Agrobacterium tumefaciens :genetic tool

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Agrobacterium: a natural genetic engineer
Agrobacterium
Agrobacterium tumefaciens is the causal agent of crown gall disease (the formation
of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil
bacterium. A. tumefaciens is an alphaproteobacterium of the family Rhizobiaceae,
which includes the nitrogen-fixing legume symbionts. Unlike the nitrogen-fixing
symbionts, tumor-producing Agrobacterium species are pathogenic and do not benefit the
plant.
History
Erwin Smith and Charles Townsend published a paper in Science (1907)
reporting that a bacterium that they named Bacterium tumefaciens caused plant tumors
or crown galls on a variety of plants. The next major discoveries on Agrobacterium and
crown gall, starting in the 1940s, were made at the Rockefeller Institute by Armin
Braun whose interests in the system were initially sparked by a surprising observation of
a student studying crown gall in Germany.
. During the 1960s :
Morel and colleagues demonstrated that various bacteria-free crown gall tumours
synthesized unusual amino acid–sugar conjugates (termed ‘opines’) whose chemistry was
specified by the strain of Agrobacterium that incited the tumour.Taken together, this
evidence suggested that a transformation of the plant cell by Agrobacterium is
responsible for the formation of opine-producing crown gall tumours.
In the mid-1970s :
‘Tumour -inducing’ (Ti) plasmid in A. tumefaciens is necessary for this bacterium to
incite tumours, and, moreover, specifies the type of opine produced by that
tumour.Further investigations revealed that a piece of DNA from this plasmid (the T-

DNA) is transferred from the bacterium into the plant cell where it integrates into the
nuclear DNA and is expressed .
Main approach:
Plant transformation mediated by Agrobacterium tumefaciens has become the most used
method for the introduction of foreign genes into plant cells and the subsequent
regeneration of transgenic plants.
The Ti plasmid
• Large conjugative plasmid or mega plasmid(200 kb)
• pTi is lost when Agrobacterium is grown above 28oC ;such curated bacteria not induce
crown galls, i.e., they become avirulent
p Ti are unique bacterial plasmids
• They contain some genes (T-DNA),which have regulatory sequences(expressed only in
plant cells) recognized by plant cells, while their remaining genes have prokaryotic
regulatory sequences(expressed only in bacterium).
• These plasmids naturally transfer their T-DNA into the host plant genome, which makes
Agrobacterium a natural genetic engineer.
Ti plasmids classification
Basis : opine produced by their genes
Octopine type
Nopalinee type
Important functional regions of p Ti plasmids
• T –DNA contains oncogens and opine synthesis genes, and is transferred into the host
plant genome
• vir region regulate the transfer of T-DNA into plant cells

• Opine catabolism regions produce enzymes necessary for the utilization of opines by
agrobacterium.
• Conjugative transfer(ori T or tra)region functions in conjugative transfer of the plasmid:
it can also function in T-DNA transfer when the T-DNA borders are deleted
• Origin of replication for propagation in Agrobacterium
P Ti plasmid structure
Organization of T-DNA
Iaa M(aux,tms 1)
Auxin biosynthesis ;encodes the enzyme tryptophan-2-mono-
oxygenase,which converts tryptophan into indole-3-
acetamide(IAM)
Iaa H(aux2,tms2)
Auxin biosynthesis ;encodes the enzyme indole-3-acetamide
hydrolase, which converts IAM into IAA(indole acetic acid)

ipt(tmr,cyt)
Cytokinin biosynthesis; encodes the 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
Sites of endonuclease action during T-DNA transfer; the only
sequence of T-DNA essential for its transfer.
Organization of vir region
Vir A(1)
Encodes a sensor protein; receptor for acetosyringone and functions as an
autokinase; also phosphorylates vir G protein; constitutive expression
Vir B(11)
Membrane proteins; possiby form a channel for T-DNA transport(conjugal
tube formation; virB 11 has ATPase activity
Vir C(2)
Helicase; binds to the overdrive region just outside the right border;
involved in unwinding of T-DNA
Vir D(4)
VIR D1 has topoisomerase activity; it binds to the border of T-DNA; vir
D2 is an endonuclease ;it nicks the right border

