Agrobacterium tumefaciens mediated gene transfer

Agrobacterium tumefaciens, agrobacterium tumefaciens mediated gene transfer, gene transfer in plants, agrobacterium mediated gene transfer

1. Presented by S.Kaayathri Devi 19PO03 II M.Sc., Biotechnology ANJAC Agrobacterium tumefaciens mediated gene transfer Agrobacterium tumefaciens mediated gene transfer Presented by S.Kaayathri Devi 19PO03 II M.Sc., Biotechnology ANJAC

2. Agrobacterium tumefaciens Introduction Smith and townsend (1907) – said bacteria caused crown gall disease Brown & stonier (1958) – proposed that not whole bacteria but some part of it causes disease Zenen.et.l (1974) – noted virulent strain Agrobacterium tumefaciens Chilton.et.al (1977)- reported Ti & Ri plsmid transfer to plant causing disease.

3. SYSTEMIC POSITION Kingdom: Bacteria Phylum: Proteobacteria Class: Alpha Proteobacteria Order: Rhizobiales Family: Rhizobiaceae Genus: Agrobacterium

4. Agrobacterium (little genetic engineer): Agrobacterium tumefaciens is the causal agent of crown gall disease Agrobacterium tumefaciens- Crown gall disease Agrobacterium rhizogenes - Hairy root disease Agrobacterium radiobacter - Avirulent strain

5. A soil-born gram negative bacterium. It is a rod shaped and motile and belongs to the bacterial family of Rhizobiaceae . It is a phytopathogen , and it is regarded as Nature’s most effective plant genetic engineer. It is the natural expert of inter-kingdom gene transfer .

6. Infects wounded or damaged part of plant causing plant tumor called crown gall. The entry of the bacterium is faciliated by the release of phenolic compounds like Acetosyringone and Hydroxysyringone . Crown gall occurs when the bacterium releases its Ti-plasmid into the plant cell cytoplasm. The T-DNA is transferred to the host cell via wounded section.

7. The T-DNA carries genes that code for proteins involved in the biosynthesis of growth hormones (auxin and cytokinin) and novel plant metabolites namely- opines and agropines. Growth hormones – plant cell proliferation Opines & agropines – source of C and energy

8. Ti Plasmid: A Ti plasmid is a circular piece of DNA found in almost all Agrobacteria Three main region: T-DNA region(Between right and left T-DNA border) Oncogene Opine( argenine derivative) Virulence region Opine catabolism region

9. VIRULENCE GENES(24 GENES): Located in 8 operons from vir A to vir H Vir A senses acitosyringone Vir G transcriptional activator of vir box Vir B conjugational pores between plant cell and bacteria Vir D1 essential for cleavage of supercoiled stranded substrate Vir E responsible for gene transfer protein Vir C helps in DNA transfer Vir B11 ATPase activity – provides energy for DNA

10. Agrobacterium mediated gene transfer 1. Signal induction to Agrobacterium – phenolic compounds and some sugars, induces biochemical changes that help in T-DNA transfer. 2. Attachment of Agrobacterium to plant cells – Agrobacterium attaches to cell through polysaccarides and cellulose fibres . 3. Production of virulence protein – signal induction causes virulence protein to form Vir A,which induces Vir G, this then induces the production of rest of the virulence proteins like Vir D1/D2, Vir E, Vir B.

11. 4. Production of single stranded T-DNA – this is recognised by vir D and thus carried forward to the host by Vir D2. 5. Transfer of T-DNA out of Agrobacterium – the DNA strand is carried out by Vir D2. Through a channel made by Vir B. 6. Transfer of T-DNA into plant cells and integration integration of the DNA into the host cell is helped by VirE2, which protects the DNA from degradation by host cell restriction modification system, while Vir D2 helps to navigate the DNA to the nucleus. This process is called ILLEGIMATE RECOMBINATION , since it does not depend on the sequence similarity.

12. Regeneration: for shoot organogenesis, cytokinin (lower amounts of auxin) are required. Selection: (two antibiotics are required) 1. An antibiotic to kill the agrobacterium , while not affecting the plant’s cell growth and division. 2. A second antibiotic allows growth of transformed shoots but inhibits growth of untransformed plant cells.

13. Detection of the “trait” gene: 1. PCR methods can detect the presence of the trait DNA. 2. protein detection methods are used where a gene product is produced that defines the trait. 3. verification of the corporation of the trait gene into the plant’s chromosome. By southern hybridization By demonstrating transfer of the trait to the original transformant’s progeny

14. Pro’s n Con’s Scientists can insert any gene they want into the plasmid in place of the tumor causing genes and subsequently into the plant cell genome By varying experimental materials, culture conditions, bacterial strains, etc. scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn This method of gene transfer enables large DNA strands to be transferred into the plant cell without risk of rearrangement whereas other methods like the Gene Gun have trouble doing this.

15. The vast majority of approved genetically engineered agriculture has been transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer Original problems existed in that Agrobacterium tumefaciens only affects dicotyledonous plants Monocotyledon plants are not very susceptible to the bacterial infection

16. References https://www.slideshare.net/NISHANTHSEKAR1/agrobacterium-mediated-gene-transfer-77002058 https://www.slideshare.net/Rahulselvaraj/agrobacterium-mediated-gene-transfer-140707990 All the images and pictures used in this presentation are taken from Google Inc.,(via google search) and from slideshare .