Agrobacterium tumefaciens

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 ).

5. 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.

6. 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.

7. 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.

8. 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.

9. 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 during T-DNA transfer; the only sequences 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. vir B (11) Membrane proteins; possibly 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. 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. vir F (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.

10. 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.

11. 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.

12. 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.

13. 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.

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