Resistance to insects, plant pathogens and herbicides

Plants as bioreactors

Chapter topics not covered

-Higher yields

-Tolerance of stress and delayed senescence

-Modification of nutritional content, taste and appearance

1. Introduce a bacterial gene encoding toxin active against insects

cry gene of Bacillus thuringiensis

Eliminates need to apply pesticide

2. Increase expression of natural plant proteins that inhibit insect digestive enzymes

Clone genes for inhibitor and place under control of strong plant promoters

Trypsin inhibitor (protease inhibitor present in cowpeas)

a-amylase inhibitor (starch hydrolysis inhibitor in present in beans)

3. Introduce a bacterial gene that encodes cholesterol oxidase

Cholesterol is a component of animal cell membranes

Oxidation kills the insect by disrupting the membrane of insect gut cells

For crops not consumed by humans or livestock:

Ex. cotton & tobacco

See Fig. 18.1

Note the location of the bacterial selectable marker Spc^r in the figure appears to be incorrect. WHY?

(Refer to Fig. 17.7 B.)

Cointegrate cloning vector derived from Ti plasmid (review Fig. 17.7)

• Contains the genetic elements:

1. cry (B.t. toxing gene)

Under control of the strong constitutive 35S CMV promoter

3' termination[polyadenylation sequence

2. T-DNA right border for tintegration into plant genome

3. Bacterial and plant selectable markers

4. Sequence homologous to disarmed Ti plasmid which contains the vir genes

5. E. coli ori

• However "simply" introducing the gene into plants did not protect them from insects

cry wasn't expressed in plants at high enough level to control insects

1. Eliminated DNA encoding amino acids not involved in toxicity

Carboxy-terminal amino acids aren't involved in toxicity

2. Codon optimization. DNA sequence of codons of bacterial gene was changed to codons preferred by plants

-These changes resulted in higher expression levels and insect-resistant transgenic tobacco, tomato and cotton plants

3. Integration of wild-type bacterial gene into chloroplast DNA

Transcription and translation in chloroplasts is prokaryotic

Bacterial promoters are functional

Bacterial codons are preferred

Manipulation of gene not needed

Each plant cell has many chloroplasts so gene dosage is higher/cell

Chloroplasts (and their DNA) not present in plant pollen

Prevents transmission of cry to other plants (weeds, crops growing in adjacent fields)

Transgenic Plants Resistant to Viral Pathogens

1. "Immunize" plants by introducing a gene for a virus coat (capsid) protein

Expression of capsid protein by plant inhibits viral proliferation in cells by unknown mechanisms

Ex. Introduction of gene for coat protein of tobacco mosaic virus into tobacco and tomato protects the transgenic plants form the virus

2. Introduce DNA that encodes viral antisense RNA into plant

Sense RNA = mRNA product of gene transcription that can be translated

Antisense RNA = RNA transcript that is complementary to sense RNA

Hybridization of antisense to sense RNA prevents translation

Also, duplex RNA is rapidly degraded in cell

Target an essential viral gene such as one that encodes a viral capsid protein gene

Functional virus can't be produced in plant cells without the coat protein

• $10 billion spent annually on chemical herbicides
• 10% of global crop lost due to weeds anyway
• Some herbicides harm crop plants
• Carry over of herbicide to next growing season may damage the following year's crop

Strategies for Producing Transgenic Herbicide Resistant Crops

I. Introduce a gene that encodes a resistant form of the herbicide's target protein

Ex. Glyphosate (Roundup).

Inhibits activity of EPSPS in plants, an enzyme involved in biosynthesis of aromatic amino acid (Tyr, Phe, Trp)

Plants die without aromatic amino acids for protein synthesis

aroA gene encoding an EPSPS resistant to glyphosate was cloned from Agrobacterium tumefaciens

Introduced into cotton and soybeans

(Roundup Ready Soybeans and Cotton; Monsanto, St. Louis)

Bacterial EPSPS compensated for the inhibited plant enzyme

Transgenic cotton and soybeans can be sprayed with glyphosate

Only weeds killed: glyphosate prevents aromatic amino acid biosynthesis

II. Metabolic inactivation of herbicide

Bromoxynil -Inhibits plant photosynthesis

Nitrilase -Bacterial enzyme that detoxifies Bromoxynil

Transgenic tobacco plants expressing the nitrilase gene cloned from Klebisella ozaenae are not killed by Bromoxynil

For production of nonplant proteins and chemicals

aka molecular farming, pharming, pharming for farmaceuticals

• Human blood coagulants (Ex. Factor VIII)

Production by 1 tobacco plant = 1,000 liter mammalian cell fermentor

1000 plants would satisfy demand for entire World

• Monoclonal antibodies ("plantibodies"): diagnostic or therapeutic (if humanized)
• Polyhydroxyalkanoates (bacterial biopolymers used to make biodegradable plastics)
• Edible vaccines ("plantigens")

Advantages and disadvantages

(+) Farming crops easier than bacterial fermentation

Requires less technical expertise and doesn't use expensive equipment and growth media as does large-scale bacterial fermentation and cell culture

(-) Product purification may be more difficult from plants than from a microbial fermentation medium

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Last updated: December 2, 2004 /jdh

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