Agrobacterium tumefaciens is a soil bacterium that can naturally infect plant wounds and transfer DNA (T-DNA) from its tumor-inducing (Ti) plasmid into the plant genome. This leads to crown gall disease in infected plants. The T-DNA contains genes that produce phytohormones causing tumors and opines that the bacteria can use. By replacing the T-DNA with a gene of interest, A. tumefaciens can be used to genetically transform plants. The process involves sensing wounded plant signals, transferring T-DNA and virulence proteins into plant cells, and integrating the T-DNA into the plant genome. A. tumefaciens-mediated transformation is

1. Dr. Divya Sharma
Assistant Professor

2. ⦿ Agrobacterium species
⦿ Ti-Plasmid
⦿ Organization of T-DNA
⦿ Genes responsible for transfer of T-DNA
⦿ Mode of action
⦿ Gene of interest
⦿ Gene cloning

3. ⦿ Genes Agrobacterium is a rod shaped, Gram negative soil
bacterium that naturally infect the dicot plants (commonly), but
also used for monocots.
⦿ Common species of Agrobacterium like A. tumefaciens,
A.rhizogenes, A. rubi and A. vitis are naturally infect plants at
wound sites and causing ‘Crown gall' and ‘Hairy root’diseases.
⦿These bacteria are natural genetic engineer due to they insert
their genes into the genome of higher plants.

4. Agrobacteriummediatedtransformationinplant

5. ⦿ The bacterium contains a plasmid (called Ti-plasmid or Tumour-
inducing plasmid), their part of T-DNA (Transfer DNA)
integrates into the host plant chromosomes.
⦿ The Ti-plasmid contains several genes including the vir genes
which control the process of infection of the plant and transfer of
the T-DNA to the chromosome.
⦿ The T-DNA contains the auxin and cytokines genes and these
gene expressed specific compounds, like opines, resulting in
tumor and changes in plant metabolism.
⦿ A. tumefaciens, now used as a tool for engineering desired genes
into plants (called as Gene cloning).

6. 1. A. tumefaciens:
 Infects wounded plant tissue which is induced plant tumor i.e.,
‘Crown gall’disease.
 Crown gall occurs when bacterium releases Ti-plasmid (Tumour-
inducing plasmid) into cytoplasm.
 Serious pathogen of walnut, grapevines, stone fruits, nut trees,
sugar beets, horse radish and rhubarb.
 It grows optimally at 28 °C. The doubling time can range from
2.5-4h depending on the media, culture format, grow aerobically,
without forming endospores.

7. Ti - Plasmid Ri - Plasmid

8. 2. A. rhizogenes:
 Responsible for inducing ‘Hairy root’diseases.
 Bacterial genes transfer its T-DNA from its Ri-plasmid
(Root-inducing plasmid) into plant through wound.
 Ri-plasmid is analogue to Ti-plasmid.
3. A. rubi:
 Cause ‘Cane gall’disease in sugarcane plant
4. A. vitis:
 Cause ‘Gall’in grapes plant

10. ⦿ Large size plasmid of 200 kbp.
⦿ The Ti-plasmid is damage when
Agrobacterium is grown above 28 °C
(curing of plasmid).
⦿ The modification of this plasmid is
very important in the creation of
transgenic plants.
⦿ Ti-Plasmid have T-DNA, Right border (RB), Left border (LB),
virulence (vir gene) region, phytochrome region, origin of
replication and opine catabolism region.

11. For T-DNA transfer
Breakdown of
opines
For gene
transfer
T-DNA region or oncogenic
region (for tumor induction):
 Plant hormone synthesis
region
 Opine synthesis region

12. NOPALINE OCTOPINE
Has one 23kb region as T-DNA Two adjoining region one is L
(13kb) and another is R (8kb)
Has 13 ORFs Has 8 (L) and 6 (R) ORFs
Nopaline and Octopine plasmids are similar; carry a variety of genes,
including T-regions that have overlapping functions

13. CompositionofOncogenicRegion
• Tumor morphology shoot
(tms) --- Produces auxin
• Shoot inhibition (shi)
Shooty
locus
• Tumor morphology root (tmr)
--- Produces cytokine
• Root inhibition (roi)
Rooty locus

15.  The transfer DNA (T-DNA) is the transferred gene, part of Ti-
plasmid of some species of Agrobacterium.
 Size of T-DNA is between 15-30 kbp.
 It has LB and RB, RB plays a important role in transfer and
integrated of T-DNA. Absence of RB will terminate the T-DNA
transfer.
 T-DNA carry genes for phytohormones (Auxin and Cytokinin)
and opine that are expressed in plant cell.

