Agrobacterium tumefaciens

1. PRESENTED BY-
RATNAKAR UPADHYAY
M.Sc.- III Sem
DEPARTMENT OF BOTANY
GUIDED BY -
Dr.SHRIRAM KUNJAM
Session- 2018-19

2. INTERNATIONAL CODE OF
NOMENCLATURE OF PROKARYOTES
Updated the Scientific name of
. Agrobacterium tumefaciens
Rhizobium radiobacter
Kingdom- Bacteria
Phylum- Proteobacteria
Class- Alphaproteobacteria
Order- Rhizobiales
Family- Rhizobiaceae
Genus- Rhizobium
species- radiobacter

3. INTRODUCTION
 Agrobacterium tumefaciens only known natural example of inter-kingdom
DNA transfer.
 Established by Sir H.J.Conn
 Soil Bacterium, Rod shaped, Gram negative, Motile, having Peritrichous
flagella(1-6).
 Broad host range pathogen (Dicots, Monocots, Gymnosperms, Fungi.)
e.g. walnuts, grape, nut trees, sugar beats, yeast.
 It is considered as Natural genetic engineer because it transforms the
plant.
 It has Ti plasmid, responsible for tumor induction (crown gall tumor) in
plants.
 To be virulent, bacterium must contain a “pTi” which contain the T-DNA.
 Agrobacterium rhizogenes has Ri plasmid, responsible for hair disease in
plants.
 Agrobacterium rubi cause cane gall in Sugarcane plant.
 Agrobacterium vitis cause gall in grapes.

4. WHY Agrobacterium IS USED TO PRODUCE
TRANSGENIC PLANT ?
 The T-DNA element is defined by its borders but not by
the sequence present within it.
 So Researchers can substitute the T-DNA coding region
with any DNA sequence without any effect on its transfer
from Agrobacterium to the plant.
 Many monocot plants can be transformed now, although
they do not form crown gall tumors.

5. CROWN GALL FORMATION

6. MOLECULAR BASIS
 The process of infection by Agrobacterium tumefaciens
culminates in the transfer of a small part of pTi into the plant
cell genome; this DNA sequence is called T-DNA.
 The infection process is governed by both chromosomal and
plasmid-borne genes of Agrobacterium tumefaciens.
 Attachment of bacteria to plant cells begins the infection,
governed by chromosomal virulence genes (chv); which are
expressed constitutively.
 The continued presence of virulent bacteria is not needed for
tumor maintenance.
 Bacteria do not penetrate the plant cells that are converted
into tumor cells.
 Only a small part of the Ti plasmid is transferred into the host
cell (The T-DNA)

7.  Large sized plasmid of 200kbp.
 Modification of this plasmid is very
important in the creation of
transgenic plant.
 The Ti plasmids are classified into
different types based on the type
of opine, namely, octopine,
nopaline, succinamopine and
leucinopine produced by their
genes.
 The different Ti plasmids can be
grouped into two general
categories: octopine type and
nopaline type.
Ti- PLASMID (TUMOR INDUCING)

9. T-DNA
 The T-DNA is the transferred DNA of the tumor inducing
plasmid of virulent species of bacteria. The size of T-DNA is
between 15-30kb.
 T-DNA contain genes for tumor induction.
 It has LB & RB (left border & right border). RB play a
important role in transfer & integration of T-DNA. Absence
of RB will terminate the T-DNA transfer.
 T-DNA carry genes for Phytohormones (Auxin, Cytokinin) &
Opine that are expressed in plant cell.
 Over production of these hormones at the site of infection is
responsible for the proliferation of wounded cell in
gall/tumor. These tumor can harbor a plenty of bacteria.

10. All the genes present in T-DNA contain eukaryotic
regulatory sequences. As a result, these genes are
expressed only in plant cells, and they are not expressed
in the Agrobacterium.

11. COMPONENTS OF T-DNA
Gene Function
iaaM (auxL,tins I) Auxin biosynthesis; encodes enzymes Tryptophan-2-
mono oxygenase, which converts Tryptophan into Indole-
3-acetamide (IAM)
iaah (aux2 tms2) Auxin biosynthesis; encodes enzymes Indole-3-
acetamide hydrolase which converts IAM into IAA
(Indole-3-acetic acid)
ipt(tmr,Cyt) Cytokinin biosynthesis; encodes enzyme Isopentenyl
transferase, which catalyses the formation of Isopentenyl
adenine.
Nos Nopaline biosynthesis; encodes the enzyme Nopaline
synthase, which produces Nopaline from arginine &
pyruvic acid.
24 bp (left & right
border sequences)
Site of endonuclease action during transfer of T-DNA, the
only sequences of T-DNA essential for its transfer.