Vir E(2)
Single strand binding proteins(SSBP); bind to T-DNA during its transfer
Vir F(1)
-
Vir G(1)
DNA binding protein ;probably forms dimer after phosphoryaltion by vir
A and induces the expression of all vir operons (operon A to H);constitutive
expression
Vir H(2)
-
Properties of crown gall cells
• Infection by A. tumefaciens produces tumour like growth from which roots and/or shoots
may sometimes be produced.
• Crown gall cells are capable of growing in culture on growth regulator(GR)-free medium,
while normal plant cells need exogenous auxin and or cytokinin.
• Crowngall cells synthesize unique nitrogenous compounds-opines
• Agrobacterium cells use opines as their carbon and nitrogen source.
• A.tumefaciens strains generally produce octopine or nopaline.
• A bacterial strain produces only one type of opine and it also catabolizes only that opine
• Plasmids carry genes for IAA and cytokinin production, which is the reason for indefinite
growth of crowngall cells on a GR-free culture medium.
Agrobacterium tumefaciens T-DNA transferprocess
Bacterial colonization
Induction of bacterial virulence system,
Generation of T-DNA transfer complex
T-DNA transfer
Integration of T-DNA into plant genome.
Bacterial colonization

The polysaccharides of the A.tumefaciens cell surface are proposed to play an
important role in the colonizing process.The LPS are an integral part of the outer
membrane and include the lipid A membrane anchor and the O-antigen polysaccharide in
their composition.The chromosomal 20kb att locus contains the genes required for
successful bacterium attachment to the plant cell.
Some chromosomal genes of Agrobacterium tumefaciens involved in cell attachment
(attachment of bacterial cells to plant cells)
Chromosomal gene
Function
chvA
encodes an inner membrane protein essential for the transport of β-
1,2-glucan from cytoplasm to periplasm
chvB
encodes an inner membrane protein most likely involved in the
synthesis of β-1,2-glucan
ChvD and chv E
Need for an optimal expression of vir genes of pTi
exo locus genes
Biosynthesis of attachment polysaccharides
exo C
Encodes an enzyme directly involved in the biosynthesis of β-1,2-
glucan
cel genes
Cellulose fibril synthesis especially during the early phases of
infection so that the bacterial cells become firmly adhered to plant
cells.
Induction of bacterial virulence system
The T-DNA transfer is mediated by products encoded by the 30-40 kb vir region
of the Ti plasmid. VirA is a transmembrane dimeric sensor protein that detects signal
molecules, mainly small phenolic compounds (acetosyringone), released from wounded

plants. Activated VirA has the capacity to transfer its phosphate to a conserved aspartate
residue of the cytoplasmic DNA binding protein VirG.VirG functions as a transcriptional
factor regulating the expression of vir genes when it is phosphorylated by VirA The
activation of vir system also depends on external factors like temperature and pH. At
temperatures greater than 32°C, the vir genes are not expressed because of a
conformational change in the folding of VirA induce the inactivation of its properties
Generation of T-DNA transfer complex
The activation of vir genes produces the generation of single-stranded (ss)
molecules representing the copy of the bottom T-DNA strand. Any DNA placed between
T-DNA borders will be transferred to the plant cell as single strand DNA and integrated
into the plant genome The proteins VirD1 and VirD2 play a key role in this step,
recognizing the T-DNA border sequences and nicking (endonuclease activity) the
bottom strand at each border.After endonucleotidic cleavage, VirD2 remains covalently
attached to the 5’-end of the ss- T-strand. This association prevents the exonucleolytic
attack to the 5’-end of the ss-T-strand and distinguishes the 5’-end as the leading end of
the T-DNA transfer complex.
T-DNA transfer
Vir B operon has 11 genes, which encode mostly membrane bound proteins; vir
B is essential for virulence. Vir B proteins ,together with vir D4 protein, participate in
conjugational tube formation between the bacterial and plant cells, which provides a
channel for T-DNA transfer. Vir B 11 has ATPase activity and generates energy needed
for the delivery of T-DNA into the plant cells.
Integration of T-DNA into plant genome
T-DNA enters plant cells in a single stranded formIt is converted ds DNA form in
the nuclei. dsT-DNA integrate at random sites in the host plant genome most likely by a
process of illegitimate recombination due to a homology in short segments of the host
DNA. T-DNA integration is accompanied by short deletions of 23-79 bp at the site of
recombination.

