Lec 3b T-DNA+supplement-b

Lec 3b T-DNA+supplement-b - Agrobacterium tumefaciens a...

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Unformatted text preview: Agrobacterium tumefaciens a natural example of genetic engineering. Crown gall tumors Jeff Schell The bacterial Ti plasmid causes crown gall disease in plants Agrobacterium tumefaciens is a soil bacteria that contains a plasmid that induces tumors in most dicotyledonous plants. The tumor inducing plasmid (pTi) contains a portion that is transferred to the plant cell (T-DNA) that contains genes that : 1.) function in the plant to produce enzymes that synthesize unique products called opines (conjugates of basic amino acids and -ketoglutarate or pyruvate); 2.) cause the plant to synthesize hormones resulting in tumors. Agrobacterium/plant interactions Acetosyringone is produced by wounded plant cells (phenolic compound). Agrobacterium in tumor at wound site transfers T-DNA to plant cell. opines Agrobacterium infection succeeds only on wounded plants. Agrobacterium in soil use opines as nutrients. Components of the Ti Plasmid 1- T-DNA: 23 kb region that is transferred to the plant (only the T-strand actually transferred) a-opine synthesis b-plant hormone synthesis 2-Virulence region (Vir): encodes all of the functions required for the transfer of the T-strand to the plant. T-DNA 23 kb vir genes Encodes genes necessary for transfer to the plant pTi ~200 kb oriV Components of the Ti Plasmid 3-Opine catabolism: allows the bacterium to use octopine and nopaline as a nutrient (most other soil bacteria lack these genes) 4-Bacterial conjugation: The Tra region is similar to Tra in other bacteria. This is for transfer of the entire pTi between different Agrobacterium. T-DNA 23 kb tra vir genes Encodes genes necessary for transfer to the plant bacterial conjugation pTi ~200 kb oriV opine catabolism For opine metabolism by the bacteria Ti plasmids can be classified according to the opines produced 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. Ti plasmids can be classified according to the opines produced 3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out. 4. Ri plasmids: induce hairy root disease on some plants and crown gall on others; have agropine-type genes and may have segments from both nopaline and octopine plasmids Ti plasmids and the bacterial chromosome act in concert to transform the plant 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. ChvE enhances sensitivity to asg. 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. oc (opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient. Agrobacterium chromosomal DNA chvE chvA pscA chvB T-DNA vir genes transfer to the plant oriV pTi tra bacterial conjugation opine catabolism inc pTi's are in the same inc group. The vir region is responsible for the transfer of T-DNA to the plant wounded plant cell. virA is the sensor. activated virG membrane constitutive virA receptor for acetosyringone virG -constitutive/inducible Note: activated virG positive causes its own regulator promoter to have a for other new start point with vir genes increased activity. Signal Sensing signal molecules = plant cell membrane Linker region periplasmic domain 1 ChvE TM1 TM2 His P Asp receiver domain response regulator DB D acetylsyringone arabinose receptor kinase (VirA) His kinase (transmitter domain) Asp inhibitory domain 2 Asp Signal Transduction activated response regulator (VirG) VirA autophosphorylates at a histidine residue and then activates the response regulator by transferring the phosphate to an aspartate residue in the receiver domain of VirG. receiver domain DNA binding domain Bacterial TwoComponent System Vir box promoters The vir region is responsible for the transfer of T-DNA to the plant wounded plant cell. 1 Asg virA is the sensor. virA bacterial membrane Asg P 2 Acetosyringone is produced by wounded plant cells (phenolic compound). triggers autophosphorylation of VirA VirA phosphorylates virG which causes virG to become activated. 3 P virG virG is the effector. virG activates transcription from other vir promoters. VirA VirA AS Autophosphorylation Autophosphorylation triggered by binding of triggered by binding of acetosyringone acetosyringone Helix C The vir region is responsible for the transfer of T-DNA to the wounded plant cell. sensor effector virA virB virG virC virD endonuclease nicks TDNA (D1&D2) virE ssDNA binding protein. Binds Tstrand. membrane Binds protein; overdrive ATP-binding DNA. Note: The virA-virG system is related to the EnzZ-OmpR system that responds to osmolarity in other bacteria. Generation of the T-strand Left Border (25 bp) T-DNA Right Border (25 bp) OVERDRIVE= enhancer of the transfer process 5' virD/virC VirD nicks the lower strand (Tstrand) at the right border sequence and binds to the 5' end. Generation of the T-strand Left border T-DNA Right border virE Synthesis of the new strand displaces the old strand T-strand D1/D2 virD1/virD2 1. Helicases unwind the Tstrand which is then coated by the virE protein. 2. ~one T-strand produced per cell. Generation of the T-strand Left border D T-DNA Right border T-strand coated with virE2 protein (T complex) virD nicks at left border sequence 1. Transfer to plant cell (VirE2 and VirD have NLS) 2. Second strand synthesis 3. Integration into plant chromosome; well defined right junction retains 1-2 bp of the right repeat; left junction is variable T-strand integration 1. T-DNA transfer resembles bacterial conjugation; Can you list some similarities? 2. T-DNA integration process is not known -Probably through nonhomologous recombination -Target sites AT-rich -T-DNA expressed at its site of integration (genes that control transformed state of the plant & genes for the opine synthesis) 3. The ability of Agrobacterium to transfer T-DNA to the plant genome makes it an ideal vector for the introduction of new genes into plants Which is the comparison between VirD1/D2 and proteins encoded by the Tra region? 1. 2. 3. 4. TraD and TraN TraJ and TraM TraI and TraY TraA and finP MiniTi T-DNA based vector for plants Disarmed vectors: do not produce tumors; can be used to regenerate normal plants containing the foreign gene. 1. Binary vector: the vir genes required for mobilization and transfer to the plant reside on a modified pTi. 2. consists of the right and left border sequences, a selectable marker (kanomycin resistance) and a polylinker for insertion of a foreign gene. miniTi MiniTi T-DNA based vector for plants a binary vector system T-DNA deleted kan LB r polylinker modified Ti plasmid RB bom vir 1 ori miniTi bom = basis of mobilization oriV 2 3 weeks after inoculation using T-DNA vector sunflower seedlings Harvest time Transformation of Arabidopsis plants Floral Dip Detergent added to allow bacteria to infiltrate the floral meristem. Dip floral buds in 1 ml of Agrobacterium culture for 5 to 15 min. Transformation of Arabidopsis plants 700 to 900 seeds per plant. Germinate on kanamycin plates to select transformants. 10 to 20 transformed plants per plant. 10 day old seedlings End of Agrobacterium Supplement ...
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