12. COMPONENTS OF VIR REGION
OPERON FUNCTION
VirA Encodes a receptor for acetosyringone that functions as an autokinase, also
phosphorylates virG protein; constitutive expression.
VirB Membrane proteins; possibly form a channel for T-DNA transport(conjugal tube
formation); also has ATPase activity.
VirC Helicase; binds to the overdrive region just outside the right border, involved in
unwinding of T-DNA.
VirD VirD1 has Topoisomerase activity it binds to the right border of T-DNA & prevent
attack by exonuclease at 5’ end of T-DNA , VirD2 is an Endonuclease (play role
in cutting phosphodiester bond), it nicks the right border.
VirE/E2 Protect T-DNA against nuclease & target T-DNA to plant cell .It act as a single
stranded binding protein.
VirG DNA binding protein; probably forms dimer after phosphorylation by Vir A &
induces the expression of all Vir operons (constitutive expression)
VirD2 &
VirE2
Have NLS (nuclear localisation signal)
VirB &
VirD4
Type IV Secretion system (mating pair formation apparatus), it’s a pore channel
formation.

13. PLANT TRANSFORMATION

14. STEPS OF AGROBACTERIUM-PLANT CELL INTERACTION
 Plant stress condition.
 Phenolic production.
 Signal to Bacteria.
 Virulence system activation.
 Generation of T-DNA complex.
 T-DNA transfer (Nuclear import).
 T-DNA integration in plant genome.

16. PLANT SIGNALS
 Wounded plants secrete sap with
acidic pH (5.0 to 5.8) and a high
content of various phenolic
compounds (lignin, flavonoid
precursors) serving as chemical
attractants to Agrobacterium and
stimulants for vir gene expression.
 Among these phenolic compounds,
acetosyringone (AS) is the most
effective.
 Sugars like glucose and galactose
also stimulate vir gene expression
when AS is limited or absent.
These sugars are probably acting
through the chvE gene to activate
vir genes.
 Low opine levels further enhance
vir gene expression in the
presence of AS.
These compounds stimulate the
autophosphorylation of a
transmembrane receptor kinase VirA
at its His-474.
It in turn transfers its phosphate group
to the Asp-52 of the cytoplasmic VirG
protein

17. PRODUCTION OF T-STRAND
 Every induced Agrobacterium
cell produces one T-strand.
 VirD1 and VirD2 are involved
in the initial T-strand
processing, acting as site-and
strand-specific endonucleases
 After cleavage, VirD2
covalently attaches to the 5’
end of the T-strand at the right
border nick and to the 5’-end
of the remaining bottom strand
of the Ti plasmid at the left
border nick by its tyrosine 29.

18. FORMATION OF T-COMPLEX
 The T-complex is composed
of at least three components:
one T-strand DNA molecule,
one VirD2 protein, and
around 600 VirE2 proteins.
 If VirE2 associates with the
T-strand after intercellular
transport, VirE1 is probably
involved in preventing VirE2-
T-strand binding.
 Judging from the size of the
mature T-complex (13nm in
diameter) and the inner
dimension of T-pilus (10nm
width), the T-strand is
probably associated with
VirE2 after intercellular
transport.

19. INTERCELLULAR TRANSPORT
 Transport of the T-complex into the
host cell most likely occurs through
a type IV secretion system.
 In Agrobacterium, the type IV
transporter (called T-pilus)
comprises proteins encoded by
virD4 and by the 11 open reading
frames of the virB operon.
 Intercellular transport of T-DNA is
probably energy dependent,
requiring ATPase activities from
VirB4 and VirB11.
 Physical contact between
Agrobacterium and the plant cell is
required to initiate T-complex
export. Without recipient plant
cells, T-strands accumulate when
vir genes are induced.

20. NUCLEAR IMPORT & CHROMATIN TARGETING
OF T-COMPLEX
 Because of the large size of T-complex (50,000 kD, ~13nm in
diameter), the nuclear import of T-complex requires active nuclear
import.
 The T-complex nuclear import is presumably mediated by the T-
complex proteins, VirD2 and VirE2. Both of them have nuclear
localizing activities.
 CAK2M and TATA-Box binding protein (TBP) both of which bind to
VirD2 and VIP1 binds to VirE2.
Which leads in chromatin targeting of T-Complex.

