Making Transgenic Plants and Animals

1. Making Transgenic Plants and Animals Why? Study gene function and regulation Making new organismic tools for other fields of research Curing genetic diseases Improving agriculture and related raw materials New sources of bioengineered drugs (use plants instead of animals or bacteria) Metabolic engineering (2,4,5); marked (GFP) plants for population studies (2), increased food production or pest resistance (4)Metabolic engineering (2,4,5); marked (GFP) plants for population studies (2), increased food production or pest resistance (4)

3. Agrobacterium - mediated Gene Transfer Most common method of engineering dicots, but also used for monocots Pioneered by J. Schell (Max-Planck Inst., Cologne) Agrobacteria soil bacteria, gram-negative, related to Rhizobia species: tumefaciens- causes crown galls on many dicots rubi- causes small galls on a few dicots rhizogenes- hairy root disease radiobacter- avirulent J. Schell in race with Mary-Dell Chilton (CIBA-GEIGY)J. Schell in race with Mary-Dell Chilton (CIBA-GEIGY)

6. Note: pili used for attachment to plant cells, and conjugation to other bacteria.Note: pili used for attachment to plant cells, and conjugation to other bacteria.

7. Infection and tumorigenesis 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

8. Tumor characteristics Synthesize a unique amino acid, called “opine” octopine and nopaline - derived from arginine agropine - derived from glutamate Opine depends on the strain of A. tumefaciens Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce. Has obvious advantages for the bacteria, what about the plant?

9. 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.

10. Ti Plasmid Large (?200-kb) Conjugative ~10% of plasmid transferred to plant cell after infection Transferred DNA (called T-DNA) integrates semi-randomly into nuclear DNA Ti plasmid also encodes: enzymes involved in opine metabolism proteins involved in mobilizing T-DNA (Vir genes)

13. Vir (virulent) genes On the Ti plasmid Transfer the T-DNA to plant cell Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes. virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

14. Vir gene functions (cont.) 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

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.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.

21. Leaf-disc transformation - after selection and regeneration with tissue culture, get plants with the introduced gene in every cell 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)

24. Chemically-Induced Transformation Usually use on cells without walls Multiple protocols (examples): Put DNA inside artificial membranes (liposomes), they will fuse with plasma membrane. Bind DNA with polycations to neutralize charge, some cells endocytose the complex. Combine (1) and (2)

32. Transgenic Plants In Use or About to be on a Large Scale Herbicide-resistant plants Pest-resistant plants Vaccine plants (just starting to be used)

33. Herbicide-resistant plants Resistant to herbicide “Round-up” (Glyphosate) Contain bacterial EPSP synthase Advantages: better weed control, less tillage soybeans, corn, rice, wheat

35. Pest-resistant plants Resistant to certain insects Lepidopterans, Coleopterans Carry gene(s) for Bacillus thuringiensis (Bt) toxin Toxin proteins produced as a parasporal crystal Complex, composed of several proteins Cry and Cyt genes encoded on a plasmid Advantage: less insecticide required, better yield corn, cotton, potatoes

38. Concerns that have been raised about cultivating and consuming GM crops They may be toxic or allergenic. They may become established in the wild and outcompete other plants. They may negatively affect insects or other organisms that use crops. They may outcross to a nearby wild relative spreading the transgene into a wild population.

39. References on release of GM crops into the environment Nap et al. (2003) Plant Journal 33, 1-18 Focuses on current status and regulations Conner et al. (2003) Plant Journal 33, 19-46 Focuses on ecological risk assessment