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Agrobact transfer.pptx

  1. Agrobacteria  soil bacteria, gram-negative, related to Rhizobia  Causative agents of “Crown gall” disease of dicoltyledones  Have ability transfer bacterial genes to plant genome  Attracted to wound site via chemotaxis in response to sugars and Phenolic molecules: acetosyringone released from damaged plant cells  Contains Ti plasmid which can transfer its T-DNA region into genome of host plants
  2. Agrobacteria • soil bacteria, gram-negative, related to Rhizobia Classification of Agrobacterium. Two basis of classification 1. Basis of pathogenicity. Agrobacterium tumefaciens- crown gall. Agrobacterium rhyzogen- hairy root Agrobacterium radiobacter- Avirulent 2. On growth pattern Biotype I Biotype II Biotype III
  3. History of Agrobacterium • Smith & Townsend (1907)- said bacteria caused crown gall disease • Brown & Stonier (1958)-proposed that not whole bacteria but some part of it causes disease • Zenen (1974)- noted virulent strain-Agrobacterium tumefaciens • Chilton (1977)-reported Ti & Ri plasmid transfer to plant causing disease
  4. History of Agrobacterium • Smith & Townsend (1907)- said bacteria caused crown gall disease • Brown & Stonier (1958)-proposed that not whole bacteria but some part of it causes disease • Zenen (1974)- noted virulent strain-Agrobacterium tumefaciens • Chilton (1977)-reported Ti & Ri plasmid transfer to plant causing disease
  5. Tumor characteristics 1. Synthesize a unique amino acid, called “opines” •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.
  6. Elucidation of the TIP (tumor-inducing principle) • 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.
  7. Ti-plasmid 1. Large : 200-kb 2. Conjugative 3. ~10% of plasmid transferred to plant cell after infection 4. Transfer DNA (called T- DNA) integrates semi- randomly into nuclear DNA 5. Contain a vir region ~ 40 kb at least 8~11 vir genes involved in mobilizing T-DNA 6. Has origin of replication 7. Has genes for the catabolism of opines
  8. Ti Plasmid (14 bp repeat) (7 bp repeat)
  9. auxA auxB cyt ocs LB 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
  10. • Two strains of Ti-plasmid: Nopaline strains- contain one T-DNA region(23 kb) Octopine strains- contains two T-DNA region: TL (14 kb) and TR ( 7 kb)
  11. 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.
  12. Ti plasmids and the bacterial chromosome act in concert to transform the plant • 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. Chv A& B major role in exopolysaccharide production psc A major role in T-DNA transport chv E glucose and galactose transport chvD, ilv, miaA and att, have virulence property. • 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. • T-DNA encodes genes for opine synthesis and for tumor production. • occ (opine catabolism) genes carried on the pTi allow the bacterium to utilize opines as nutrient.
  13. Virulence genes. • Virulence region consists of 24 genes in total • Virulence genes are located in 8 operons from vir A-vir H • vir A,F and G are monocistronic operons , where as vir B,C,D,E,H are polycistronic • virA - transports AS into bacterium, activates virG post-translationally • virG –transcriptional activator; 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, conjugational pores between plant cell and bacteria; gets T-DNA through bacterial membranes
  14. Agrobacterium tumefaciens T-DNA transfer process Steps of gene transfer in plant:- 1. Bacterial colonization 2. Induction of virulence system 3. Generation of T-DNA transfer complex 4. T-DNA transfer 5. Integration of T-DNA into plant.
  15. Generation of the T-strand overdrive Right Borde r 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’
  16. Generation of the T-strand Right bord er Left borde r 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.
  17. 1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome Right bord er Left borde r D T-strand coated with virE T-DNA virD nicks at Left Border sequence
  18. VirE2 may get DNA-protein complex across host PM Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485
  19. Overview of the Infection Process
  20.  Monocots don't produce AS in response to wounding.  Put any DNA between the LB and RB of T-DNA it will be transferred to plant cell. Engineering plants with Agrobacterium: Two problems had to be overcome:  Ti plasmids large, difficult to manipulate  couldn't regenerate plants from tumors
  21. Limitations of Agrobacterium as cloning vector Ti plasmids are large (approximately 200 to 800kb).--------remove large segments of DNA that are not essential for a cloning vector •The production of phytohormones by transformed cells growing in culture prevents them from being regenerated into mature plants---remove cytokinin and auxin genes •A gene encoding opine synthesis is not useful to a transgenic plant and may lower the final plant yield by diverting plant resources into opine production---- remove opine synthesis gene •Ti plasmid does not replicate in E.coli, the convenience of perpetuating and manipulating Ti plasmids carrying inserted DNA sequences in that bacterium is not available. •Transfer of the T-DNA,which begins from the rightborder, does not always end at the left border. Rather, vector DNA sequences past the left border are often transferred, although the transfer of these sequences is not often tested for.