22. T-DNA INTEGRATION

24. REQUIREMENTS ADVANTAGES DISADVANTAGES
The plants explant must
produce acetosyringone
or other related
compounds.
Natural means of
transfer, hence plant
friendly
Limited host range. Can
not infect cereal plants.
The induced bacteria
should give access to
cells that are competent
for transformation
It is capable of infecting
intact plant cells.
Sometimes cells in a
tissue that are able to
regenerate are difficult to
transform.
Transformation
competent cells &
tissues should be able to
regenerate into whole
plants.
Capable of transferring
large fragments of DNA
very efficiently without
substantial
rearrangements.
The stability of gene
transferred is excellent.
Agrobacterium MEDIATED GENE TRANSFER

25. Wild type Agrobacterium Ti plasmid cannot be use as
gene cloning vectors because of :-
 Large size of Ti plasmid, difficult to handle.
 Presence of oncogenes or tumor causing genes
( Auxin & Cytokinin).
 Lack of unique restriction sites & makes sites within
T-DNA.
There are two genetically engineered Ti plasmid
based vectors. They are Cointegrate & Binary
vector.
Ti plasmid based vectors: Co-integrate &
Binary vectors

26. MAKING OF CO-INTEGRATE VECTORS
 Both the T-DNA with our gene of interest & Vir region are
present in the same vector used for transformation.
 At first, an intermediate vector is made using E.coli plasmid
+ Vir region + T-DNA borders + Ori + pBR322 sequences.
 Second vector is a disarmed pTi vector + gene of interest +
some markers + pBR322 sequences.
 Both intermediate vector & disarmed pTi has some
sequences in common (pBR322 sequences). Therefore, by
homologous recombination, cointegration of two plasmids
will takes place within Agrobacterium.
 Result- A cointegrate vector that has both T-DNA with our
gene of interest within the T-DNA borders & Vir regions.
This complete vector is used for Transformation
eg.pGV2260

27. BINARY VECTOR STRATEGY: TWO VECTOR
STRATEGY
 Here two vectors are used, based on the knowledge that
Vir region need not be in the same plasmid alongwith T-
DNA for T-DNA transfer.
 Binary vector consists of pair of plasmids.
1.A disarmed Ti plasmid: This plasmid has T-DNA with
gene of interest + Ori for both E.coli & Agrobacterium.
Also called as mini-Ti or micro Ti plasmid e.g- Bin 19.
2.Helper Ti plasmid has virulence region that mediates
transfer of T-DNA in micro Ti plasmid to the plant

28. ADVANTAGES OF BINARY VECTOR OVER
COINTEGRATE VECTOR
 Binary vector do not need in vivo recombination.
 Binary vectors require only an intact plasmid vector to be
introduced into the target bacterium, making the process
of gene transfer more efficient & quicker.
 Confirmation of the transformation event is accomplished
very easily.

29. UPDATE
 Recent work has shown that the plant factor KU80 is
involved in the T-DNA integration process, most likely by
bridging between double-stranded T-DNAs.
 In addition, Shaked et al. reported that Over expression of
the yeast Rad54 protein led to high frequency gene
targeting in transgenic plants. These two reports further
support the notion that integration of T-DNA molecules is
promoted by host cellular factors and open a new direction
for plant gene targeting by genetic manipulation of the host
genome.

30. CONCLUSION & FUTURE PROSPECTS
 With an ever-expanding host range that includes many
commercially important crops, flowers, and tree species,
Agrobacterium is guaranteed a place of honor in nearly
every plant molecular biology laboratory and biotechnology
company for a long time to come.
 Furthermore, its recent application to the genetic
transformation of non-plant species, from yeast to cultivated
mushrooms, and even human cells, places Agrobacterium
at the forefront of future biotechnological applications.

31. REFERENCES
 Bhojwani, S.S,Razdan, M.K, Plant tissue culture,
1996,ELSEVIER,New York.
 Snustad, D.Peter, Simmons, Michael J, Principles of
Genetics, Sixth Edition, John wiley & sons, USA.
 Karp, Gerald, Cell & Molecular Biology, Sixth Edition, John
Wiley & Sons, USA.
 Tzvi Tzfira and Vitaly Citovsky, www.Sciencedirect.com,
 Sharma, Vandana, Meena, Rishikesh, Biotechnologyu &
genetic engineering of plants,Vardhman Mahaveer open
university kota (Raj.)

32. THANK YOU