Mechanism involved in Agrobacterium

2. ROLE OF AGROBACTERIUM IN
PLANT PATHOLOGY
M.PRADEEP
2015 631 505

3. CONTENTS :

4. History
• In 1907 Smith and Townsend determined that
Gram negative soil bacterium Agrobacterium
tumefaciens .
• In the 1940s Braun and colleagues
demonstrated that the uncontrolled
proliferation of the tumor cells was not
dependent on the continuous presence of the
inciting bacteria.

5. • In 1960 Morel and colleagues demonstrated
that various bacteria free crown gall tumor
synthesis unusual amino acid-suagrs
conjugates termed opines.
• In mid 1970s several groups discovered that
a large tumor inducing (Ti) plasmid in
A.tumefaciens is necessary for this bacterium
to incite tumor.

6. Agrobacterium
Scientific classification
Kingdom Bacteria
Phylum Proteobacteria
Class Alphaproteobacteria
Order Rhizobiales
Family Rhizobiaceae
Genus Agrobacterium
Species A.tumefaciens

7. Agrobacterium
• Gram negative soil
bacterium
• Causes crown gall
disease in wounded
dicot plants .
• A ”natural genetic
engineer capable of
integrating its DNA into
plant genome

8. Disease caused by Agrobacterium

9. Characteristics of
 Agrobacterium is called the natural genetic
engineer.
 Size of the plasmid: ~200 kbp.
 Contains one or more T-DNA region.
 Contains a region enabling conjugative transfer.
 Contains regions for opine synthesis and
catabolism.
 Responsible for crown gall disease in plants.

14. Ti Plasmid

15. • Virulence region consists of 24 genes in total.
• Virulence genes are located in 8 operons from
Vir A to Vir H.
• Vir A vir F and Vir G are monocistronic
operons, where as Vir B,C,D,E,H are
polycistronic

16. NAME OF THE Vir GENES CONTRIBUTION OF TRANSFORMATION
Vir A Sense acetosyringone secreted by wounded plant cells
Vir G Transcriptional activator of vir box
Vir D2 Protects 5’ end from being cleaved by endonuclease
Vir E Protein helps in gene transfer
Vir c1c2 Forms overdrive sequence and helps in DNA transfer
Vir D Excise T-DNA
Vir B Forms conjugational pore between plant and bacteria
Vir B11 ATPase activity-provides energy for movement of DNA
Vir H Detoxify other adversely affecting components during the
DNA transfer

22. Induction of Virulence Function
(initiation of T-DNA transfer)
Virulence functions are transcriptionally regulated
by 2 component gene regulatory system belonging
to a large family of bacterial chemosensors that
respond to the chemical environment. Optimal vir
gene induction occurs at acidic pH and in the
presence of phenolic inducers such as
acetosyringone (AS) that are released by wounded
plant cells. The vir gene regulatory system
operates through two monocistronic virulence
genes: vir A and vir G.

23. vir A gene is constitutively expressed. Vir A protein is located in the inner
membrane and responds to the chemical environment [acidic pH and
acetosyringone (AS)]. In the presence of the stimulants, it is auto-
phosphorylated.
NH2
COOH
cytoplasmperiplasm
Inner membrane
receiver
sensor
kinase
linker
AS
(pH)
Linker responds to pH and interacts with ChvE (a sugar-binding
protein encoded by Agrobacterium genome). At sub-optimal AS levels,
VirA can be further stimulated by sugars, opines or amino acids.
Auto-phosphorylation
Vir A

24. VIR G
vir G gene is also constitutively expressed. Vir G protein is freely available in the
cytoplasm. The activated (phosphorylated) Vir A in turn phosphorylates Vir G
protein at aspartic acid residue . Phosphorylated Vir G becomes the
transcriptional activator of the remaining vir genes. Promoters of vir genes
possess one of more “vir box” of 12 bp sequence.
AS
P
P
P
Vir A Vir G
A mutant that expresses its vir genes constitutively, contains a
vir G mutation called virG-N54D. This mutation leads to a
conformation of protein that is similar to phosphorylated Vir G.

