Agrobacterium

A unique bacterial species

Plant-Fungal-Animal Transformation

Agrobacterium tumefaciens

1. Soil bacterium closely related to Rhizobium.

2. Causes crown gall disease in plants (dicots).

3. Infects at root crown or just below the soil line.

4. Can survive independent of plant host in the soil.

5. Infects plants through breaks or wounds.

6. Common disease of woody shrubs, herbaceous plants, particularly problamatic with many members of the rose family.

7. Galls are spherical wart-like structures similar to tumors.

Only known natural example of DNA transport between Kingdoms

1. (Virulent) strains of A. tumefaciens contain a 200-kb tumor inducing (Ti) plasmid

2. Bacteria transfer a portion of the plasmid DNA into the plant host (T-DNA).

T-DNA 🡪

The T-DNA is transferred from the Bacteria into the Nucleus of the Plant

1. Stably integrates (randomly) into the plant genome.

2. Expression of genes in wild-type T-DNA results in dramatic physiological changes to the plant cell.

3. 🡪 Synthesis of plant growth hormones (auxins and cytokinins) 🡪 neoplastic growth (tumor formation)

Opine Biosynthesis

1. Within tumor tissues, the synthesis of various unusual amino acid-like compounds are directed by genes encoded on the integrated plasmid.

2. The type of opine produced is specified by the bacterial T-DNA

3. Opines are used by the bacteria as a carbon (nutrient) source for growth.

4. Opine catabolism within bacteria is mediated by genes encoded on the Ti plasmid.

Overview of the Infection Process

How is the signal recognition (acetosyringone and other plant phenolics) converted to gene activation and other cellular responses?

Bacterial 2-Component Signal Transduction Systems

1. Component 1 : Sensor kinase

i) Substrate receptor, signal recognition domain, input domain (periplasmic)

ii) Signal transduction domain, membrane spanning region

iii) Autokinase domain, phosphorylation domain (cytoplasmic)

a) ATP binding (sub) domain

b) phosphorylation-phosphotransfer (sub)-domain

2. Component 2 : Response regulator

i) Phosphorylation domain

ii) DNA binding domain

Simplest case: transcriptional activator when phosphorylated

First component is typically (auto)-phosphorylated on a His residue and transfers to a Asp group on the response regulator (second component).

Agrobacterium tumafaciens senses acetosyringone via a 2-component-like system

3 components: ChvE, VirA, & VirG

1. ChvE

periplasmic protein binds to sugars, arabinose, glucose

• binds to VirA periplasmic domain

🡪 amplifies the signal

2. VirA : Receptor kinase

1. Membrane protein five functional domains:

a) Periplasmic binds ChvE-sugar complex does NOT bind acetosyringone

b) Transmembrane domain

c) Linker region BINDS acetosyringone NOTE this is on the cytoplasmic side!

d) Transmitter domain (His) auto- phosphorylates and then transfers to the response regulator protein VirG

e) Inhibitory domain 🡪 in absence of analyte will bleed off the phosphate from the His in the transmitter domain (to an Asp)

3. VirG : Response Regulator

a) Receiver domain that is phosphorylated on an Asp residue by the His on the transmitter domain of VirA

b) Activates the DNA binding domain to promote transcription from Vir-box continaing promoter sequences (on the Ti plasmid)

Periplasmic domain

acetosyringone

ChvE

VirA

VirG

sugars

Transmitter

Inhibitory domain

receiver

DNA-binding

Crown gall tumors

a natural example of genetic engineering.

Agrobacterium/plant interactions

opines

Agrobacterium at wound site transfers T-DNA to plant cell.

Agrobacterium in soil use opines as nutrients.

1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface.

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2. Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer.

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Ti plasmids and the bacterial chromosome act in concert to transform the plant

3. T-DNA encodes genes for opine synthesis and for tumor production.

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4. occ (opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient.

vir genes

opine catabolism

pTi

tra

for transfer to the plant

bacterial conjugation

Agrobacterium chromosomal DNA

chvA

chvB

pscA

oriV

T-DNA-inserts into plant genome

Generation of the T-strand

overdrive

Right Border

Left Border

T-DNA

virD/virC

VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.

5’

Generation of the T-strand

Right border

Left border

D

virD/virC

gap filled in

T-strand

T-DNA

virE

1. Helicases unwind the T-strand which is then coated by the virE protein.

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2. ~one T-strand produced per cell.

