Agrobacterium mediated gene transfer in plants
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1) Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in dicot plants by transferring oncogenic DNA (T-DNA) from its Ti plasmid into the plant genome. 2) The T-DNA contains genes that produce hormones causing uncontrolled cell division and the formation of tumors. 3) By removing the oncogenic genes and inserting foreign DNA between the border repeats, the Ti plasmid can be used as a vector to stably introduce foreign genes into plant genomes through the natural plant-Agrobacterium interaction and T-DNA transfer process.
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Agrobacterium mediated gene transfer in plants
- 1. Agrobacterium Mediated Gene Transfer In Plants AAMIR RAINA M Sc, Ph. D (Genetics and Plant Breeding)
- 2. Gene Transfer Recombinant DNA technology is based on the insertion of a DNA fragment (Gene of interest) into a suitable cloning vector and then its introduction into a suitable host to propagate the recombinant DNA.
- 3. Gene Carrier Vehicle If a gene is to be introduced into a host cell,a carrier molecule that can transport the gene into the host cell is required. Such a molecule is called a cloning vehicle or carrier molecule or a vector.
- 4. Gene Carrier Vehicle Following are few gene carrier vehicles. Plasmids Bacteriophages Cosmids Viruses Virus vector for animal cells. Simian Virus
- 5. Making Transgenic Plants—Why? 1. Study gene function and regulation 2. Making new organismic tools for other fields of research 3. Curing genetic diseases 4. Improving agriculture and related raw materials 5. New sources of bioengineered drugs.
- 6. Must get DNA: 1. into the cells 2. integrated into the genome (unless using transient expression assays) 3. expressed (everywhere or controlled) For (1) and (2), two main approaches for plants: 1. Agrobacterium - mediated gene transfer 2. Direct gene transfer For (3), use promoter that will direct expression when and where wanted – may also require other modifications such as removing or replacing introns. Genetic Engineering of Plants
- 7. Agrobacteria soil bacteria, gram-negative, related to Rhizobia species: It invades many dicot plants when they are injured at the soil level and causes crown gall disease Agrobacterium tumefaciens-Nature’s smallest genetic engineer
- 8. The ability to cause crown gall disease is associated with the presence of Ti Plasmid within bacterial cell A remarkable feature of Ti Plasmid is that after infection part of molecule is integrated into plant chromosomal DNA. This segment called T- DNA is between 15 and 30 kb in size It is maintained in a stable form in plant cellc and is passed onto daughter cells as an integral part of chromosome Agrobacterium tumefaciens-Nature’s smallest genetic engineer
- 9. Crown galls caused by A. tumefaciens on nightshade. More about Galls: http://waynesword.palomar.edu/pljuly99.htm http://kaweahoaks.com/html/galls_ofthe_voaks. html
- 10. the species of choice for engineering dicot plants; monocots are generally resistant some dicots more resistant than others (a genetic basis for this) complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genes Agrobacterium tumefaciens
- 12. Infection occurs at wound sites 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 Infection and tumorigenesis
- 13. 1. Synthesize a unique amino acid, called “opine” octopine and nopaline - derived from arginine agropine - derived from glutamate 2. Opine depends on the strain of A. tumefaciens 3. Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce. 4. Has obvious advantages for the bacteria, what about the plant? Tumor characteristics
- 14. It was recognized early that virulent strains could be cured of virulence, and that cured strains could regain virulence when exposed to virulent strains; suggested an extra- chromosomal element. Large plasmids were found in A. tumefaciens and their presence correlated with virulence: called tumor-inducing or Ti plasmids. Elucidation of the TIP (tumor-inducing principle)
- 15. 1. An extrachromosomal double stranded circular DNA molecule 2. Tumour inducing 3. Large (200-kb in size) and conjugative type 4. ~10% of plasmid transferred to plant cell after infection 5. Transferred DNA (called T-DNA) integrates semi-randomly into nuclear DNA 6. Ti plasmid also encodes: enzymes involved in opine metabolism proteins involved in mobilizing T-DNA (Vir genes) Ti Plasmid
- 16. auxA auxB cyt ocsLB RB LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin Ocs – octopine synthase, produces octopine T-DNA These genes have typical eukaryotic expression signals!
- 17. auxA auxB Tryptophan indoleacetamide indoleacetic acid (auxin) cyt AMP + isopentenylpyrophosphate isopentyl-AMP (a cytokinin) • Increased levels of these hormones stimulate cell division. • Explains uncontrolled growth of tumor.
- 18. 1. On the Ti plasmid 2. Transfer the T-DNA to plant cell 3. Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes. 4. virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid. Vir (virulent) genes
- 19. virA - transports AS into bacterium, activates virG post-translationally (by phosphoryl.) virG - promotes transcription of other vir genes virD2 - endonuclease/integrase that cuts T- DNA at the borders but only on one strand; attaches to the 5' end of the SS virE2 - binds SS of T-DNA & can form channels in artificial membranes virE1 - chaperone for virE2 virD2 & virE2 also have NLSs, gets T-DNA to the nucleus of plant cell virB - operon of 11 proteins, gets T-DNA through bacterial membranes Vir gene functions (cont.)
