In part 1 we went through restriction enzymes and plasmid vector construct. Here in part 2, we will go through the bio-data of Agrobacterium tumefaciens and what properties make it natural plant genetic engineer!

1. Introduction to Plant Biotechnology
Part 2
Bio-Data of Natural Plant Genetic Engineer
Somnath Mondal

2. • Agrobacterium tumefaciens or Rhizobium radiobacter or Agrobacterium radiobacter is a
Gr -ve, rod shaped soil bacterium, which infects many plants.
• It causes crown gall disease, a plant tumor.
• It transforms plant cells at wound sites, causing the cells to grow and divide to form a
tumor.
• It contains Ti plasmid, which means “Tumor inducing” plasmid.
• Unique genetic configuration of Ti plasmid makes Agrobacterium tumefaciens a potent
plant genetic engineer.
• Agrobacterium rhizogenes or Rhizobium rhizogenes, another species causes hairy root
disease of plants.
Somnath Mondal

3. Somnath Mondal
C
P
Ti Plasmid

4. C
P
Ti Plasmid
T-DNA REGION
T-DNA REGION • About 23-24 kb in length.
• LB & RB T-DNA: Flanked by 24-25bp direct repeat
sequences, called Left Border T DNA and Right Border T
DNA respectively. It is the site of endonuclease cleavage
during T-DNA Transfer.
• Auxin Production: iaaM and iaaH genes are present. They
produce Tryptophan-2-mono-oxygenase (a) and Indole-3-
acetamide hydrolase (b) enzymes respectively.
Tryptophan (a) Indole-3-acetamide (b) IAA
• Cytokinin Production: ipt gene Produces the enzyme
Isopentenyltransferase, responsible for isopentenyl adenine
biosynthesis.
• Opine Synthesis: Nos gene produces Nopaline Synthase.
Arginine + Pyruvic Acid Nopaline.
Somnath Mondal

5. Ti Plasmid
vir REGION•virA (1): Receptor of Acetosyringone and
phosphorylates virG.
•virB (11): Forms channel for T-DNA transfer.
•virC (2): It has helicase activity and helps in
unwinding of T-DNA.
•virD (4): virD1 and virD2 have topoisomerase and
endonuclease activity respectively. They bind to RB
T-DNA and cuts it.
•virE (2): It codes Single Strand Binding Proteins,
which bind to the T-DNA during transfer.
•virF (1): -
•virG (1): virG Protein is phosphorylated by virA
protein and then induces expression of all vir genes.
•virH (2): - Somnath Mondal

6. Somnath Mondal
Mechanism of T DNA Transfer into Plant Cell
Plant Wound Acetosyringone + α hydroxyl acetosyringone
P
vir Operon- virA To virH genes
vir
promoter
Transcription, followed by translation and formation of all vir genes in
Agrobacterium cell
virA
virA
virG
virG
virG
1. Activation of vir operon

7. Somnath Mondal
vir Region T-DNA
LB RB
virD1
T-DNA
LB RB
virD2
T-DNA
LB RB
T-DNA
LB RB
3'
5'
Contd.
D1 D2 E2 B D4
2.TDNACUT&TRANSFER

8. Somnath Mondal
virE2
5'
3'
LB
T-DNA
RB
5'
3'
vir Region
virB & virD4
proteins together form the
conjugal tube in between
bacterium & plant cell
Contd.
Contd.
Into The Plant Cell
virE2
virE1T DNA
WITH
virD2

9. Somnath Mondal
virE2
virE1
T DNA Inserted into Plant DNA
Contd.
Plant Cell Cytoplasm
3.TDNAIntegration

10. Molecular Mechanism of T-DNA Integration into Plant
Genome
• Two models:
I. Strand Invasion Model.
II. Double Strand Break Model.
Somnath Mondal
• In Double Strand Break (DSB) Model, Non-Homologous End Joining (NHEJ)
occurs.
• NHEJ is a dsDNA-break repair mechanism.
• A number of proteins take part in this process, viz. :
i. Ku70/Ku80 heterodimer: This protein complex binds to the DNA double strand
breaks & allows proteins involved in NHEJ to bind.
ii. Xrcc4: Full form- X-Ray Repair Cross Complementing 4. It performs NHEJ.
iii. Lig4: It is an ATP dependent DNA Ligase.
*Saika et al, doi: 10.3389/fpls.2014.00560

11. DSB Model- Non-Homologous End Joining
3' 5'
3' 5'
3'5'
3' 5'
3'5'
3'
Plant Cell Nucleus
T DNA + virD2
+ virE1/E2
DNA Pol+ RNA
Primer
Ku80/Ku70
3'
Ku80/Ku70
5'
3'
3'
5'
Lig4/Xrcc4 Lig4/Xrcc4
3' 5'
3'5'
Part of plant
DNA
Somnath Mondal