T-DNA Mutagenesis

1. T-DNA Mutagenesis Purpose: Determine gene function to produce better plants for society

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

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

4. X X Mutagenesis- larger mutations • Delete a segment of DNA - delete many nucleotides Insert a segment of DNA = “Insertional”

5. Insertion tagging • Principle: A DNA fragment (with a known sequence) is allowed to insert into the genome (when it lands in a gene, it usually causes a recessive, loss of function mutation).

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

7. Is it useful? • Highly and broadly useful • Applied to most organisms. • Mice, bacteria, yeast and plants have had their genes inactivated by knockouts.

8. 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.

9. Agrobacterium tumefaciens - and Ti Plasmid Soil Bacterium infects plants through wounds & openings And causes crown gall tumors…. by expressing genes on a Ti plasmid - Tumor Inducing Plasmid

10. Ti Plasmid • Contains genes for: Plant growth hormones - cytokinins and auxins. - stimulate undifferentiated growth • Opine biosynthesis - food for Agrobact. • Opine catabolism • Acetosyringone receptors

11. Plant wound produces acetosyringone Bacterial T-plasmid produces receptors for acetosyringone Bacteria is attracted to wound - receptor tells bacteria to swim to wound

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

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

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

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

16. 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

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

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

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

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

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

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

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

24. Genome-wide insertional mutagenesis of Arabidopsis thaliana (2003) • 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. • > 225,000 independent T-DNA integration events thus far.

25. Arabidopsis • Genome size = 125,000 kb; Average gene length = 2 kb • Random distribution of insertion events, predicts 96.6% probability of finding an insertion in an average 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 location • Of the 29,454 annotated genes, 21,799 (74%) were hit. • Create a catalog and allow researchers to order seeds for their favorite gene disruption on-line.

26. 125332 142332 CNGC10 2000 bp Not all genes can be knocked out. T-DNA

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

28. N T N T N T Obtaining Homozygous - 2 T-DNAs in same gene Heterozygous is self-pollinated NN NT TN TT

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

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

32. Non-perfect, but usable, results