Agrobacterium Ti-Plasmid Transfer for Biotechnology & Botany Sem-5

Agrobacterium Ti-Plasmid Transfer for Biotechnology & Botany Sem-5

1. 1
Agrobacterium is soil borne Gram-
negative phytopathogen known to
cause crown gall disease (Smith and
Townsend, 1907) in dicotyledonous
plants like grape vines, stone fruit, apple,
peach, cherry, almond, raspberry, and
nut trees. Agrobacterium has been referred
to as nature’s genetic engineer because of
its natural ability to transfer its genetic
material to eukaryotic cells and it is the
only known example of trans-kingdom
DNA transfer because of which it has been used extensively in genetic engineering
of plants for the introduction of foreign genes into plant cells. Agrobacterium-
mediated plant transformation has revolutionized agricultural biotechnology and has
led to improved yield and quality of crops thus aiding in world food security.


Agrobacterium Ti-Plasmid Transfer

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Structure of the Ti-Plasmid
The Ti plasmid is a large,
double-stranded, circular
DNA molecule, typically
ranging from 200 to 800 kb. It
consists of several important
regions:
1. T-DNA (Transferred
DNA) Region
o Contains genes
responsible for
plant
transformation.
o Flanked by left
border (LB) and right border (RB) sequences, which are essential
for transfer.
o Carries genes that code for:
▪ Oncogenes (e.g., iaaM, iaaH, and ipt) → induce plant tumors
(crown galls).
▪ Opine biosynthesis genes → produce special nutrients (opines)
for the bacterium.
2. Vir (Virulence) Region
o Contains genes required for the transfer of T-DNA into the plant cell.
o Key genes include VirA, VirG, VirB, VirC, VirD, VirE, and VirF.
3. Opine Catabolism Genes
o Enable Agrobacterium to utilize opines (e.g., nopaline, octopine) as
nutrients.
4. Origin of Replication (Ori)
o Allows the plasmid to replicate within Agrobacterium cells.
5. Conjugation (tra) Genes
o Facilitate horizontal gene transfer between Agrobacterium cells.

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Mechanism of Ti Plasmid Transfer
The transfer of T-DNA from Agrobacterium to a plant cell involves several steps:
1. Recognition of Wounded Plant Cells
• Agrobacterium detects plant wound signals such as acetosyringone and
other phenolic compounds released by injured plant tissues.
• The VirA-VirG two-component regulatory system in the Vir region gets
activated.
2. Activation of Vir Genes
• VirA (sensor kinase) detects plant signals and phosphorylates VirG
(response regulator).
• VirG then activates transcription of other Vir genes (VirB, VirC, VirD,
VirE, etc.).
3. Processing of T-DNA
• VirD1/D2 endonuclease complex recognizes LB and RB sequences,
making a single-stranded nick at the RB.
• The T-strand (single-stranded T-DNA) is released, with VirD2 covalently
attached to its 5’ end.
• VirE2 coats the T-strand to protect it from degradation.
4. T-DNA Transfer to the Plant Cell
• The VirB/D4 type IV secretion system (T4SS) forms a pilus-like
structure that transports the T-DNA into the plant cell cytoplasm.
• VirD2 guides the T-DNA into the nucleus of the plant cell.
5. Integration into the Plant Genome
• The T-DNA is integrated into the plant’s genome randomly.
• The oncogenes in T-DNA cause overproduction of auxins (IAA) and
cytokinins, leading to uncontrolled cell division and tumor (crown gall)
formation.
• The opine synthesis genes allow the plant cells to produce opines, which
serve as an exclusive food source for Agrobacterium.

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Applications of Ti Plasmid in Plant Biotechnology
The Ti plasmid has been engineered for plant genetic modification, replacing
tumor-inducing genes with genes of interest:
1. Development of Transgenic Crops
o Introduction of disease resistance, herbicide resistance, pest
resistance genes (e.g., Bt crops).
o Genetic modifications for improved nutritional quality (e.g., Golden
Rice).
2. Plant Functional Genomics
o Study of gene functions by knockout or overexpression in model
plants.
3. Production of Pharmaceutical Compounds
o Expression of therapeutic proteins, vaccines, and antibodies in plants.
4. Bioremediation
o Engineering plants to detoxify pollutants using transgenic
technology.

Modified Ti Plasmid for Genetic Engineering
• Disarmed Ti Plasmids
o The oncogenes in T-DNA are removed to prevent tumor formation.
o A gene of interest (e.g., resistance genes, marker genes) is inserted
instead.
o The binary vector system is commonly used for plant transformation.
• Binary Vector System
o The T-DNA region and Vir genes are separated into two different
plasmids:
1. Binary Vector – Contains T-DNA with the gene of interest.
2. Helper Plasmid – Provides Vir genes for T-DNA transfer.

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Key Terms to Remember
• Ti Plasmid – Tumor-inducing plasmid in Agrobacterium.
• T-DNA – The DNA region transferred into the plant genome.
• Vir Genes – Genes responsible for T-DNA processing and transfer.
• Opines – Special nutrients synthesized by transformed plant cells.
• Binary Vector System – A method for plant genetic engineering using two
plasmids.