Agrobacterium is tool for genetic engineering. Let's study about its mode of infection to plants at molecular level.

1. Prepared by: Darshan T. Dharajiya
Ph.D. (Plant Molecular Biology and Biotechnology)
Dept. of Plant Molecular Biology and Biotechnology,
CPCA, S. D. Agricultural University. 1

2. Content
o Agrobacterium tumefaciens
o Molecular Biology of Agrobacterium Infection
• The Ti-Plasmid
• Organization of T-DNA
• Steps of Agrobacterium-plant cell interaction
o References
2

3. Biology of the Agrobacterium - plant interaction
The only known natural example of
inter-kingdom DNA transfer
The only known natural example of
inter-kingdom DNA transfer
3

4. 4

5. Crown gall disease 5

6. Agrobacterium tumefaciens
• Gram negative, short
rod, soil bacterium
• established by H. J.
Conn
• broad-host range
pathogen,
• initiating tumours on
plants of most
dicotyledonous genera
and some monocots
6

7. The genus Agrobacterium has
a wide host range
• Overall, Agrobacterium can transfer T-DNA to a
broad group of plants.
• Yet, individual Agrobacterium strains have a
limited host range.
• The molecular basis for the strain-specific host
range is unknown.
• Many monocot plants can be transformed (now),
although they do not form crown gall tumors.
• Under lab conditions, T-DNA can be transferred
to yeast, other fungi, and even animal and
human cells.
7

8. Why Agrobacterium is used for
producing transgenic plants?
• The T-DNA element is defined by its borders
but not the sequences within.
• So researchers can substitute the T-DNA
coding region with any DNA sequence without
any effect on its transfer from Agrobacterium
into the plant.
8

9. Molecular Biology of
Agrobacterium Infection
• 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.
9

10. The Ti Plasmid
• The Ti plasmid is a large conjugative plasmid or megaplasmid
of about 200 kb (range 150-250 kb).
• The pTi is unique bacterial plasmids in the following two
respects:
– They contain some genes, located within their T-DNA,
which have regulatory sequences recognized by plant
cells, while their remaining genes have prokaryotic
regulatory sequences.
– These plasmids naturally transfer a part of their DNA, the
T-DNA, into the host plant genome, which makes
Agrobacterium a natural genetic engineer.
• The Ti plasmids are classified into different types based on
the type of opine, namely, octopine, nopaline, succinamopine
and leucinopine, produced by their genes.
10

11. The Ti Plasmid
• The different Ti plasmids can be grouped into two general
categories: octopine type and nopaline type.
11

12. 12

13. The Ti Plasmid
• Ti Plasmids contain the following important functional
regions:
1. T-DNA contains oncogenes and opine 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 utilization of opines by Agrobacterium.
4. Conjugative transfer (oriT or tra) region functions in
conjugative transfer of the plasmid.
5. Origin of replication (ori) for propagation in
Agrobacterium.
13

14. Agrobacterium-induced plant tumors
contain high concentrations of
• Plant hormones (auxin, cytokinin)
• Opines (octopine, nopaline)
14

15. Organization of T-DNA
• T-DNA (transferred DNA) is the 23 kb segment
of Ti plasmids, 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 contains
the genes for tumour/hairy root induction and
those for opine biosynthesis.
15

16. Organization of T-DNA
16

17. T-DNA
• T-DNA carries genes involved in the synthesis of
plant growth hormones (auxin, auxin synthesis; cyt,
cytokinin synthesis) and the production of low
molecular weight amino acid and sugar phosphate
derivatives called opines (ocs, octopine; mas,
mannopine; and ags, agropine).
• Agrobacterium are usually classified based on the
type of opines specified by the bacterial T-DNA.
• 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.
17

18. Organization of vir Region
• The vir region contains 8 operons (designated
as virA, virB, virC, virD, virE, virF, virG and
virH), which together span about 40 kb of DNA
and have 25 genes.
• This region mediates the transfer of T-DNA
into plant genomes, and hence is essential for
virulence or production of crowngall/hairy
root disease; therefore, it is called the
virulence region or vir region.
18

19. • 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.
• Of the 8 vir operons, 4 operons, viz., virA, virB,
virD and virG, are essential for virulence, while
the remaining 4 operons play an accessory role.
• The operons vir A and virG are constitutive,
encode one protein each, and are concerned
with the regulation of all the vir operons.
Organization of vir Region
19

