lecture 6.plant transformation -...

Plant transformationObjectives:

1. What is plant transformation?

2. What is Agrobacterium? How and why does it transform plant cells?

3. How is Agrobacterium used as a tool in molecular genetics?

References:

Hooykaas and Schilperoort. 1992. Agrobacterium and plant genetic engineering. Plant Molecular Biology 19: 15-38.

Westhoff et al. Molecular Plant Development:from gene to plant. Chapter 7, 236-243.

Plant transformation

• Transient – no incorporation of exogenous DNA into the genome

• Stable – incorporation of introduced exogenous DNA into the genome

Introduction of exogenous DNA into a plant cell

Transformation of multicellular organisms:

• Cannot directly transform every cell - Transformation involves one cell which then regenerates an entire organism

Agrobacterium tumefaciens:a natural tool for plant transformation

Soil gram positive bacterium

Agrobacterium tumefaciens attached to a plant cell

Martha Hawes


• Causes Crown Gall disease - tumors (galls) form at base of stem in many dicotyledonous plants (dicots)

Photographs supplied by Sharon von Broembsen, Oklahoma State University

• production of tumors is caused by the transfer of bacterial DNA to the plant, which integrates into theplant genome


• Genes involved in crown gall disease are not present on the chromosome of A. tumefaciens but on a large plasmid, called the Ti (tumor-inducing) plasmid.

Ti

A. tumefaciens

Circular chromosome

virgenes

LB

RBT-DNA

Ti plasmid~ 120 kbp

ori

A. tumefaciens T-DNA Structure

LB and RB – 25 bp direct repeatsNos - nopaline synthase – opine biosynthetic gene*Shi - shoot inducing - 2 genes for auxin synthesis*Roi - root inducing - gene for cytokinin synthesis*

Shi NosShi Roi

*have eukaryotic promoters – these genes are not expressed in Agrobacterium!!!

RBLB

T-DNA transfer into plants

•T-DNA transfer process is activated when Agrobacterium gets in contact with damaged plant tissue

• T-DNA is nicked at the RB, T-DNA gets replicated to the LB and moved intothe plant cell – these processes are catalyzed by products of vir genes

http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF

•T-DNA is inserted into plant nuclear genome at random sites.

• Transformed cell starts proliferating upon DNA integration resulting in tumor formation. Why?

• Transformed cells make opines = N-rich nutrients (amino acid derivatives) for bacterium (“Genetic colonization”)

T-DNA transfer into plants

http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF

Agrobacterium tumefaciens as a tool for genetic engineering

Problem: tumor

How can we engineer Agrobacterium to make it useful for genetic engineering?

• Delete auxin, cytokinin and opine genes

• Retain vir genes, LB&RB, ori

• Ti plasmid is huge (~120 kb) – need to make it smaller

Binary vector

LB

T-DNARB


• vir genes and T-DNA can be on separate plasmids

• only left and right borders (LB & RB) are required for T-DNA to be transferred

virgenes

Ti plasmid

ori(Agrobacterium)

ori(E.coli)

Selectable marker

(Bacteria)

Selectable marker(Plants)

Selectable marker

(Bacteria)

Cloning site for plant genes

ori(Agrobacterium)

Steps in plant transformation

1. Propagate binary vector in E. coli

3. Re-introduce engineered binary vector into E. coli to amplify

4. Isolate engineered binary vector from E. coli and introduce into Agrobacterium already containing a modified (smaller) Ti plasmid with vir genes

5. Infect plant tissue with engineered Agrobacterium(T-DNA containing the gene of interest gets inserted into a plant cell genome at random sites)

2. Isolate binary vector from E.coli and engineer (introduce a gene of interest)

• In each cell T-DNA gets integrated at a different site in the genome


• Consequences of the insertion: - Foreign DNA is inserted- Insertional mutagenesis (does not kill the cell – the organism is diploid!)

• Each cell is hemizygous for the insertion – only one of the homologous chromosomes gets the insertion

Problem:

We want to transform the whole organism, not one cell!!!


This is done by:

• Transforming plant cells in culture, selecting transformed cells and regenerating the entire plant from the transformed cell (eg. tobacco)


Solanum chacoense

http://en.wikipedia.org/wiki/File:Transformation_with_Agrobacterium.JPG

Problem:



This is done by:


• In planta transformation of Arabidopsis- Dip flowering plants into Agrobacterium suspension


In planta transformation of Arabidopsis(Floral dip method)

Systemic infection in Arabidopsis is accomplished by transformation of female gametes!

plbio.life.ku.dk

Problem:



This is done by:


• In planta transformation of Arabidopsis- Dip flowering plants into Agrobacterium suspension- Harvest seed and select for transformants – (they are hemizygous!)

Binary vector

LB

T-DNARB


• vir genes and T-DNA can be on separate plasmids

• only left and right borders (LB & RB) are required for T-DNA to be transferred

virgenes

Ti plasmid

ori(Agrobacterium)

ori(E.coli)

Selectable marker

(Bacteria)

Selectable marker(Plants)

Selectable marker

(Bacteria)

Cloning site for plant genes

ori(Agrobacterium)

Selection of transformants

http://krauthammerlab.med.yale.edu/imagefinder/

Screening for transformants

DsRed selection using green light excitation

http://www.isb.vt.edu/articles/jan0803.htmhttps://www.emsdiasum.com/microscopy/technical/datasheet/sfa-2.aspx

lecture 6.plant transformation -...

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