Gene delivery techniques

Genetic Engineering of Plants Must get DNA: into the cells integrated into the genome Expressed For (1) and (2), two main approaches for plants: Direct gene transfer Agrobacterium - mediated gene transfer For (3), use promoter that will direct expression when and where wanted.

Commonly used gene delivery techniques Microinjection Electroporation Microprojectiles Agrobacterium tumifaciens mediated transfer

Microinjection Mechanical method of gene insertion Very fine micropipette used to inject DNA molecule directly into the nucleus Most commonly used for animal embryos Not very successful in case of plants

Microinjection Source:www.translatingscience.pbwork.com

Electroporation Electric current is provided to plant cells Tiny holes are created DNA molecules present in the liquid medium Diffusion into the cells Cells allowed to recover Regeneration into whole plant

Microprojectile/Gene Gun Most commonly used for monocots Tungsten or gold particles coated with DNA Shot into the cells http://passel.unl.edu/pages/animation.php?a=GeneGun.swf

Agrobacterium tumefaciens Mainly for engineering dicot plants; monocots generally Some dicots more resistant than others (a genetic basis for this) Complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genes 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 Agrobacterium tumefaciens: The Gram-negative soil bacterium as pathogen results in crown gall tumors in plants. T-DNA, a part of the bacterial tumor-inducing (Ti) plasmid, is transferred from the bacterium to the plant cell with the aid of a number of Virulence (Vir) proteins encoded by the Ti-plasmid. Agrobacterium is the only known prokaryote to transfer genes to a eukaryote. Agrobacterium is used throughout microbiology to transfer DNA into plant cells

A. tumefaciens Ti (Tumor inducing) Plasmid A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. Most commonly found as small circular, double-stranded DNA molecules in bacteria

Structure of Ti Plasmid

Ti Plasmid containing T-DNA region

T-DNA auxA auxB cyt ocs LB RB LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin Ocs – octopine synthase, produces octopine

Agrobacterium mechanism of plant infection The transferred genes form the T-DNA (transferred DNA) region of the Ti plasmid (tumour inducing). T-DNA is less than 10% of the whole plasmid, encodes only 3 or so genes and enters a plant that has at least 25000 genes Tumor formation is the result of the transfer, integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA (transferred DNA) Agrobacteria usually infects plants from their wounds, which occurred quite frequent after frost. In practice, protection from subfreezing winter temperatures and control of chewing insects and nematodes can prevent infection by agrobacteria

Effect of T-DNA genes on plant cells Changes the plant’s metabolism to produce food materials (opines) that only the bacterium can use Produces the plant hormones auxin and cytokinin that remove the controls that normally limit cell division and cell expansion. Result - cells showing altered metabolism multiply uncontrollably

RESULT Even if the T-DNA originally change only one cell in the plant, because that cell divides uncontrollably and passes on those bacterial genes to all the new cells, there are soon millions of cells feeding the bacterium at the expense of the plant.

Infection of a plant with A Infection of a plant with A. tumefaciens and formation of a crown gall tumor. Agrobacterium tumefaciens and A. rhizogenes infect wounded plants and transfer plasmid DNA (T-DNA) and virulence (Vir) proteins into plant cells. The Plasmid shown here in the plant cell codes for T-DNA and Virulence proteins, this T-DNA is then transferred to the host with the aid of the virulence proteins. You can see a cellular view in which a portion of the plasmid DNA and the Vir proteins are transferred here This dna then encodes for oncogenes and enzymes that aid in the synthesis of opines, which is an amino acid and sugar complex that the bacteria can metabolize and use as an energy source. Thus the bacterium has the plant make food for it.

EM of A. tumefaciens attached to plant cell

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.

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 get T-DNA to the nucleus of plant cell virB - operon of 11 proteins, gets T-DNA through bacterial membranes

A. tumifaciens mediated gene transfer Removal & Replacement of T-DNA region

Engineered A. Tumefaciens Ti Plasmid vector Use engineered instead of wild-type A. tumefasciens Ti plasmid Still possesses virulence genes (allow transfer of T-DNA to target cell) but lacks opine and PGR synthesis genes

Common Transformation Protocols 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)

T-DNA transfer mechanism ANIMATION http://highered.mheducation.com/sites/dl/free/0072835125/126997/animation39.html