20. Gene/Operon Function
T-DNA
iaaM (auxL tins I)*
Auxin biosynthesis; encodes enzyme tryptophan-2-mono-oxygenase, which converts
tryptophan into indole-3-acctamide (IAM).
iaaH (aux2, tms2)
Auxin biosynthesis; encodes enzyme indole-3-acetamide hydrolase, which converts
IAM into IAA (indole-3-acetic acid).
ipt (tmr, Cyt )
Cytokinin biosynthesis; encodes enzyme isopentenyl transferase, which catalyzes the
formation of isopentenyl adenine.
Nos
Nopaline biosynthesis; encodes the enzyme nopaline synthase, which produces
nopaline from arginine and pyruvic acid.
24 bp left and right border
sequences
Site of endonuclease action during T-DNA transfer; the only sequences of T-DNA
essential for its transfer.
vir Region
vir A(1)
Encodes a receptor for acetosyringone that functions as an autokinase; also
phosphorylates VirG protein; constitutive expression.
virB (11)
Membrane proteins; possibly form a channel for T-DNA transport (conjugal tube
formation); VirB 11 has ATPase activity.
virC (2)
Helicase; binds to the overdrive region just outside the right border; involved in
unwinding of T-DNA.
virD (4)
VirDl has topoisomerase activity; it binds to the right border of T-DNA; VirD2 is an
endonuclease; it nicks the right border.
virE (2) Single-strand binding proteins (SSBP); bind to T-DNA during its transfer.
virF(l) Not well understood.
virG (l)
DNA binding protein; probably forms dimer after phosphorylation by VirA, and
induces the expression of all vir operons; constitutive expression.
virH (2) Not well known.
20

21. Steps of Agrobacterium-plant cell
interaction
• Cell-cell recognition and binding
• Signal transduction and transcriptional
activation of vir genes
• Conjugal DNA metabolism
• Intercellular transport
• Nuclear import
• T-DNA integration
21

22. 22

23. 23

24. 24

25. Agrobacterium-host cell recognition is a
two-step process
1. Loosely bound step: acetylated polysaccharides
are synthesized.
2. Strong binding step: bound bacteria synthesize
cellulose filaments to stabilize the initial
binding, resulting in a tight association between
Agrobacterium and the host cell.
25

26. 26

27. 27

28. 28

29. Receptors are involved in initial
binding
• Plant vitronectin-like protein (PVN, 55kDa) was
found on the surface of plant cell. This protein is
probably involved in initial bacteria/plant cell
binding.
• PVN is only immunologically related to animal
vitronectin.
• Animal vitronectin is an important component of
the extracellular matrix and is also an receptor
for several bacterial strains.
• Aside from PVN, rhicadhesin-binding protein was
found in pea roots.
29

30. 30Electron microscopy of Attachment of Agrobacterium cells to plant cell

31. 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.
31

32. Representative phenols and sugars (pyranose forms) capable of serving as signals to induce vir
gene expression. Active versus inactive enantiomers of DDF and MMCP are shown. AS,
acetosyringone; DDF dehydrodiferulate dimethylester; MMCP, 1-hydroxymethyl-2-(4-hydroxy-3-
methoxyphenyl)-cyclopropane. 32

33. • 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.
Plant signals
33

34. • 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.
Plant signals
34

35. 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.
35

36. • 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.
Production of T-strand
36

37. Formation of the 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.
37

38. • Whether VirE2
associates with T-
strand before or
after the
intercellular
transport is not
clear.
Formation of the T-complex
38

39. • 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.
Formation of the T-complex
39

40. 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.
40

41. • 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.
Intercellular transport
41

42. Nuclear Import
• Because 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.
42

43. • VirD2 is imported into
the cell nucleus by a
mechanism conserved
between animal, yeast
and plant cells.
• VirE2 has a plant-
specific nuclear
localization
mechanism. It does not
localize to the nucleus
of yeast or animal cells.
Nuclear Import
43

44. • In host plant cells VirD2
and VirE2 likely
cooperate with cellular
factors to mediate T-
complex nuclear
import and integration
into the host genome.
Nuclear Import
44

