agrobacterim vector
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The document summarizes how Agrobacterium tumefaciens is used for plant genetic engineering. It describes the key components of the Ti plasmid, including the T-DNA, vir genes, and opine catabolism genes. It explains that the Ti plasmid is modified by deleting tumor-causing genes and inserting a gene of interest, along with selectable markers. The modified plasmid is transferred to A. tumefaciens via triparental mating and then used to transform plant cells. Transformed cells are selected using antibiotics and regenerated into transgenic plants.
![Agrobacterium tumefaciens
• How is T-DNA modified to allow genes of
interest to be inserted?
– In vitro modification of Ti plasmid
• T-DNA tumor causing genes are deleted and replaced with
desirable genes (under proper regulatory control)
• Insertion genes are retained (vir genes)
• Selectable marker gene added to track plant cells
successfully rendered transgenic [antibiotic resistance
gene geneticin (G418) or hygromycin]
• Ti plasmid is reintroduced into A. tumefaciens
• A. tumefaciens is co-cultured with plant leaf disks under
hormone conditions favoring callus development
(undifferentiated)
• Antibacterial agents (e.g. chloramphenicol) added to kill A.
tumefaciens
• G418 or hygromycin added to kill non-transgenic plant cells
• Surviving cells = transgenic plant cells](https://image.slidesharecdn.com/10agrobacterimvector-190603174344/85/agrobacterim-vector-21-320.jpg)
agrobacterim vector
- 1. What determines the choice vector?What determines the choice vector? • Insertion size • Vector size • Restriction sites • Copy number • Cloning efficiency • Ability to screen for inserts • What downstream experiments do you want
- 2. Agrobacterium A unique bacterial species used for Plant-Fungal-Animal Transformation
- 3. Agrobacterium • Gram - • A natural genetic engineer • 2 species – A.tumefaciens (produces a gall) – A. rhizogenes (produces roots) • Dicots • Worldwide
- 4. 1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas). 2. Octopine plasmids: carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue. 3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out. Ti plasmids can be classified according toTi plasmids can be classified according to the opines producedthe opines produced
- 5. Ti plasmid of A. tumefaciens
- 6. vir genes opine catabolism pTipTi tra for transfer to the plant bacterial conjugation Agrobacterium chromosomal DNAAgrobacterium chromosomal DNA chvA chvB pscA oriV T-DNA-inserts into plant genome
- 7. 1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface. 2.Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer. 3.T-DNA encodes genes for opine synthesis and for tumor production. 4. occ (opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient. Ti plasmids and the bacterial chromosomeTi plasmids and the bacterial chromosome act in concert to transform the plantact in concert to transform the plant
- 8. Overview of the Infection Process
- 9. The infection process: 1. Wounded plant cell releases phenolics and nutrients. 2. Phenolics and nutrients cause chemotaxic response of A. tumefaciens 3. Attachment of the bacteria to the plant cell. 4. Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene transcription and allow for T-DNA transfer and integration into plant chromosomal DNA. 5. Transcription and translation of the T-DNA in the plant cell to produce opines (food) and tumors (housing) for the bacteria. 6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use opines as a C, H, O, and N source. Figure 18.2 and 18.3 Ti plasmid structure and function. Note the wound- induced plant phenolics induce the vir genes on the Ti plasmid.
- 10. TheThe virvir region is responsible for the transfer of T-region is responsible for the transfer of T- DNA to the wounded plant cell.DNA to the wounded plant cell. receptor for acetyl- syringone positive regulator for other vir genes virA constitutive virG virA is the sensor. membrane activated virG Note: activated virG causes its own promoter to have a new start point with increased activity.
- 11. virA is the sensor. bacterial membrane Acetylsyringone is produced by wounded plant cells (phenolic compound). triggers auto- phosphorylation of virA 1 2 P 3 virG virA virG activates transcription from other vir promoters. VirA phosphorylates virG which causes virG to become activated. virG is the effector. Asg Asg P
- 12. Generation of the T-strandGeneration of the T-strand overdrive Right Border Left Border T-DNA virD/virC VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end. 5’
- 13. Generation of the T-strandGeneration of the T-strand Right borderLeft border D virD/virC gap filled in T-strand T-DNA virE 1. Helicases unwind the T-strand which is then coated by the virE protein. 2. ~one T-strand produced per cell.
- 14. 1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome Right borderLeft border D T-strand coated with virE T-DNA virD nicks at Left Border sequence
- 16. VirE2 may get DNA-protein complex across host PM Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485
- 17. TheThe virvir region is responsible for the transfer of T-region is responsible for the transfer of T- DNA to the wounded plant cell.DNA to the wounded plant cell. ssDNA binding protein. Binds T- strand. virA virGvirB virC virD virE sensor effector endo- nucleas e nicks T- DNA Binds overdrive DNA. membrane protein; ATP- binding Note: The virA-virG system is related to the EnzZ-OmpR system that responds to osmolarity in other bacteria.