1. Transfer to plant cell.

2. Second strand synthesis

3. Integration into plant chromosome

Right border

Left border

D

T-strand coated with virE

T-DNA

virD nicks at Left Border sequence

The vir region is responsible for the transfer of T-DNA to the wounded plant cell.

receptor for acetyl-syringone

positive regulator for other vir genes

Note: activated virG causes its own promoter to have a new start point with increased activity.

virA is the sensor.

bacterial

membrane

Acetylsyringone is produced by wounded plant cells (phenolic compound).

triggers auto-phosphorylation of virA

1

2

3

virG

virA

virG activates transcription from other vir promoters.

VirA phosphorylates virG which causes virG to become activated.

virG is the effector.

Asg

Asg

The vir region is responsible for the transfer of T-DNA to the wounded plant cell.

ssDNA binding protein. Binds T-strand.

virA

virG

virB

virC

virD

virE

sensor

effector

endo-

nuclease nicks T-

DNA

Binds overdrive DNA.

membrane protein; ATP-binding

Note: The virA-virG system is related to the EnzZ-OmpR system that responds to osmolarity in other bacteria.

Generation of the T-strand

overdrive

Right Border

Left Border

T-DNA

virD/virC

VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.

5’

Generation of the T-strand

Right border

Left border

D

virD/virC

gap filled in

T-strand

T-DNA

virE

1. Helicases unwind the T-strand which is then coated by the virE protein.

​

2. ~one T-strand produced per cell.

1. Transfer to plant cell.

2. Second strand synthesis

3. Integration into plant chromosome

Right border

Left border

D

T-strand coated with virE

T-DNA

virD nicks at Left Border sequence

Assembly of the Agrobacterium T-Complex Transport Apparatus

6. VirD4, VirB4 and VirB11 have nucleotide-binding motifs that are essential for their activity.

7. The T-complex, consisting of a ss copy of T-DNA bound to VirD2 and coated with VirE2, is exported through the transport apparatus.

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SP, signal peptide; SPI, signal peptidase I.

(a) The pilus has not contacted the surface of the recipient plant cell and the apparatus is unable to transport T-complex.

(b) The pilus has contacted a receptor (?) on the surface of the recipient plant cell. This induces the VirB transporter, perhaps via a change in conformation, so that it is now competent to transfer the T-complex to the plant cell cytoplasm.

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OM, outer membrane; IM, inner membrane; CW, plant cell wall; PM, plasma membrane.

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Model for contact-dependent activation of the T-complex transport apparatus

1. The VirB and VirD4 proteins are grouped according to probable functions:

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1. exocellular proteins mediating attachment (VirB1*, VirB2 pilin and VirB5)
2. channel proteins (VirB3, VirB6, VirB7, VirB8, VirB9 and VirB10)
3. ATPases (VirB4, VirB11 and VirD4).

Locations of the Vir Protein Components of the T-DNA transfer system

Agrobacterium can be used to transfer DNA into plants

pTi-based vectors for plant transformation:

2. Early shuttle vectors integrated into the T-DNA; still produced tumors.

1. Shuttle vector is a small E. coli plasmid using for cloning the foreign gene and transferring to Agrobacterium.

E. coli

Agrobacterium

pTi

Shuttle plasmid

conjugation

Several hundred tumors containing foreign gene can be grown for experimental purposes.

Transformed sunflower seedlings

Harvest time!

3 weeks after inoculation

Transformation of Arabidopsis plants

Dip floral buds in 1 ml of Agrobacterium culture for 5 to 15 min.

Detergent added to allow bacteria to infiltrate the floral meristem.

Transformation of Arabidopsis plants

700 to 900 seeds per plant.

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Germinate on kanamycin plates to select transformants.

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10 to 20 transformed plants per plant.

10 day old seedlings

MiniTi T-DNA based vector for plants

1. Binary vector: the vir genes required for mobilization and transfer to the plant reside on a modified pTi.

2. consists of the right and left border sequences, a selectable marker (kanomycin resistance) and a polylinker for insertion of a foreign gene.

Disarmed vectors: do not produce tumors; can be used to regenerate normal plants containing the foreign gene.

miniTi

MiniTi T-DNA based vector for plants

modified Ti plasmid

a binary vector system

oriV

vir

T-DNA deleted

LB

RB

ori

kan^r

polylinker

miniTi

bom

bom = basis of mobilization