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

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 lead to changes in DNA sequence passed on to the next generation. Mutagenesis

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

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

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).

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.

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

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.

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

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 Agrobact. Opine biosynthesis - food for Agrobact. Opine catabolism Opine catabolism Acetosyringone receptors Acetosyringone receptors

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

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”

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.

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

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

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

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

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

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

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

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

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

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

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.

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.

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

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 T-DNA - heterozygous - 1 normal gene - 1 disrupted gene

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

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

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

Non-perfect, but usable, results