- 19. (a) The pilus has not contacted the surface of the recipient plant cell and the apparatus is unable to transport T-complex. (b) The pilus has contacted a receptor (?) on the surface of the recipient plant cell. This induces the VirB transporter, perhaps via a change in conformation, so that it is now competent to transfer the T-complex to the plant cell cytoplasm. OM, outer membrane; IM, inner membrane; CW, plant cell wall; PM, plasma membrane. Model for contact-dependent activation of the T-complex transport apparatus
- 20. Agrobacterium and genetic engineering: Engineering the Ti plasmid
- 21. Agrobacterium tumefaciens • How is T-DNA modified to allow genes of interest to be inserted? – In vitro modification of Ti plasmid • T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control) • Insertion genes are retained (vir genes) • Selectable marker gene added to track plant cells successfully rendered transgenic [antibiotic resistance gene geneticin (G418) or hygromycin] • Ti plasmid is reintroduced into A. tumefaciens • A. tumefaciens is co-cultured with plant leaf disks under hormone conditions favoring callus development (undifferentiated) • Antibacterial agents (e.g. chloramphenicol) added to kill A. tumefaciens • G418 or hygromycin added to kill non-transgenic plant cells • Surviving cells = transgenic plant cells
- 22. Co-integrative and binary vectors Binary vector LB RB Co-integrative
- 25. Plant genetic engineering with the binary Ti plasmid system Clone YFG (your favorite gene) or the target gene in the small T-DNA plasmid in E. coli, isolate the plasmid and use it to transform the disarmed A. tumefaciens as shown. Transgenic plant (disarmed) Disarmed Ti plasmid
- 26. MiniTi T-DNA based vector for plants 1.1. Binary vectorBinary vector: the: the virvir genesgenes required for mobilization andrequired for mobilization and transfer to the plant reside on atransfer to the plant reside on a modified pTimodified pTi.. 2. consists of the2. consists of the right and leftright and left border sequencesborder sequences, a, a selectableselectable markermarker (kanomycin resistance)(kanomycin resistance) and aand a polylinkerpolylinker for insertion offor insertion of a foreign gene.a foreign gene. Disarmed vectors: do not produce tumors; can be used to regenerate normal plants containing the foreign gene. miniTi
- 27. MiniTi T-DNA based vector for plants modified Ti plasmid a binary vector system oriVoriV virvir T-DNA deleted 2 LB RB oriori kanr polylinker miniTiminiTi bombom1 bom = basis of mobilization
- 28. Transfer of miniTi from E. coli to Agrobacterium tumefaciens Triparental mating:Triparental mating: bombom site forsite for mobilizationmobilization miniTi;miniTi; kan resistancekan resistance E. coli Agrobacteriumstr resistant pRK2013;pRK2013; kan resistancekan resistance contains tratra genes modified pTimodified pTi 15A ori;15A ori; E. coli or Agrobact.E. coli or Agrobact. ColE1 oriColE1 ori tra bom Ti oriVTi oriV
- 29. Steps in the mating 1-2: Triparental mating:Triparental mating: pRK2013;pRK2013; kan resistancekan resistance contains tratra genes tra ColE1 oriColE1 ori bom tra 1 2 E. coli Helper plasmid (pRK2013) mobilizes itself into 2nd E. coli strain containing miniTi. miniTi;miniTi; kan resistancekan resistance
- 30. Steps in the mating 2-3: E. coli miniTi;miniTi; kan resistancekan resistance Agrobacterium Helper plasmid mobilizes itself and the miniTi into Agrobacterium. 2 miniTi 3 pTi pRK2013 miniTi pRK2013 can not replicate. pRK2013
- 31. Selection of Agrobacterium containing the miniTi on strep/kan plates miniTi;miniTi; kankan resistanceresistance pRK2013;pRK2013; kankan resistanceresistance modified pTimodified pTi Agrobacterium str resistant Agrobacterium str resistantplate on str and kan media tra str r bom can not replicate pTi miniTi pRK2013 kanr str r
- 33. 33 References: 1.Www. Wikipedia.org.in 2.Gustavo A. de la Riva1 , Joel González-Cabrera , Roberto Vázquez-Padrón , Camilo Ayra-Pardo (1998)Agrobacterium tumefaciens: a natural tool for plant transformation. *. Journal of Bacteriology 3. Baron, C., Llosa, M., Zhou, S. and Zambryski, P.C.(1997). VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens is processed to a C-terminal secreted product VirB1*. Journal of Bacteriology 179:1203-1210. 4. Beaupre, C.E., Bohne, J., Dale, E.M. and Binns, A.N. (1997). Interactions between VirB9 and VirB10 proteins involved in the movement of DNA from Agrobacterium tumefaciens into plant cells. Journal of Bacteriology 179:78-89. 5. Binns, A.N. and Thomashow, M.F. (1988). Cell biology of Agrobacterium infection and transformation of plants. Annual Review of Microbiology 42: 575-606.
Editor's Notes
- #17: VirE1 chaperones VirE2 in Agro. Cytoplasm, but complex can also bind the SS T-DNA. VirE2 may help the T-DNA cross the plant cell PM, as it can form a channel in artificial bilayers by itself.
- #23: Co-integrative vectors require the genes that are transferred from bacteria to go into the plasmid DNA by homologous recombination. For binary vectors, the plasmid containing the t-DNA is able to replicate in E.coli and can be mobilised into Agrobacterium by a triparental mating with a helper strain of E.coli. This greatly simplifies the process of plasmid construction.