1. Agrobacterium Mediated Gene Transfer Presented by: Anisur Rahman ASH1313008M Md. Homaion Kaber ASH1413075M Munia Akter BKH1413077F Urmi Das BKH1413074F Md. Shahidullah Bhuiyan ASH1313014M Presented to: Md. Anwar Hossain Assistant Professor BGE, NSTU. Razia Sultana Assistant Professor BGE, NSTU. Asma Talukder Lecturer BGE, NSTU. Course Name: Plant Genetic Engineering Practical Course Code: BGE-3206

2. Agrobacterium - mediated Gene Transfer ▪Most common method of engineering dicots, but also used for monocots ▪Pioneered by J. Schell (Max-Planck Inst., Cologne) ▪Agrobacteria – soil bacteria, gram-negative, related to Rhizobia – species: tumefaciens- causes crown galls on many dicots rubi- causes small galls on a few dicots rhizogenes- hairy root disease radiobacter- avirulent

3. Infection and Tumorigenesis ▪Infection occurs at wound sites as a response of Acetosyringone and α-hydroxy-acetosyringone released by wound sites of the plant ▪Involves recognition and chemotaxis of the bacterium toward wounded cells ▪galls are “real tumors”, can be removed and will grow indefinitely without hormones ▪genetic information must be transferred to plant cells

4. Tumor Characteristics The Plants cells in the tumor acquire two new properties 1.They show phytohormone independent growth 2.Synthesize a unique amino acid, called “opine” – octopine and nopaline - derived from arginine – agropine - derived from glutamate ▪ Opine depends on the strain of A. tumefaciens ▪ Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce.

5. Ti-plasmid Features Size is about 200 kb (Range from 150-250 kb) Has a central role in Crown-gall formation Has T-DNA region which contains oncogens and opine synthesis genes, and is transferred into the host plant Contain a vir region ~ 40 kb at least 8~11 vir genes which regulates the transfer of T-DNA into plant cells Has origin of replication Contains a region enabling conjugative transfer Has genes for the catabolism of opines

6. A Ti-plasmid

7. T-DNA iaaH LB (24 bp) iaaM ipttml nos RB (24 bp) LB, RB – left and right borders (24 bp direct repeat) iaaM – Encodes Tryptophan-2-monooxygenase (auxin biosynthesis) iaaH - Encodes indole-3-acetamide hydrolase (auxin biosynthesis) Ipt - Encodes isopentenyl transferase (cytokinin biosynthesis) tml – large tumor locus nos – Encodes nopaline synthase (nopaline biosynthesis)

8. Role of T-DNA Products IAAM IAAH Tryptophan  indole-3-acetamide  indoleacetic acid (auxin) IPT AMP + isopentenylpyrophosphate  isopentyl-AMP (cytokinin) Increased levels of these hormones stimulate cell division. Explains uncontrolled growth of tumor.

9. Organization 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. Functions of vir Genes virA – transports acetosyringone into bacterium, activates virG post-translationally (by phosphorylation) virB – membrane protein, which form channel during T-DNA transfer. virB11 has ATPase activity virC – Helicase. virD1 – topoisomerase activity virD2 – endonuclease activity virE – single stranded binding protein (SSBP) bind to T-DNA during its transfer. virG – promotes transcription of other vir genes

11. 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 as follows

12. Transfer of T-DNA (contd..) Acetosyringone and α- hydroxyacetosyringone bind with VirA protein which has autokinase activity to phosphorylate itself by ATP. Phosphorylated Vir A protein then phosphorylates Vir G protein which then dimerises. Phosphorylated Vir G protein induces the trnascription of all vir operons

13. Transfer of T-DNA (contd..) VirD1 binds to right border sequence of T-DNA and facilitate the action of VirD2 protein which is also endonuclease and nicks at the right border and remains bound to 5’end so generated. 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 E2 protein (SSBP ) bind for its protection against exonucleases VirB operon consisting of 11 genes encode membrane bound VirB proteins. These along with VirD4 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 to integrate the T DNA into plant genome.

14. Overview of the Infection Process

15. References o Bevan, M. (1984) “Binary Agrobacterium vectors for plant transformation”. Nucleic Acids Res 12: 8711 -8721. o Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell J., Van Montagu M. and Leemans J. (1985) “Efficient octopine Ti plasmid-derived vectors for Agrobacteriummediated gene transfer to plants”. Nucleic Acid Research 13:4777-4788. o Chilton, M.D. (1983) “A vector for introducing new genes into plants”. Scientific American, 248, 50-9. o Nadolska-Orczyk, A., Orczyk, W. and Przetakiewicz, A. (2000) “Agrobacteriummediated transformation of cereals – from technique development to its application”. Acta Physiologiae Plantarum, 22, 77-8. o Kakkar, A. and Verma, V.K.,(2011) Agrobacterium mediated biotransformation. Journal of Applied Pharmaceutical Science 01 (07); 2011: 29-35.