2. AGROBACTERIUM is a soil borne gram negative
bacteria and it is ubiquitous in nature .it is rod in
shaped and motile .
It belongs to bacterial family of Rhizobiaceae.
It is a phytopathogen.
There are mainly two species of Agrobacterium.
1. A. tumefaciens that induces crown gall disease.
2. A. rhizogenes that indiuces hairy root disease.
The uniqueness of this soil microorganism lies in
its capability of transfer of DNA to eukaryotic cells
.
Agrobacterium mediated transformation is one of
the most efficient methods for gene transfer and
takes advantage of the naturally evolved crown
gall inducing mechanism of DNA present in A.
tumefaciens.
3. Agrobacterial tumefaciences chromosomal genes
:chvA,chvB,PscA required for initial binding of the
bcterium to the plant cell and code for
polysaccharide on bacterial surface.
4. Agrobacterium vectors are two types
1. CO-integrative vector
2. Binary agrobacterium vector
Both hairy root and crowngall cells (
) are capable of growing in
culture on a growth regulator free medium while
normal plant cells need exogenous auxin and /or
cytokinin. Thus these plant cells have undergone
cancerous or oncogenic alteration ;they generally
induce tumor when grafted onto healthy plant .
5. These cells also synthesise unique nitrogenous
compounds called opine,which are neither produced
by normal plant cells nor utilised by them.
Agrobacterium cells use opines as their carbon and
nitrogenous source; the bacteria are usually present
in the intercellular space of crown galls. Different
types of opines are produced .
A. Agrobacterium tumeficiens strains generally
produce octapine or nopaline .
B. Agrobacterium rhizogenes produce either agropine or
mannopine
• a bacterial strain produces only one
type of opine and it also catabolises only that opine
the concerned genes are present in its pTi or pRi.
• these plasmids also carry gene IAA and cytokinin
production ,which is the reason for indefinite growth
6.
7. of crowngall cells on GR free culture medium.
Ti plasmid
Ti plasmid is a large conjuctive plasmid or
megaplasmid of about 200 kb (range 150-250 kb)
The pTi and pRi are unique bacterial plasmid in
the following two respects.
1. They contain some genes,located within their T-
DNA,which have regulatory sequences
recognised by plant cells,while their remaining
genes have prokaryotic regulatory sequence.
2. these plasmid naturally transfer a part of their
DNA,the T-DNA,into the host plant genome,which
makes Agrobacterium a natural genetic engineer.
8. The diiferent Ti plasmids can be grouped into two
general categories .
1. octapine types (derived from arginine)
2. nopaline types (derived from arginine) but
agropine derived from glutamate . They contain
the following important functional regions.
1)T-DNA contain oncogene and opines synthesis
genes and is transferred into the host plant genome
.
2)Vir region regulates the transfer of T-DNA into
plant cells.
3)Opine catabolism regions produce enzymes
necessary for the utilisation of opine by
Agrobacterium.
4)Conjugative transfer (oriT or tra ) region functions
in conjuctive transfer of plasmid .
9. ORGANISATION OF t-DNA:
T-DNA is that 23 kb segment of Ti/Ri plasmid,which
is transferred into the plant genome during
Agrobacterium infection .
T-DNA is defined on both its sides by a 24 bp direct
repeat border sequence and contain the genes for
tumor/hairy root induction and those for opine
biosynthesis.
, which are involved in crowngall
formation .two of these genes encode enzymes,
which together convert tryptophan into IAA.
the ipt gene encodes an enzyme which
produces the zeatin type cytokinin isopentenyl
adenine .
10. In addition,genes involved in opine biosynthesis are
located near the right border of T-DNA .
The T- DNA is organised in two distinct regions called
TL (left T-DNA ) and TR (right T- DNA).
in case of noplaine type of plasmid , both TL and TR
are always transferred together and integrated into the
plant genome as a single segment .
But in case of octapine types of plasmids ,the TL and
TR are transferred independently so that a single cell
may contain one or both of these segments.
The vir region contains 8 operons (designated as
VirA,VirB,VirC,VirD,VirE,VirF,VirG and VirH).
Length of vir dna is about 40 kb and have 25 genes.
The genes of vir region are not transferred ,
11. themselves ,they induce the trasfer of T-DNA.
on the other hand ,the genes present in T-DNA are
not required for its transfer ;only the 24 bp direct
repeat left and right borders of T-DNA are
essential for the transfer .
4 genes VirA,VirB,VirD and Vir G are essential for
virulence .
Other 4 are accessory role .
Vir region is activated by the phenolic signal
molecules and
,which is produced by
wounded tissues of virtually all dicot plant
species and constitute the wound response.
VirA acts as a receptor and binds with phenolic sugar.
12.
13. virA activated after binding with phenolic
compound and cause autophosphorylation due
to . (on histidine residue of vir
A).
activated Vir-A cause phosphorylation to VirG
protein and bind and then
induce the expression of other operons.
VirD1 has .
VirD2 has .
Vir E2 is a nonspecific single stranded binding
protein ,it bind to the single stranded DNA and
protect it from nuclease action .
VirB has 11 genes (mostly encode memebrane
bound proteins).
Vir E2 and Vir D2 contain nuclear localization
signal.
14. VirC1 specifically binds to overdrive sequence
and stimulates the transfer process.Agrobacterium
tumefaciens uses type IV secretion system to transfer
T-DNA complex to its host cells.
Type IV secretion system also known as
is a cell envelope spanning
complex(11-13 core proteins ).
T4SS form a pore or channel .
VirB and VirD4 cause
between plant cell and bacteria,
is assembled from 11 proteins (VirB1
to VirB11) encoded by the virB operon and VirD4.
VirB11 has ATPase activity and generate ATP needed
for the delivery of T-DNA into the plant cells.
VirF ,which directs the T-complex [virD4,virB1,virB2,
T4SS(mpf)] protein for destruction in proteosomes.
15. ?????why monocot plant is not
infected by agrobacterium
tumefaciens?????
/////because monocoats are not
able to produce phenolic
compounds .
Agrobacterium tumefaciens
mediated transformation :
there are two strategy for gene
17. CO-integration vector strategy:-
Although disarmed wild type Ti-plasmid can be
used as vector ,they are not easy to manipulate ,
because their large size makes them difficult to
manipulate in vitro and there are no unique
restriction sites in the T-DNA.
this problem can be overcomed by the
construction of
In this strategy, the gene of interest to be
introduced into Ti plasmid vector is the first sub-
cloned in a conventional E.coli plasmid vector
(such as pBR322) for easy manipulation
producing a so called .
The insertion of gene of interest into a Ti plasmid
result from the recombination of
and a .
18. takes place in
both plasmids.
Homologous recombination between T-DNA
sequences of Ti plasmid and intermediate vector
forms a large
BINARY VECTOR STRATEGY
The T-DNA doesnt need to be physically
associated with vir gene in order to become
integrated into plant genome.
could be split onto
two separate replicons.
As long as both of these replicons are located
within the , protein
encoded by vir genes could act upon T-DNA in
19. trans to mediate its processing and export to the
plant.
System in which T- DNA and vir genes are located on
separated replicons were eventually termed
.
Thus,in binary vector strategy ,
and both complement each other in same bacterial
cell.
The is transferred to
the plant chromosomal DNA by proteins coded by vir
genes carried by