  22. To overcome these constraints, Ti plasmid-based vectors are engineered which are similarly organized and contain the following components. • A selectable marker gene, such as neomycin phosphotransferase(npt), that confers kanamycin resistance on transformed plant cells. Because the npt gene, as well as many of the other marker genes used in plant transformation, is prokaryotic in origin, it is necessary to put it under the control of plant (eukaryotic) transcriptional regulation signals, including both a promoter and a termination–polyadenylation sequence, to ensure that it is efficiently expressed in transformed plant cells. •An origin of DNA replication that allows the plasmid to replicate in E. coli. In some vectors, an origin of replication that functions in A.tumefaciens has also been added. •The right border sequence of the T-DNA region(although most cloning vectors include both a right and a left border sequence) •A polylinker (multiple cloning site) to facilitate insertion of the cloned gene into the region between T-DNA border sequences. •A “killer” gene encoding a toxin downstream from the left border to prevent unwanted vector DNA past the left border from being incorporated into transgenic plants. If this incorporation occurs, and the killer gene is present, the transformed cells will not survive.
  23. Cointegrate Vector The Cloning or intermediate vector has a plant selectable marker gene, the target gene, the right border, an E. coli origin of DNA replication, and a bacterial selectable marker gene. The intermediate vector recombines with a disarmed Ti plasmid that lacks both the tumor- producing genes and the right border of the T- DNA within A. tumefaciens. The intermediate vector and the disarmed helper Ti plasmid both carry homologous DNA sequences that provide a shared site for in vivo homologous recombination; normally these sequences lie inside the T-DNA region. Following recombination, the cloning vector becomes part of the disarmed Ti plasmid, which provides the vir genes necessary for the transfer of the T-DNA to the host plant cells.
  25. Binary vector system 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.
  26. Binary vector system: • E. coli and A. tumefaciens origins of DNA replication, i.e., an E. coli–A. tumefaciens shuttle vector, or a single broad-host-range origin of DNA replication(RK2). •Selectable marker genes for plants and bacteria •T DNA border sequences •Multiple cloning site
  27. •All the cloning steps are carried out in E. coli before the vector is introduced into A. tumefaciens. The T-DNA border sequences are sub cloned on a small E. coli plasmid.This plasmid, called mini-Ti or micro-Ti, can be introduced into an Agrobacterium strain •The recipient A. tumefaciens strain carries a disarmed Ti plasmid which acts as a helper plasmid as it contains a complete set of vir genes enabling the T-DNA from the binary cloning vector to be inserted into the plant chromosomal DNA.
  29. Plant transformation based on direct DNA delivery 1.Polyethylene glycol (PEG)-mediated protoplast transformation. 2.Particle bombardment 3.Electroporation 4.Microinjection 5.Ultrasound mediated 6.Pollen-mediated 7.Cell penetrating peptides
  30. Gene gun or Particle Bombardment or Biolistic or Balistic method Used for most of Monocotyledons Usefullto introduce DNA into mostly all tissues: •Shoot apical meristems •Monocotyledons •Leaf blades •Immature and mature embryos •Root and shoot sections •Plant cell suspensioni •Callus culture •Pollen •Mitochondria and chloroplast
  31. Particle bombardment PDS-1000 First particle gun was developed by Sanford and colleagues in 1987. They introduced CAT (chloremphenicol acetyl transferase) gene into onion epidermal cells and detected transient gene activity. First transgenic plants were produced in 1988 using soybean and tobacco tissue culture. Christou (1988) and Klein (1988) bombarded soybean shoot meristem and tobacco leaf, respectively. Christou recovered chimeric transgenic soybean plants that transmitted the gene into next generation. Particle bombardment is the method of choice for chloroplast transformation.
  32. METHOD 1. Precipitate DNA onto 1- 2 um tungsten or gold particles. 2. Accelerate particles to high to velocities that allow their entry into plant cells/nuclei 3.Accelaration is achieved by devices which vary in design and function •Pressurized helium gas •Electrostatic energy released by a droplet of water exposed to high voltage
  33. Gold or tungsten particles are used gold tungsten •Tungsten particles are cheaper but are of irregular shape and size •Toxic to certain cell types and show surface oxidation leading to precipitation of DNA •Tend to form aggregates after addition of DNA which reduces paticle dispersion •Gold particles are uniform in size and shape , show low toxicity but are costlier and show variable coating of DNA
  34. • Coating of DNA to microprojectiles is by precipitation • One approach is to mix 1.25 – 1.8 mg of microparticles with 0.5 – 70 ug DNA in CaCl2 and spermidine solution • Vortexed to ensure uniform coating • After coating microprojectiles are transferred to macrocarrier membrane , allowed to dry and immediately used
  35. The Helium Gas Gun – Circa 2000
  36. DNA is bound to the microprojectiles, which impact the tissue or immobilized cells at high speeds. J. Sanford & T. Klein, 1988 Original 22-caliber biolistic gun
  37. Helium particle gun
  38. Gene Gun