- 20. From Covey & Grierson
- 21. Gauthier, A. et al. (2003) J. Biol. Chem. 278:25273-25276 Type IV Secretion Sys. • many pathogens, also used in conjugation • promiscuous • forms T-Pilus • B7-B10 span OM & IM • B7-B9 in OM interacts w/B8 & B10 of IM to form channel • 3 ATPases • D4 promotes specific transport • B2 can form filaments
- 22. VirE2 may get DNA-protein complex across host PM Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485
- 23. T-DNA is excised from the TI Plasmid and transferred to the nucleus of the plant cell. Here the T-DNA gets integrated into the plant DNA . The T-DNA can be passed onto daughter cells as an integral part of plant chromosome Transfer of tumour inducing Principle
- 24. Only small segment of Ti Plasmid is transferred to the host plant cell and gets integrated with the genome It contains genes for tumour formation(Tum) and nopalina biosynthesis(NOS) Tum genes encode enzymes that catalyse the synthesis of phytoharmones like IAA and the Cytokinin, Isopentenyl adenosine that causes tumourous growth of cells in crown galls The T-DNA is bordered by 25 bp repeats, required for the excision and transfer of T-DNA The T-DNA
- 25. Recognition of susceptible wounded plant cell - Plant exudates act as signals by inducing genes in the Vir genes of the Ti Plasmid - Acetosyringone (AS) ,alpha – hydroxy acetosyringone - -Binding to wounded cells, controlled by two chromosomal genes of agrobacterium-chv-A and chv-B Tumour induction by Agrobacterium
- 26. Excision, transfer and integration - The border repeats of the T-DNA play an important role - Any DNA sequence located between the border repeats is transferred to the post plant - The T-DNA region is excised from the plasmid by the enzymes encoded by the Vir region These enzymes specifically recognize the T-DNA borders The T-DNA enters the plant cell and integrates into the host genome , mediated by host enzymes Tumour induction by Agrobacterium
- 27. The Ti plasmid has an innate ability to transmit bacterial DNA into plant cell. This potential is exploited by the genetic engineers to use this as a vector The gene of a donnor organism can be introduced into the Ti Plasmid at the TDNA region This plassmid now becomes a recombinant plasmid By Agrobacterium infection , the donor genes can be transferred and integrated into the genotype of host This results in the production of a transgenic plant Ti plasmid as a vector
- 28. A mature plant regenerated from transformed cells will contain the cloned gene in every cell and will pass on the cloned gene onto its offsprings However regeneration of transformed plant can occure only if the Ti plasmid has been disarmed so that transformed cells donot display cancerious properties. Ti plasmid as a vector
- 29. Disarming is possible because cancer genes are not needed for infection Infectivity is controlled by virulence region of Ti plasmid. Infactonly parts ofTDNA are involved in infection are two 25bp repeat sequance Disarmed Ti plasmid
- 30. Any DNA placed between these two repeat sequences will be treated as T- DNA and transferred to plant It is therefore possible to remove all the cancer genes from normal T-DNA and replace them with an entirely new set of genes with out disturbing the infection process. Disarmed Ti plasmid
- 31. Removal of genes responsible of auxin cytokinin and nopaline synthesis. Disarmed Ti plasmids Deletion of T-DNA REGION. PGV3850 is constructed from pTiC58 It has pBR322 with AmpR Has Border repeats and NOS genes Disarmed Ti plasmid
- 32. Agrobacterium having this PGV 3850 can transfer the modified T-DNA into plant cells. But the recipient cells will not produce tumour, but could produce nopaline. This can be used as an efficient vector for introducing foreign genes into plants. Disarmed Ti plasmid
- 33. The main problem is of course that a unique restriction site is an impossibility with a plasmid 200Kb in size. Novel strategies have to be developed for inserting new DNA into plasmid. LIMITATIONS
- 34. Strategy: 1. Move T-DNA onto a separate, small plasmid. 2. Remove aux and cyt genes. 3. Insert selectable marker (kanamycin resistance) gene in T-DNA. 4. Vir genes are retained on a separate plasmid. 5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids. 7. Infect plant with the transformed bacteria. Binary vector system
- 36. A foreign gene cloned into an appropriate plasmid (pBR322) can be integrated with the disarmed Ti plasmid by a homologous recombination A compound plasmid called cointegrate is formed. Construction of Cointegrate
- 37. 1. Leaf-disc transformation - after selection and regeneration with tissue culture, get plants with the introduced gene in every cell 2. Floral Dip – does not require tissue culture. Reproductive tissue is transformed and the resulting seeds are screened for drug- resistant growth. (Clough and Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743) 2 Common Transformation Protocols
- 38. Making a transgenic plant by leaf disc transformation with Agrobacterium. S.J. Clough, A.F. Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743.
- 39. Employing method, genes from entirely unrelated plants can be transferred to other dicots transgenic plants with the following attributes were developed. Resistance – To antibiotics Pesticides and insect pests Drought and herbicides Uses
- 40. Thank You