2. T-DNA Mutagenesis and Plant Genetic Engineering Purpose: Determine gene function to produce better plants for society

3. Mutagenesis: Chemical or physical treatment that changes the nucleotide sequence of DNA. The altered DNA sequence may be passed on to the next generation. Mutant: An organism that differs from the “normal” or wild type by one or more changes in its DNA sequence. Mutagenesis

4. - Single nucleotide change G --> A Mutagenesis- creating mutants ATTAGGCTACCGT TAATCCGATGGCA ATTAGACTACCGT TAATCTGATGGCA -Or delete or add a nucleotide Normal: Wild type Mutant Mutagenesis

5. - Delete a segment of DNA - many nucleotides Mutagenesis- larger mutations Insert a segment of DNA = “Insertional” XX

6. Insertion tagging Principle: A DNA fragment (with a known sequence) is allowed to insert into the genome (it usually causes a recessive, loss of function mutation). Principle: A DNA fragment (with a known sequence) is allowed to insert into the genome (it usually causes a recessive, loss of function mutation). Similar to ligating an insert into LacZ-alpha Similar to ligating an insert into LacZ-alpha

7. Insertion tagging Advantages: Advantages: – tags or marks the gene. – Provides a powerful way to identify or fish the gene out. Disadvantages: Disadvantages: – Cannot knock out essential genes. – Other redundant genes mask loss of disrupted gene. – May disrupt non-functional sub-region of gene.

8. Is it useful? Highly and broadly useful Highly and broadly useful Applied to most organisms. Applied to most organisms. Mice, bacteria, yeast and plants have had their genes inactivated by DNA insertions Mice, bacteria, yeast and plants have had their genes inactivated by DNA insertions -> knockouts. -> knockouts.

9. T-DNA Mutagenesis: A method of disrupting genes in plants with a “T-DNA” to “knock-out” gene function and activity. T-DNA = Transfer DNA a segment of DNA derived from the Ti plasmid contained inside the bacterium, Agrobacterium tumefaciens. “Agro” = plant pathogen Transferred from the bacterium to the plant. Randomly integrated into chromosomal sites in the nuclei. A type of insertion mutagenesis

10. Agrobacterium tumefaciens - and Ti Plasmid  Soil Bacterium infects plants through wounds & openings  Causes crown gall tumors….  Expresses genes on a Ti plasmid - Tumor inducing Plasmid

11. Ti Plasmid Contains genes for: Contains genes for: Plant growth hormones Plant growth hormones - cytokinins and auxins. - cytokinins and auxins. - stimulate undifferentiated growth - stimulate undifferentiated growth Opine biosynthesis - food for Agro. Opine biosynthesis - food for Agro. Opine catabolism - convert opines into E Opine catabolism - convert opines into E Acetosyringone receptors Acetosyringone receptors

12. Plant wound produces acetosyringone Bacteria is attracted to wound - receptor tells bacteria to swim to wound = Bacterial T-plasmid encodes receptors for acetosyringone Bacterial cell

13. T-DNA is excised from Ti plasmid and integrates into plant genome. Genes on T-DNA are activated and stimulate cell proliferation. Opine genes produce bacterial nutrients “Opines”

14. Tumor- producing genes Virulence region Opine catabolism ORI T-DNA region IDEA: Ti- Plasmid, Tumor producing genes can be Replaced with other genes. New genes will be transferred! Left & right borders must be retained.

