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

Crown gall tumors

a natural example of genetic engineering.

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

opines

Agrobacterium in soil use opines as nutrients.

Genes required to breakdown opines for use as a nutrient source are harbored on the Ti plasmid in addition to vir genes essential for the excision and transport of the T-DNA to the wounded plant cell. cell.
T-DNA 23 kb

tra vir genes

for transfer to the plant

pTi ~200 kb

bacterial conjugation

opine catabolism

Ti plasmids can be classified according to the opines produced

1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas). 2. Octopine plasmids: carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue.

3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out.

H2N

(Nopaline)

CNH(CH2)2CHCO2H HN NH HO2C(CH2)2CHCO2H

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

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

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

Asg

virA is the sensor.

P

virA
bacterial membrane

Asg

Acetylsyringone is produced by wounded plant cells (phenolic compound). 3

P

triggers autophosphorylation of virA

VirA phosphorylates virG which causes virG to become activated.

virG

virG activates transcription from other vir promoters.

virG is the effector.

Agrobacterium chromosomal DNA

pscA

chvA chvB T-DNA-inserts into plant genome tra

for transfer to the plant

pTi
vir genes

bacterial conjugation

opine catabolism oriV

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

virA

virG virB

virD virC

virE
ssDNA binding protein. Binds Tstrand.

endomembrane Binds protein; overdrive nucleas e nicks ATP-binding DNA. TDNA Note: The virA-virG system is related to the EnzZOmpR system that responds to osmolarity in other bacteria.

Generation of the T-strand TLeft Border Right Border overdrive

T-DNA

5 virD/virC VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5 end.

Generation of the T-strand TLeft border Right border

T-DNA

gap filled in

virE virD/virC
D

T-strand

1. Helicases unwind the T-strand which is then coated by the virE protein. 2. ~one T-strand produced per cell.

Left border D

T-DNA

Right border

T-strand coated with virE virD nicks at Left Border sequence

1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome

The vir region is responsible for the transfer of T-DNA to the wounded plant cell. TvirA is the sensor. activated virG membrane
constitutive

virA
receptor for acetylsyringone

virG Note: activated virG positive regulator causes its own promoter for other to have a new start point vir genes with increased activity.

Left border D

T-DNA

Right border

T-strand coated with virE virD nicks at Left Border sequence

1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome

Assembly of the Agrobacterium T-Complex Transport Apparatus 1. VirB1 may have local lytic activity that allows assembly of the transporter at specific sites in the cell envelope. 2. The processed VirB1* peptide is secreted through the outer membrane by an unknown mechanism. 3. The structural components of the pilus are VirB2 and VirB5. 4. Complexes of VirB7/9, formed by disulfide bridges, may initiate assembly of the VirB channel. 5. The exact role of VirB3, 4, 6, 8, 10 and 11, and VirD4 in the transporter apparatus is unknown.

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. SP, signal peptide; SPI, signal peptidase I.

Model for contact-dependent activation of the Tcomplex transport apparatus (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.

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

Agrobacterium can be used to transfer DNA into plants

pTipTi-based vectors for plant transformation: 1. Shuttle vector is a small E. coli plasmid using for cloning the foreign gene and transferring to Agrobacterium. 2. Early shuttle vectors integrated into the TDNA; still produced tumors. pTi Agrobacterium

Shuttle plasmid E. coli conjugation

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

Transformed sunflower seedlings

3 weeks after inoculation

Harvest time!

Transformation of Arabidopsis plants

Detergent added to allow bacteria to infiltrate the floral meristem.

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

Transformation of Arabidopsis plants

700 to 900 seeds per plant. Germinate on kanamycin plates to select transformants. 10 to 20 transformed plants per plant. 10 day old seedlings

Summary Agrobacteria are biological vectors for introduction of genes into plants. Agrobacteria attach to plant cell surfaces at wound sites. The plant releases wound signal compounds, such as acetosyringone. The signal binds to virA on the Agrobacterium membrane. VirA with signal bound activates virG.

Activated virG turns on other vir genes, including vir D and E. vir D cuts at a specific site in the Ti plasmid (tumor-inducing), the left border. The left border and a similar sequence, the right border, delineate the T-DNA, the DNA that will be transferred from the bacterium to the plant cell Single stranded T-DNA is bound by vir E product as the DNA unwinds from the vir D cut site. Binding and unwinding stop at the right border.

The T-DNA is transferred to the plant cell, where it integrates in nuclear DNA. T-DNA codes for proteins that produce hormones and opines. Hormones encourage growth of the transformed plant tissue. Opines feed bacteria a carbon and nitrogen source.