Response of agrobacterium tumefaciens to plant signaling molecules

1. RESPONSE OF AGROBACTERIUM
TUMEFACIENS TO PLANT SIGNALLING
MOLECULES
Presented by:-
Rajpal Choudhary

2. • Agrobacterium is a rod shaped plant pathogenic soil
bacteria having two strains.
• A. tumifaciens cause crown gall (tumor) and
A. rhizogenes cause hairy root disease in dicot plants by
infecting through wounds on roots or stem at the soil
surface.
Agrobacterium

3. • The bacterium contains Ti (Tumor inducing ) and Ri (Root
inducing) plasmids. Both these plasmids can transfer
part of their DNA (T-DNA) into plant cell chromosome by
which Plant cells become transformed by expression of
T-DNA gene which induce disease.
Ti plasmid

4. COMPONENTS OF Ti PLASMID
• T- (Transferable) DNA region
• Vir (Virulence ) region
• Host Specificity Region
• Ori (origin of Replication) region
The Ti plasmids are classified into different ( about 14)
types depending upon the specific opine being synthesized.
( octapine/nopaline/Agropine ).

6. • It is ~200 kb mega plasmid.
• T-DNA ( 15-40 kb) region contains genes for synthesis of
Auxins, Cytokinin and Opines.
• Auxins and cytokinin genes are expressed in plant tissue
inducing disease.
• Opines (unusual amino acids ) produced by infected cells
are used as nutrients by Agrobacterium.
• T-DNA region is bordered on both sides by 25bp repeat
which helps in its transfer to plant genome.

7. • Virulence Region contains about 8 operons having about
24- 25 genes
• These genes help in transfer of T-DNA.
• Host specificity region has gene for conjugative transfer
and opine catabolism.
• Ti plasmid also has origin of replication.

8. ORGANISATION OF T-DNA
• T-DNA is ~ 23kb ( 15-40kb) segment bordered on both
sides by 25bp direct repeat sequences.
• T-DNA contains genes for tumor induction (IAAM, IAAH &
IPT) by forming auxins, cytokinins and Opines.
• All the genes in T-DNA region contain eukaryotic
regulatory sequences, so are expressed only in plant
cells.

9. ORGANISATION OF Vir REGION
• Vir region contains 8 operons (VirA,B,C,D,E,F ,G &H) which
together have 25 genes.
• Vir region mediates transfer of T-DNA into plant genome. It is
itself not transferred.
• VirA and Vir G are constitutive operons encoding Vir A and VirG
Proteins.
• Other Vir operons encode various proteins involved in T-DNA
transfer.

10. 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 duringT-DNA transfer; the onlysequences 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)
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.

11. MECHANISM OF TRANSFER OF T-DNA
• Transfer of T-DNA is a step wise process.
• Vir region of Ti plasmid becomes activated by the phenolic
signal molecules Acetosyringone and α-
hydroxyacetosyringone released by wounded tissue of dicot
plants which constitute wound response.

12. • Acetosyringone and α-hydroxyacetosyringone bind with Vir
A protein (located in the inner membrane) and activates it.
It start functioning as autokinase to phosphorylate itself
by ATP. Phosphorylated Vir A protein then phosphorylates
Vir G protein which then dimerises.
• Phosphorylated Vir G protein has DNA binding function. It
induces expression of rest of Vir operons.
• Vir D1 protein has topoisomerase and endonuclease
activity. It binds to right border sequence of T-DNA and
facilitate the action of Vir D2 protein which is also
endonuclease and nicks at the right border and remains
bound to 5’end.

14. • The 3’end produced at the site of nick serves as a primer for DNA
synthesis in 5’----3’ direction as a result of which one strand of T-
DNA is displaced from the DNA duplex.
• The T-DNA strand is again nicked at the left border to generate a
single strand copy of T-DNA.
• To this single strand copy Vir E 2 protein (single strand DNA
binding proteins ) bind for its protection against exonucleases.
• Vir B operon consisting of 11 genes encode membrane bound Vir
B proteins. These along with Vir D4 proteins participate in
conjugal tube formation between bacterial and plant cells for
transfer of T-DNA.
• Vir D2 which remains bound to 5’end of T DNA has a signal
sequence which drives it into the nucleus of plant cell.

16. INTEGRATION OF T-DNA INTO PLANT GENOME
• T-DNA enters plant cell as a single stranded structure which is
immediately converted into double stranded form.
• Vir E2 also has nuclear localization sequence and is
responsible for transfer of T DNA into plant cell nucleus.
• Double stranded T-DNA integrate at random sites in the host
plant genome.
• For integration 23-79 base pair deletion takes place at the
integration or target site.
• After integration of T-DNA into plant genome ,the genes for
auxins, cytokinins and opines express themselves which result
in uncontrolled growth in the form of tumor.

17. THANKYOU