Agrobacterium and genetic engineering: Engineering the Ti plasmid
The use of wild type pTi as vector presents the following three problems
1. Presence of oncogenes (iaaM,iaaH and ipt) in T-DNA, which causes a disorganized
growth and a loss of regeneration potential of the cells having T-DNA in their genomes.
2. Their large size makes the handling procedures during cloning tedious and cumbersome.
3. A general lack of unique cloning sites within the T-DNA ,which are needed for the
insertion of DNA segments to be cloned.
The deletion of genes governing auxin and cytokinin production(the oncogenes) from T-
DNA of a Ti plasmid is known as disarming.
Co integrative pTi vectors:
A co-integrative vector is produced by integrating the modified E.coli plasmod
into a disarmed pTi. The co-integration of the two plasmids is achieved within
Agrobacteium by homologous recombination.

BINARY VECTOR:
A binary vector consists of a pair of plasmids of which one plasmid contains
disarmed T-DNA sequences, while the other contains the vir region, and ordinarily lacks
the entire T-DNA including the border.
Agrobacterium and genetic engineering: Engineering the Ti plasmid

The Agrobacterium mediated transformation process involves a number of steps
1) Isolation of the genes of interest from the source organism
2) Development of a functional transgenic construct including the gene of interest;
promoters to drive expression; codon modification, if needed to increase successful
protein production; and marker genes to facilitate tracking of the introduced genes in the
host plant
3) Insertion of the transgene into the Ti-plasmid
4) Introduction of the T-DNA-containing-plasmid into Agrobacterium
5) Mixture of the transformed Agrobacterium with plant cells to allow transfer of T-DNA
into plant chromosome.
6) Regeneration of the transformed cells into genetically modified (GM) plants.
7) Testing for trait performance or transgene expression at lab, greenhouse and field
level.
Two methods
• Co-culture with tissue explants :
The appropriate gene construct is inserted within the T-region of a disarmed Ti
plasmid; either a co-integrate or a binary vector is used. The recombinant DNA is placed
in Agrobacterium, which is then co-cultured with the plant cells or tissues to be
transformed for about 2 days.

• In planta transformation:
Imbibitions of Arabidopsis genome; the majority of about 14,000 transformants
screened represent independent and unique events.
CASE STUDY -1
GIST OF THE RESEARCH
They developed an effective floral dip, Agrobacterium-mediated transformation method
for rice (Oryza sativa L.) cultivar RD41. A. tumefaciens strain AGL1 harboring the binary vector
pCAMBIA1304 carrying the gusA gene (AGL1-1304) was used to infect rice spikelets via the
floral-dip method. The tip-cut spikelets of the rice inflorescence stage 51 were dipped in the
Agrobacterium AGL1-1304 suspension and co-cultivated at 25 °C for 3 d. The target sites of
transformation were detected by histochemical GUS assay.
MATERIALS AND METHODS
• Rice (Oryza sativa L.) cultivar RD41 obtained from the Phitsanulok Rice Research
Center, Thailand was used in this study.
• The RD41 seeds were soaked in water for 2 d and then placed in a moist filter cloth until
germination.
• The germinated seeds were planted in a pot containing fertile and wet clay.
• The 1-2 week-old seedlings were moved to a new pot, three plants per pot.
• The rice plants were grown in the greenhouse .
• The inflorescences at stage 51 (the beginning of panicle emergence: tip of inflorescence
emerged from sheath) according to the BBCH-scale were used for the floral-dip
transformation.
• Agrobacterium strain and plasmid
• A. tumefaciens strain AGL1 harboring pCAMBIA1304 was used for the floral-
dip transformation.
• The binary vector pCAMBIA1304 contains the hygromycin
phosphotransferase (hptII) gene, and the reporter genes, green fluorescent protein
(gfp) and β-glucuronidase (gusA), under the control of the CaMV35S promote .

