Download Agro bacterium-mediated Transformation

UNIT I
PLANT TRANSFORMATION
Lecture 1&2
Agro bacterium-mediated Transformation
The most promising method of transforming plant cells makes use of a plasmid
called the Ti plasmid, which is found within the bacterium Agrobacterium tumefaciens.
Fraley et al. (1983) and An et al. (1985) exploited the natural ability of Agrobacterium
tumefaciens to transfer DNA into plant chromosomes. This gram-negative, rod-shaped,
motile bacterium lives in soil and invades many dicotyledonous plants and some
gymnosperms when they are damaged at soil level. The bacterium enters the fresh wound
and attaches itself to the wall of an intact cell, after which it transfers a relatively small
part of its Ti plasmid into the nucleus of the cell. This plasmid integrates some of its
DNA ;( T-DNA) into the chromosome of its host plant cells. This is a unique
morphogenetic phenomenon wherein a permanent incorporation of a portion of bacterial
genome completely diverts the host cells from their predetermined path of development.
This infection results in a crown gall, i.e., a lump or callus of tumor tissue that
grows in an undifferentiated way at the site of infection. The cells of the crown gall
acquire the properties of independent, unregulated growth. When crown gall cells are
cultured, they grow to form a callus even in media devoid of the plant hormones that
must be added to induce normal plant cells to grow in culture.
Ti plasmids have evolved solely for the benefit of the bacterium. The potential of
the Agrobacterium Ti plasmid as a vector arises from the ability of the bacterium to
somehow transfer and stably integrate a piece of the plasmid DNA into the plant nuclear
genome; here is a case of a natural vector system. The transferred DNA is known as
T-DNA and carries several genes which are expressed within the plant and which have
dramatic effects on its metabolism.
Characteristics of Agro bacteria

related to Rhizobium

contain large plasmids: Ti (A. tumefaciens); Ri (A. rhizogenes)

integration of a part of the plasmid (T-DNA) induces the tumorous or rooty
growth T-DNA;

this DNA directs the synthesis of opines, which are sources of carbon and
nitrogen molecules used for the continued bacterial growth

each bacterial strain can utilize one of the three opine classes: octopine, nopaline
and agropine

host range of A. tumefaciens
o


331 genera; 643 species
variability is seen for infection between
o
varieties of a species
o
the organ of the plant
monocot species except for some members of the Liliales and Arales genera are
not susceptible
Characteristics of the Ti-Plasmid


large circular plasmid contains genes for:
o
virulence
o
catabolism of specific opines
o
host-directed opine synthesis
o
host-directed, bacterial-type plant hormones
virulence genes are grouped genes for synthesis of opines and plant hormones are
contained on the T-DNA; T-DNA is bordered by 25 bp direct repeats; only the TR
border is required for transfer although the Ti border increased the efficiency
when it is present; no sequences other that the borders are required for transfer
Transfer of T-DNA to Plant Cells
The T-DNA that is transferred to the plant cells contains genes which encode for proteins
involved in opine and plant-type phytohormone biosynthesis. Although these genes
reside on a bacterial plasmid they are only active in a plant cell. To be transcribed in the
plant cell, though, they must contain typical eukaryotic controlling sequences. All of
these genes have been shown to contain TATA and CAAT boxes and typical plant
polyadenylation signals. Further, portions of the promoters and the polyadenylation
regions have been added to marker genes as a method of following the transformation
events.
Gene transfer and the Ti plasmids
Agrobacterium has the ability to transfer DNA into the plant cells

the DNA from the bacterium is integrated into a plant chromosome

the transferred DNA (T-DNA) is a portion of a large plasmid

in Agrobacterium tumefaciens, this is the Ti (=tumor-inducing) plasmid
The T-DNA includes genes for enzymes involved in IAA, cytokinin, and opine
biosynthesis

two genes, iaaM and iaaH, encode enzymes for the production of IAA

one gene, iptZ, encodes an enzyme for the production of cytokinin

these genes are different from the plant genes for hormone synthesis enzymes

these genes on the bacterial Ti plasmid have eukaryotic plant-type promoters

Another gene encodes octopine synthetase, an enzyme for the production of
opine, a modified amino acid.

The opines are synthesized and secreted within the crown gall. The three opines
include:

Octopine(condensed product of arginine and pyruvic acid)

Nopaline(condensed product of arginine with α-ketoglutaraldehyde)

Agropine(bicyclic sugar derivative of glutamic acid)
The overproduction of hormones causes cell proliferation, while the opine produced
serves as a source of nitrogen for the bacterium, but not for the plant.
Mutations in individual genes of the T-DNA can cause different types of tumors to grow

mutations in the iaa genes result in shooty tumors

mutations in the ipt gene causes rooty tumors
How Agrobacterium and the Ti plasmid have been used as a system for plant
transformation?

the genes for hormone and opine synthesis are removed from the Ti plasmid

they are replaced with a selectable marker, a gene to allow for growth of only
transformed cells, usually a drug- or herbicide-resistance gene (kanamycin
resistance)

can also add in another gene, from any source, as long as it has a plant promoter
(such as herbicide resistance, insecticidal proteins, vitamin synthesis, etc.)