25. Vir D1, D2
LB RB
LB RB
LB RB
Vir D2
T-DNA Processing

26. Ti plasmid (octopine type) encoded proteins required for T-DNA
processing and transfer (vir genes)
vir operons: virA, -B, -C, -D, -E, -G.
vir F and vir H
Vir D1 and D2 :
D2 is a site-specific endonuclease.
However, both D1 and D2 are required for nicking borders
on a supercoiled or relaxed double stranded DNA. Whereas
D2 can cleave border sequence on a single stranded DNA
without the help of D1. This suggests that D1 could be
involved in ripping ds DNA into ss form for D2 to act upon the
ss border sequences.

27. • During infection, A. t. carrying an octopine-type Ti
plasmid transfers two fragments of DNA to plant
cell. These fragments are designated as TL-DNA
and TR-DNA and are 13 and 7.8 kb long,
respectively. A nopaline type Ti plasmid transfers
a single DNA fragment (T-DNA) that is about 20 kb
long.
• TL-DNA and TR-DNA or T-DNA is each flanked by
cis-acting 25 bp direct repeats called border
sequences (LB and RB or A, B, C and D). The
left border is dispensable for T-DNA transfer but
right border is essential and acts in polar fashion.

28. • In the presence of Vir proteins, T-region undergoes
following processing steps:
1. Each border is cleaved exactly 4 nt from its left end
catalyzed by VirD2 protein, which remains covalently
bound to the 5’ end of each cleaved strand.
2. Bottom strands are recovered as single-stranded (ss)
form, referred to as T strand.
LB RB
cleavage
TL-DNA
LB RB
TR-DNA

29. • Important: Putting any DNA between the
LB and RB of T-DNA it will be transferred
to plant cell!

30. Expression of virE2 in planta complements
a virE2 mutant strain of A. tumefaciens

31. Vir C1 and C2
C1 mutants display lower virulence
C2 function is unknown.
C1 binds to the overdrive site.
It is not clear exactly how binding of C1 on
overdrive helps increase the efficiency of
T-strand transfer to plants.
Overdrive is absent in nopaline type Ti
plasmid.

32. cytoplasm
VirB2
VirB7
VirB4
VirB11
VirB9
VirB pore
Other VirB proteins [B3,
B5, B6, B8, B10] are
minor constituents of the
pore.
VirB1
T-DNA Transfer Apparatus: pilus or pore

33. Vir H
Non-essential. May be involved in detoxification of plant
phenolics. VirH exhibits sequence homology with
cytochrome P450 like gene. Cytochrome P450 enzymes
catalyze NADH-dependent oxidation of aromatic substrates.
Vir F
Host range factor. Possible interaction with Skp1 proteins to
regulate plant cell division cycle.

34. Transgenic plants

35. All stable transformation methods
consist of three steps:
• Delivery of DNA into a
single plant cell.
• Integration of the DNA
into the plant cell
genome.
• Conversion of the
transformed cell into a
whole plant.

36. Productionof transgenic plants
Isolate and clone gene of interest
Add DNA segments to initiate or enhance gene
expression
Add selectable markers
Introduce gene construct into plant cells
(transformation)
Select transformed cells or tissues
Regenerate whole plants

37. Drawbacks of transgenic palnts
1) Auxine/Cytochine made by T-DNA do not allow proper
plant regeneration
2)Opine is not usefull for plant
3)Ti plasmids are big (200-800Kb)
4) couldn't regenerate plants from tumors

38. Agrobacterium for TRANGENIC
PLANTS
Agrobcaterium for transgenic plants are of two
types :-
 Binary vector system
 Co-integrated vector system

39. Binary vector system
1. Move T-DNA onto a separate, small
plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin
resistance) gene in T-DNA.
4. Vir genes are retained on a separate
plasmid.

