use opines specifically as a nutrient source is
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5
Agrobacterium tumefaciens-mediated Transformation of Plant Cells
Octopine
H2N
C
NH
(CH2)3
CH
HN
COOH
NH
CH3
CH
COOH
CH
COOH
Nopaline
H2N
C
NH
(CH2)3
HN
NH
HOOC
(CH2)2
CH
COOH
Mannopine
CH2OH
(CHOH)4
CH2
NH
HOOC
CH
O
(CH2)2
expression. The opine that induces conjugation is the same
opine synthesized by the T-DNA of that Ti or Ri plasmid.
This conjugation process is regulated by an autoinducer
similar to those involved in a variety of different ‘quorum
sensing’ (cell density sensing) mechanisms in prokaryotes.
The production of Agrobacterium autoinducer (AAI), an
acyl homoserine lactone, is induced by the opines, and
AAI, in turn, induces expression of the Ti (or Ri) localized
tra genes that are required for conjugal DNA transfer
between agrobacteria. This opine dependency of conjugation results in the transfer of the inciting Ti or Ri plasmid to
plasmid-free agrobacteria in the soils and ensures that the
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
the Ti or Ri plasmid results in a greater proportion of the
Agrobacterium population carrying the selective advantage of being able to utilize opines produced by the
transformed plant cells.
C
NH2
Summary
Agropine
CHOH
(CHOH)3
NH
O
(CH2)2 CONH2
O
Figure 4 The chemical composition of some well-characterized opines.
believed to give A. tumefaciens its selective advantage in the
crown gall environment because, with only a few exceptions, other soil bacteria cannot metabolize these molecules. Moreover, recent studies have shown that opines can
move out of the tumour, via roots, into the surrounding
soil, thus serving as a nutrient source to promote,
selectively, the growth of Agrobacterium within the rhizosphere.
Agrobacterium tumefaciens and Agrobacterium rhizogenes
are nature’s most successful plant genetic engineers. These
bacteria have evolved the capacity to deliver DNA into
plant cells. The expression of this T-DNA not only results
in the proliferation of the cell carrying it, but causes such
transformed cells to produce nutrients that serve as
dedicated carbon and nitrogen sources to the inciting
bacterium. Plant and agricultural scientists have been able
to harness the DNA transfer activity of Agrobacterium so
that it is now possible to genetically engineer a wide variety
of plants. In addition to this remarkable technical advance,
the study of Agrobacterium continues to provide novel
insights into the general mechanisms whereby both plant
and animal pathogens transfer macromolecules into host
cells, resulting in disease states in the host organisms.
Further Reading
Ti Plasmid Conjugation
While strains that contain the Ti plasmid can metabolize
opines, the majority of strains isolated in nature do not
contain a Ti plasmid. This leads to the question, how does
the genetic engineering of a plant cell by A. tumefaciens to
form an opine-producing tumour result in a selective
advantage if most of the A. tumefaciens in the surrounding
environment cannot metabolize these compounds? The
answer lies in the ability of strains that contain the Ti (or
Ri) plasmid to conjugate it into strains lacking such a
plasmid. Intriguingly, Ti and Ri plasmid conjugation is
induced by opines and occurs independently of vir gene
6
Binns AN and Howitz VR (1994) The genetic and chemical basis of host
recognition by Agrobacterium tumefaciens. Current Topics in Microbiology and Immunology 192: 119–138.
Christie PJ (1997) Agrobacterium tumefaciens T-complex transport
apparatus: a paradigm for a new family of multifunctional transporters in Eubacteria. Journal of Bacteriology 179: 3085–3094.
Kado CI (1998) Agrobacterium-mediated horizontal gene transfer.
Genetic Engineering 20: 1–24.
Spaink H, Kondorosi A and Hooykaas PJJ (eds) (1998) The
Rhizobiaceae. Dordrecht: Kluwer Press.
Winans SC, Burns DL and Christie PJ (1996) Adaptation of a conjugal
transfer system for the export of pathogenic macromolecules. Trends
in Microbiology 4: 64–68.
Zupan J and Zambryski P (1997) The Agrobacterium DNA transfer
complex. Critical Reviews in Plant Science 16: 279–295.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net