transform Agrobacterium with this modified plasmid, then dip leaf disks in a
suspension of the Agrobacterium

the bacterium transfers the T-DNA with these genes into the plant cells and the
genes become integrated into the plant chromosome

the leaf disks are placed on tissue culture medium with the drug, so only drugresistant cells grow

with the appropriate addition of hormones, leaf cells can grow and eventually
develop into shoots and roots, regenerating a whole plant that is transgenic
Two primary steps in transformation

binding of Agrobacterium to a plant cell (up to 200/cell can attach)

transfer of DNA to the plant cell (multiple T-DNAs can be transferred)
Each step involves a different set of genes. Binding to plant cell requires three
chromosomal Agrobacterium genes:
chvA and chvB - mutants at these two loci result in a marked reduction in Agrobacterium
binding to plant cells; chvA may encode a transport factor and chvB encodes a protein
involved in 2-linked beta-glucan synthesis
pscA - required for the synthesis of the major neutral and acid extracellular
polysaccharides
Vir Region
Initially it was thought that wounding, an absolute prerequisite for Agrobacterium
transformation, was required for the plant cell and bacteria to come in contact with each
other. Actually, wounded cells secrete low-molecular weight molecules that stimulate the
vir genes. These molecules are acetosyringone and hydroxy-acetosyringone. These two
molecules stimulate the synthesis of several vir genes. Further, acetosyringone can act as
a chemical attractant in vitro and thus may act as chemotactic agent in nature.
The vir region contains six genes. virA and virG are the only two monocistronic loci, the
other four are polycistronic and encode several proteins. The regulation of these
important genes is integrated and involves a cascade of transcriptional events. The
cascade begins with virA which is constitutively expressed. The protein encodes a
transmembrane protein that senses the chemical environment. It is assumed that this
protein senses the presence of acetosyringone. VirG produces two transcripts that differ at
the 5' end of the mRNA, one which is constitutively expressed and a longer mRNA that
only is expressed in the presence of plant inducible compounds. VirB and virE
polypeptides are the two vir products that are highly induced by the wound induced plant
phenolic compounds.
Vir
Gene
VirA
Function
encodes a single protein which resembles a transmembrane chemoreceptor
found in other bacteria; constitutively expressed; monocistronic
VirG
a positive regulatory protein which relays environmental information (when
plant inducible factors are present) to other vir loci; requires virA; strongly
affects virB,C,D, E: monocistronic
VirE
a single-stranded DNA binding protein that appears to coat the T-strand during
transfer to the plant cell; polycistronic
VirC
site-specific endonuclease that cleaves at the 25 bp direct repeats borders of the
and virD T-DNA; produces a T-strand that is the intermediate molecule that is
transported to the plant cell; polycistronic
VirB
may play a role in directingT-DNA transfer events at the bacterial cell surface
Types of Ti Plasmids
Two classes of Ti-plasmids exist. The nopaline and octopine Ti-plasmids have T- DNA
regions but the structure of these regions differs.
Nopaline T-DNA - single continuous segment of about 22kb
Octopine T-DNA - three segments; left DNA (TL) = 13 kb; center DNA (TC) DNA =
1.5 kb; right DNA (TR) = 7.8 kb; TL DNA contains the oncogenic function and TR
contains the opine synthetic genes
The only structural requirement for T-DNA transfer is the TR direct repeat border, but
the T-DNA region also contains a TL border repeat. The consensus sequence of the TR
repeat is:
TG Pu G
TGGCAGGATATAT
N
NC C
AT
TGTAA
T
TC
In this repeat, two domains of 13 and 5-7 bp are conserved. But deletion of the first 6 bp
or the last 10 bp blocks T-DNA transfer.
If the TL border is deleted transformation can be detected, but if the TR border is deleted
transformation is eliminated. This suggested that transfer was in the right to left direction.
Further the orientation of the repeat is important because if the border is reversed,
transformation is greatly attenuated. Thus, when the right border is used for transfer, the
genes required for the oncogenic functions are transferred, but when the left border is
used in lieu of the right border, transfer does not include this gene and the transformation
phenotype is not detected.
T-strand - a single stranded product that is derived from the T-DNA; on average, one
strand per bacterium is produced; T-strands are derived from the bottom strand of the
nopaline plasmid; the 5' end corresponds to the TR border and the 3' end corresponds to
the TL border; the nick occurs between the 3rd and 4th base; the 5' end is always within a
few bases of the TR border but the 3' border ranges from 100 bases before TL to right at
the TL border.
Integration into the Plant Genome
A single integration is the most frequent event, but on average 3 copies are integrated. An
analysis of 161 transformants of Arabidopsis was performed to determine the type of
integration events.

55% single event

20% two unlinked events

6% three unlinked events

1% four unlinked events

12% non-Mendelian ratios
In tomato it was shown that in ten different transformants, integration occurred at nine
different chromosomes. The target DNA sites do not appear specific except for the fact
they are AT rich.
Only one detailed analysis of a T-DNA insertion site has been performed. It was
determined that a 158 bp repeat was developed at both the TR and TL borders. Other
events that occur are short deletions and insertions at the end of the 158 bp segment.
Generalized Model of T-DNA Transfer and Integration
1. T-strand is produced and transferred to the plant cell as a DNA/protein complex.
Requires vir (Ti-plasmid) and chromosomal genes
2. A protein that is at the 5' end of the T-strand interacts with a nick in the plant
DNA.
3. A single-stranded DNA attaches to one strand of the plant DNA and a torsional
change occurs in the plant DNA which generates a second nick.
4. Each strand of the T-strand is ligated to the plant DNA and the homologous strand
is produced.
5. Repair and replication of the staggered nick in the plant DNA results in
duplication and rearrangement of the target DNA.
Ti Plasmid
T-DNA
region
DNA between
L and R borders is
transferred to plant
as ssDNA;
Tumorproducing
genes
Opine catabolism
Virulence region
ORI
T-DNA encoded
genes can be
substituted by
target genes