Making Transgenic Plants and AnimalsGenetic Engineering of PlantsAgrobacterium - mediated Gene TransferSlide 4Agrobacterium tumefaciensSlide 6Infection and tumorigenesisTumor characteristicsElucidation of the TIP (tumor-inducing principle)Ti PlasmidSlide 11Slide 12Vir (virulent) genesVir gene functions (cont.)Slide 15Slide 16Slide 17Slide 18Binary vector systemSlide 20Slide 21Slide 22Making Transgenic Plants and Animals •Why?1. Study gene function and regulation2. Making new organismic tools for other fields of research3. Curing genetic diseases 4. Improving agriculture and related raw materials 5. New sources of bioengineered drugs (use plants instead of animals or bacteria)Genetic Engineering of Plants•Must get DNA:1. into the cells2. 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 transfer2. 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.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 dicotsrubi- causes small galls on a few dicotsrhizogenes- hairy root disease radiobacter- avirulentCrown galls caused by A. tumefaciens on nightshade. More about Galls: http://waynesword.palomar.edu/pljuly99.htmhttp://kaweahoaks.com/html/galls_ofthe_voaks.htmlAgrobacterium tumefaciens• the species of choice for engineering dicot plants; monocots are generally resistant (but you can get around this)•some dicots more resistant than others (a genetic basis for this)•complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genesAgrobacterium tumefaciensInfection 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 cellsTumor characteristics1. Synthesize a unique amino acid, called “opine”–octopine and nopaline - derived from arginine–agropine - derived from glutamate2. Opine depends on the strain of A. tumefaciens3. 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?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.Ti Plasmid1. Large (200-kb)2. Conjugative3. ~10% of plasmid transferred to plant cell after infection4. Transferred DNA (called T-DNA) integrates semi-randomly into nuclear DNA 5. Ti plasmid also encodes:–enzymes involved in opine metabolism–proteins involved in mobilizing T-DNA (Vir genes)auxA auxB cyt ocsLB RBLB, RB – left and right borders (direct repeat)auxA + auxB – enzymes that produce auxincyt – enzyme that produces cytokininOcs – octopine synthase, produces octopine T-DNA These genes have typical eukaryotic expression signals!auxA auxBTryptophan indoleacetamide  indoleacetic acid (auxin) cytAMP + isopentenylpyrophosphate  isopentyl-AMP (a cytokinin)• Increased levels of these hormones stimulate cell division.• Explains uncontrolled growth of tumor.Vir (virulent) genes1. On the Ti plasmid2. Transfer the T-DNA to plant cell3. 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 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 membranesFrom Covey & GriersonGauthier, A. et al. (2003) J. Biol. Chem. 278:25273-25276Type 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 filamentsVirE2 may get DNA-protein complex across host PMDumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485• Monocots don't produce AS in response to wounding.• Important: 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:(1) Ti plasmids large, difficult to manipulate(2) couldn't regenerate plants from tumorsBinary vector systemStrategy: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 system1. 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 ProtocolsMaking 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,