15. Tumor- producing genes Virulence region Opine catabolism ORI T-DNA region Ti- Plasmid - delete genes for tumor and Agro nutrients X X X X

16. Virulence region Opine catabolism ORI T-DNA region Ti- Plasmid - delete genes for tumor and Agro nutrients New Gene

17. New foreign genes can be carried as passengers when the T-DNA integrates into plant genome. No tumors formed when auxin and cytokinin genes are replaced - plant has taken up T-DNA but no disease! = Disarmed Ti Plasmid

18. What kind of genes can be added to T-DNA? - Any gene - Selectable marker - Selectable marker Kanamycin Resistance Hygromycin R “ Hygromycin R “ - reporter gene, marks cells - reporter gene, marks cells to show they are transformed. to show they are transformed. Not always used. Not always used. - genes for crop improvement, - genes for crop improvement, disease & insect resistance, new proteins, Vitamins, many possibilities

19. Left border Right border HygR GFP Plants will be hygromycin resistant and express green fluorescent protein. Modified T-DNA for GFP Expression

20. Green fluorescent protein (GFP) From luminescent jellyfish Aequorea victoria. Produces green fluorescence under blue and UV light

21. Root Root Hair cotyledon Light Dark Redistribution of GFP-2SC in the Light

22. GFP-2SC moves from vacuole to ER and golgi, from Dark to Light Protoplasts: plants with cell walls removed.

23. Left border Right border KanR Plants will be Kanamycin resistant. Might disrupt a gene or spacer DNA. Modified T-DNA for Mutagenesis

24. Transformation with Disarmed Ti-plasmid in Agrobacterium - Mix Agro containing Ti-plasmid with: - Wounded leaf - Wounded leaf - Plant cells in culture - Plant cells in culture - Floral dip under vacuum - Floral dip under vacuum -plant cells or seeds on growth media containing selection antibiotic (i.e. Kan). -Only engineered plants grow

25. Genome-wide insertional mutagenesis of Arabidopsis thaliana (2003) Objective: create loss of function mutations for all genes.Objective: create loss of function mutations for all genes. Strategy: use T-DNA (with kanamycin-resistance gene as selectable marker) to generate collection of 150,000 T1 transformants.Strategy: use T-DNA (with kanamycin-resistance gene as selectable marker) to generate collection of 150,000 T1 transformants. > 225,000 independent T-DNA integration events thus far.> 225,000 independent T-DNA integration events thus far.

26. Arabidopsis Genome size = 125,000 kb; Avg gene length = 2 kbGenome size = 125,000 kb; Avg gene length = 2 kb Random distribution of insertion events, predicts 96.6% probability of finding an insertion in a gene,Random distribution of insertion events, predicts 96.6% probability of finding an insertion in a gene, To determine the site of integration of each T-DNA, junction sequences were analyzed and 88,122 sites were proven to be at a single genomic locationTo determine the site of integration of each T-DNA, junction sequences were analyzed and 88,122 sites were proven to be at a single genomic location Of the 29,454 annotated genes, 21,799 (74%) were hit,Of the 29,454 annotated genes, 21,799 (74%) were hit, Create catalog and allow researchers to order seeds for their favorite gene disruption on-line.Create catalog and allow researchers to order seeds for their favorite gene disruption on-line.

27. 2000 bp 125332 142332 CNGC10 Not all genes can be knocked out. T-DNA

28. Distribution of T-DNAs showed hot spots (in gene-rich regions) and cold spots (in centromere and Peri-centromeric regions) T1 generation - first generation after T-DNA insertion Single T-DNA insertion Single T-DNA insertion T-DNA - heterozygous - 1 normal gene - 1 normal gene - 1 disrupted gene - 1 disrupted gene

29. Obtaining Homozygous - 2 T-DNAs in same gene Heterozygous is self-pollinated N T N TN T NN NT TN TT 25% homozygous TT

30. Need homozygous - both copies knocked out T-DNA - Homozygous Screen for homozygotes by PCR using combinations of primers to the T-DNA combinations of primers to the T-DNA and to the target gene to be knocked out

31. Want to know precise location of the T-DNA T-DNA - Homozygous Where is it exactly within a gene or near a gene?

32. Normal gene T-DNA How can PCR be used to verify copy # and location of the T-DNA?

33. Gene 3’ Gene 5’ PCR screen T-DNA mapping No PCR product with this primer with this primer T-DNA Normal gene

34. Non-perfect, but usable, results