16.  Over production of these hormones at the sites of infection is
responsible for the proliferation of wound cell into a gall/tumor.
These tumor can harbor a plenty of bacteria.
 Opines synthesis is a unique characteristics cells. some opines
are showing high plant species specificity.
 Opines are low molecular weight compounds found in plant’s
crown gall tumors or hairy root diseases produced by parasitic
bacteria of the genes Agrobacterium.
 Different types of opines like: Nopaline, Octapine, Agropine and
Succinamopine types.
 These opines are important source of nitrogen, carbon and
energy. These opines are condensation product of : a). An amino
acid and a keto acid; and b).An amino acid and a sugar.
 Naturally occuring T-DNA codes for: (i) Opine synthesis; and
(ii) Phytohormones to induce tumor genesis.

17. Structureofvariousopines

18. FunctionsofT-DNAgenesinTi-plasmid
vir All DNA transfer into plant
shi All Shoot induction
roi All Root induction
ocs Octapine Octopine synthesis
occ Octapine Octopine catabolism
nos Nopaline Nopaline synthesis
noc Nopaline Nopaline catabolism
tra All Bacterial transfer genes
Inc All Incompatibility genes
oriV All Origin of replication
tms1 Trytophane-2-mono-oxygenase Auxin synthesis
tms2 Indoleacetamide hydrolase Auxin synthesis
tmr Isopentyl transferase Cytokinin synthesis
frs Fructopine synthase Opine synthesis
mas Mannopine synthase Opine synthesis
ags Agropine synthase Opine synthesis
Genes Ti-plasmid Functions

20. Virulence
Gene
Function in Agrobacterium Function in plant
Essential genes
vir A • Kinase protein in bacterial membrane;
• Receptor of phenolic compounds, sensor of a 2
component regulatory system
-
vir Aand G Phenolic response regulator of a 2 component
regulatory system (acetosyringone)
-
vir G Activates other vir genes
vir D1 • Endonuclease
• Required for T-DNA processing in-vivo and for
ds T-DNAborder nicking in-vitro
-
vir B/D4 • Type IV secretion system (T4SS - binding
system apparatus)
• Synthesis and assembly of the T-pilus (vir B2
encodes a prepropilin)
• Formation of pore channel to transfer T-DNA
from bacterium to plant cell
-
vir C1 • Stimulate transfer
• Putative “overdrive” binding protein;
• Enhancement of T-DNAtransfer
-
vir D2 • T-DNAborder specific endonuclease; • Nuclear targeting of the T-stranded;

21. Virulence
Gene
Function in Agrobacterium
vir D2 • Prevent attack of exonuclease at 5’ end of T-
DNA;
• Cutting phosphodiester bond
• Putative ‘pilot protein’ that leads the T-strand
through the transfer apparatus and into the plant
Function in plant
• Protection of the T-strand from 5’
the plant
exonucleolytic degradation;
• T-strand integration into
genome
vir E/E2 Act as single stranded binding protein Protect T-DNA against nuclease and
target T-DNAto plant cell
vir E1 • Required for vir E2 except from Agrobacterium;
• Chaperone for vir E2
-
vir E2 • Formation of
Agrobacterium
• Have nuclear localization signal (NLS)
a putative “T-complex” in • Formation of a putative “T-complex” in
plant;
• Protection of the T-strand from
nucleolytic degradation;
• Nuclear targeting of the T-strand;
• Passage of the T-strand through the
nuclear pore complex
Non Essentials Genes
vir F - • Host range factor;
• Possible interaction with skip proteins
to regulate plant cell division cycle
vir H Putative cytochrome P450 enzyme -
vir J Putative T-strand binding proteins T-strand export
from Agrobacterium
-

22. Stepsoftransformationprocess
Plant stress conditions
Production of phenolics compounds form plant cell
Attachment of A. tumefaciens to the plant cells
Sensing plant signals by A. tumefaciens and regulation of virulence
genes in bacteria following transduction of the sensed signals
Generation and transport of T-DNAcomplex and virulence proteins
from the bacterial cells into plant cells
Nuclear import of T-DNA and effecter proteins in the plant cells
T-DNAintegration and expression in the plant genome

23. ⦿ T-DNA region get replaced by any gene of interest and then
targeted to Plant cell for transformation.
Note:
⦿ Monocot are not good host for Agrobacterium.
⦿ There is a hypothetical believe that monocot are resistant to
Agrobacterium because they do not produce phenolics that can
induce Virulence genes.

24. S.No. Requirements Advantages Disadvantages
1. The explants of plant must
produce acetosyringone or
other related phenolic
compounds
Natural means of transfer
hence plant friendly
• Limited host range.
• Can not infect
cereal plants.
2. The induce bacteria should
have access to cell 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
3. Transformation competent
cells and tissue should be
able to regenerate into
whole plants
• Capable of transferring
fragmented of
very
large
DNA
without
efficiently
substantial
rearrangements.
• The stability of gene
transferred is excellent.