40. Cont..
5. Put foreign
gene between T-
DNA borders.
6. Co-transform
Agrobacterium
with both
plasmids.
7. Infect plant with
the transformed
bacteria.

41. Co-integrated vector system

43. CASE STUDY
The Agrobacterium tumefaciens Ti
plasmids virulence gene virE2 reduce
Sri Lankan cassava mosaic infection in
transgenic Nicotiana benthamiana

44.  Cassava mosaic disease is a major
constraint to cassava cultivation worldwide.
In India the disease is caused by Indian
cassava mosaic virus and Sri Lankan cassava
mosaic virus.
 The Agrobacterium Ti plasmid virulence
gene Vir E2, encoding a nuclear localized
single stranded DNA binding protein, was
introduced into Nicotiana benthamiana to
develop tolerance against SLCMV.

45. • Several transgenic approaches based on viral
and non-viral genes have been used to
achieve geminivirus resistance .
• The efficacy of a non-viral protein, A.
tumefaciens VirE2, to develop tolerance
against SLCMV. VirE2 is a ssDNA binding
protein which binds to ssDNA in a cooperative
manner and ensures that the complete ssDNA
is coated with the protein.

46. • This protects the Agrobacterium T-strand from
nuclease attack during the transfer process.
VirE2 contains two bipartite nuclear
localization signals and both are required for
targeting VirE2 to the nucleus .
• These properties of virE2 prompted to select
the gene for engineering tolerance against
SLCMV which has a ssDNA genome.

47. • Local and systemic spread of geminiviral DNA
is essential to establish infection in different
parts of a plant.
• In bipartite begomoviruses, nuclear shuttle
protein (NSP) and movement protein (MP)
play important roles in viral movement. NSP
helps in the transport of geminivirus DNA
from the nucleus to the cytoplasm, whereas
MP facilitates the viral movement between
the cells(relay race model ) and the model
have been proposed for geminivirus
movement.

48. • As per the model, the viral ssDNA bound to
NSP shuttles between the nucleus and the
cytoplasm. The complex then interacts with
MP to cross the cell boundary.
• The Tomato leaf curl virus (ToLCV) genome
modified to express the M13 phage ssDNA
binding protein g5p developed only mild
symptoms and did not spread efficiently in N.
benthamiana plants.
• Binding of g5p with the ssDNA of ToLCV may
have competed with the NSP binding to ssDNA
and thereby reduced the spread of the viral
DNA.

49. • As in the case of g5p, the ssDNA binding
protein VirE2 also might cooperatively bind to
SLCMV ssDNA in the nucleus, thus preventing
NSP binding and shuttling to the cytoplasm for
cell to cell and systemic movement.
• This show that A. tumefaciens VirE2, with the
unique features of ssDNA binding and nuclear
localization, is very effective in both MYMV
and SLCMV and holds promise to develop
broad spectrum geminivirus tolerance.

50.  Agrobacterium is pathogenic in nature
 The continued presence of viable bacteria is not needed for tumor
maintenance
 Bacteria do not penetrate in to the plant cell that are converted in
to the tumor cell
 Only a small part of the ti plasmid is traansferred in to the host cell,
the segment is called T-DNA

51.  Scientists can insert any gene they want into the plasmid in
place of the tumor causing genes and subsequently into the
plant cell genome.
 By varying experimental materials, culture conditions,
bacterial strains, etc. scientists have successfully used A.
tumefaciens Gene Transfer to produce plants.
 This method of gene transfer enables large DNA strands to
be transferred into the plant cell without risk of
rearrangement whereas other methods like the Gene Gun
have trouble doing this

52. The vast majority of approved genetically
engineered agriculture has been transformed
by means of Agrobacterium tumefaciens
Mediated Gene Transfer.
 Original problems existed in that
Agrobacterium tumefaciens only affects
dicotyledonous plants.
Monocotyledon plants are not very
susceptible to the bacterial infection.