45. 45

46. 46

47. Chromatin targeting of T-Complex
• 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.
47

48. Targeted proteolysis and T-DNA uncoating
• It is necessary to expose
T-strand for integration.
• Vir F2- a bacterial host
range factor exported
into host cell.
• Vir F2 possess F-box
domain which binds to
Ask protein family.
48

49. • F-box and Skp1
represent conserved
components of E3
ubiquitin ligases called
SCF (Skp1-Cullin-F-box
Protein) complex that
mediate protein
destabilization by
targeted proteosomal
degradation.
Targeted proteolysis and T-DNA uncoating
49

50. • Annealing of LB and RB of
T-DNA to the host
genome.
• Insertion & ligation of T-
strand into nick.
• Conversion of T-strand
into double stranded by
gap repair mechanism.
50
DNA repair and Integration

51. DNA repair and Integration
• DSB induction at specific
site in host DNA by rare
cutting restriction
enzymes.
• Insertion of T-DNA in this
site.
51

52. DNA repair and Integration
o In Arabidopsis,
• KU80 – factor of NHEJ,
Form complex with KU70
and DNA protein Kinase
during integration.
52

53. T-DNA integration is not highly
sequence-specific
• There is no obvious site preference for
integration of T-DNA throughout the genome.
• About 40% of the integrations are in genes
and more of them are in introns.
• The mechanism of NHEJ makes deletions after
T-DNA integration a common phenomenon.
• DSB mechanism
53

54. Events of NHEJ in Agrobacterium
T-DNA integration
• Integration is initiated by the 3’(LB) of the T-
DNA invading a poly T-rich site of the host
DNA.
• A duplex is formed between the upstream
region of the 3’-end of T-DNA and the top
strand of the host DNA.
• The 3’-end of T-DNA is ligated to the host DNA
after a region downstream of the duplex is
degraded.
54

55. Events of NHEJ in Agrobacterium
T-DNA integration
• A nick in the upper host DNA strand is created
downstream of the duplex and used to initiate
the synthesis of the complementary strand of
the invading T-DNA.
• The right end of the T-DNA is ligated to the
bottom strand of the host DNA. This pairing
frequently involves a G and another
nucleotide upstream of it.
55

56. Importance to Biotechnology &
Genetically Modified Organisms
• Oncogenes and opine-creating genes can be removed from the Ti-Plasmid that is
transferred to the plant cell by T-DNA.
• Scientists can insert any gene they want into the plasmid in place of the tumor
causing genes and subsequently into the plant cell genome.
• Original problems existed in that Agrobacterium tumefaciens only affects
dicotyledonous plants.
• Monocotyledon plants like corn are not very susceptible to the bacterial infection.
• By varying experimental materials, culture conditions, bacterial strains, etc.
scientists have successfully used A. tumefaciens Gene Transfer to produce BT
Corn .
• 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.
• The vast majority of approved genetically engineered agriculture has been
transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer.
56

57. References
• Tzvi T. and Vitaly C., (2006). Agrobacterium – mediated
genetic transformation of plants biology and
biotechnology. Current Opinion in Biotechnology. 17:
147-154.
• Colleen A.M. and Andrew N.B., (2006). Agrobacterium
tumefaciens and plant cell interactions and activities
required for interkingdom macromolecular transfer.
Annu. Rev. Cell Dev. Biol. 22: 101-127.
• Vitaly C., Stanislav V.K., Benoit L., Adi Z., Mery D.Y.,
Sachi V., Andriy T. and Tzvi T., (2006). Biological
systems of the host cell involved in Agrobacterium
infection. Cellular Microbiology. 9: 9-20.
57

58. • De la Riva,G.A., Gonzalez-Cabrera,J., Vazquez-
Padron,R., Ayra-Pardo,C. (1998). Agrobacterium
tumefaciens: a natural tool for plant transformation.
Electronic J of Biotech. 1(3): 1-18.
• http://en.wikipedia.org/wiki/Agrobacterium
• http://www.bio.davidson.edu/people/kabernd/semi
nar/2002/method/dsmeth/ds.htm
• B.D. Singh. (2008). Biotechnology. Third edition.
Kalyani Publisher, New Delhi (India). pp: 355-363.
58
References

59. 59