Schematic diagram of the pCAMBIA1304 T-DNA region
• A. tumefaciens strain AGL1 harboring pCAMBIA1304 was grown in 5 ml of Luria broth
(LB) medium supplemented with kanamycin (50 mg/l) and rifampicin (40 mg/l) at 28°C
and 250 rpm for 2 d.
• The culture was grown at 28°C and 250 rpm until reaching the stationary phase OD600
0.8–1.0. Bacterial cells were collected by centrifugation at 5600xg for 3 min.
• The cell pellets were resuspended in the inoculation medium. The inoculation medium
was composed of basal medium Murashige and Skoog (MS) medium ,5% (v/v) sucrose,
44 nM benzylaminopurine, and 0.075% (v/v) of the surfactant Tween-20. The pH of the
inoculation medium was adjusted to 5.7 for dipping
Floral dip transformation
• Rice inflorescences at stage 51 as mentioned above were used for transformation.
• The tips of selected rice spikelets were cut off before the inflorescence was dipped in
the Agrobacterium inoculation medium. Dipping was carried out for 1 min.
• Each dipped inflorescence was covered with a plastic bag (to maintain humidity) at
25°C for 3 d.
• The dipped rice spikelets were immersed in GUS staining solution containing 50 mg/ml
5-bromo-4-chloro-3-indolyl-β-D glucuronidase (X-gluc), 0.5 mM potassium
ferrocyanide, 0.5 mM potassium ferricyanide, 1 M Na2PO4, 0.5 M EDTA (pH 7.0),
0.5% Triton X-100 and 20% Methanol.
• The GUS staining spikelets were incubated in the dark overnight at 37°C.
• The stained spikelets were immersed in methanol: acetic acid (3:1) and 70% ethanol
overnight at room temperature to remove the chlorophyll.
• The transient GUS activity of spikelets was recorded as blue spots (irrespective of size)
using a microscope and photographed.

Analysis of GUS activity in rice spikelets after floral dip transformation:
a)-c). The dipped spikelet with no GUS expression in any tissue, d) the dipped spikelets
with GUS activity in lemma (Le), e) the dipped spikelets with GUS activity in anthers
(An) and filaments(Fi), f) the dipped spikelets with GUS activity in lodicules (Lo), ovary
(Ov), stigma and style (S). Bar = 1 mm.

GUS expression in pollens of the dipped spikelets:
(a), pollens from the GUS-negative anthers
(b), pollens from the GUS-positive anthers.
The photographs were taken under a compound microscope at 200x
magnification. Blue-stained pollens indicate GUS expression.
Casestudy-2
In the present study, an efficient Agrobacterium-mediated stable transformation
protocol for sugarcane (Saccharum officinarum L.) was optimized using embryogenic
calli and green fluorescent protein (gfp) as explants and reporter gene, respectively.
Parameters studied were optical density of bacterial culture and agro-infiltration under
different pressure regimes. Results indicated that out of four different treatments,
inoculation of calli with bacterial culture of OD6000.4 through vacuum infiltration at -50
kPa produced maximum number of transgenic plants. This newly developed protocol of
Agrobacterium-mediated transformation of sugarcane has shown significant
improvement over conventional procedure in terms of its transformation efficiency. This
protocol can be employed to develop transgenic sugarcane plants, having
tolerance/resistance against various biotic and abiotic stresses.

Materials and Methods
• The sugarcane (Saccharum officinarum) plants of commercial cultivar CPF-246 were
obtained from the Sugarcane Research Institute (SRI), Ayub Agriculture Research
Institute, Faisalabad, Pakistan
• Embryogenic Callus Cultures
• Vector Construction
Map of expression construct pGFP35S used for
harboring gfp as a reporter gene
Detail of inoculation treatments and in vitro culturing of sugarcane explants
T1: inoculation of embryogenic calli with A.tumefaciens culture (OD600 1.0) through
vacuum infiltration for 5 min at -50 kPa,
T2: inoculation of calli with A. tumefaciens culture (OD600 0.4) through vacuum
infiltration for 10 min at -50 kPa
T3: inoculation of calli with A. tumefaciens culture (OD600 1.0) at room temperature
(100 kPa) for 25 min
T4: inoculation of calli with A. tumefaciens culture (OD600 0.4) at room temperature
(100 kPa) for 45 min

C1: inoculation of calli with liquid LB media. Values with different alphabets indicate
that they are significantly different.
Different steps involved in in vitro culturing of Agrobacterium mediated transformation of
sugarcane
a )Non-transgenic control (C1), callus inoculated with LB media and then placed on
callus induction medium with Selection
b) Embryogenic callus placed in callus induction Medium
c) Callus regeneration after inoculation on regeneration medium
d )Putative transgenic plant on rooting medium with selection
Expression of GFP in PCR positive transgenic sugarcane plants under UV light with and
without microscope

a) Comparison of gfp expression in both transgenic (right) and non-transgenic (left C2)
plants under direct UV light, b) Non-transgenic sugarcane plant (C2) under UV light, c)f
Microscopic view of transgenic sugarcane plants expressing GFP under UV light ,d), e)
Non transgenic sugarcane plants.

References
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