(3)Transgenic Plants Notes

Insect Resistance

• Insects are diverse and often considered pests, leading to crop damage and disease transmission.

• Humans have used pesticides since before 2500 BC, with early examples including elemental sulfur.

• DDT, discovered in 1939, was a highly effective insecticide.

• Insecticides are classified based on chemistry: chlorinated hydrocarbons, organophosphates, and carbamates.

• Drawbacks of chemical insecticides include:

  • Side effects on ecosystems and humans.

  • Insects developing resistance.

  • Harm to beneficial insects and natural enemies.

Bacillus thuringiensis (Bt)

• Gram-positive soil bacterium producing insecticidal proteins.

• Effective insecticides are crystalline proteins produced by B.t.

• Different strains produce toxins specific to certain insects (e.g., subsp. tenebrionis for coleoptera).

• Insecticidal activity is in the parasporal crystal, produced during sporulation.

• This toxic protein kills insects, providing nutrients for spore germination.

Bt Mode of Action

• Crystal is a protein aggregate that dissociates into subunits via mild alkali.

• Subunits further dissociate in vitro with ß-mercaptoethanol.

• Each subunit is ~250 kD, consisting of two 130 kD polypeptides.

• The parasporal crystal contains a protoxin, a precursor to the active toxin.

• Genetic engineering allows crops to produce functional insecticides, reducing the need for spraying chemical pesticides.

Increasing Expression of B. thuringiensis Protoxin

• Use functional toxin portion of the gene coupled with codon matching to achieve highest expression.

• Minimum sequence encoding toxin is determined.

• N-terminal is highly conserved (~98%), C-terminal is variable.

• Toxicity resides in the first 646 amino acids from the N-terminus.

• Shortened protoxin in transgenic tomato plants demonstrated effectiveness.

B. thuringiensis Protoxin: Vector Construction

• Short protoxin under strong constitutive 35S promoter.

• Nopaline synthase transcription termination-polyadenylation site.

• Cloned into T-DNA region of a cointegrate-type Ti plasmid vector.

• Vector contains:

  • Spectinomycin resistance gene

  • E. coli origin of replication

  • Neomycin phosphotransferase gene (kanamycin selection)

  • T-DNA right border from nopaline Ti plasmid.

B. thuringiensis Protoxin: Transformation Process

• Plasmid constructed and manipulated in E. coli.

• Transferred to A. tumefaciens (disarmed Ti plasmid).

• Recombination in A. tumefaciens transfers short protoxin to tomato plant chromosomal DNA.

Alternative Approaches for Protoxin Expression

• Express fully modified protoxin gene under a promoter with a chloroplast transit peptide sequence.

• Protoxin localized within chloroplasts.

• High expression levels (~1% of total leaf protein).

• Introduction of protoxin gene into chloroplast DNA:

  • Gene flanked by chloroplast genes.

  • Integration via homologous recombination.

  • Transcription under chloroplast promoter.

  • May reach 2-3% of leaf protein.

  • 10-20 fold increase possible with chaperon protein for folding.

Advantages & Disadvantages of Chloroplast Protoxin Incorporation

• Advantages:

  • No gene modification needed for chloroplast machinery.

  • High copy number due to many chloroplasts.

  • Chloroplast transmitted through egg, not pollen, preventing protoxin spread.

• Disadvantages:

  • Ineffective against insects attacking stems or fruit.

Other Insect Protection Strategies

• Protease Inhibitors:

  • Example: Cowpea Trypsin Inhibitor (CpTI).

  • Prevents insects from hydrolyzing proteins causing starvation.

  • Modified with stronger promoter to increase production.

• Alpha Amylase Inhibitor:

  • Effective against cowpea weevil and azuki bean weevil.

  • Gene cloned and expressed in pea plants.

Cholesterol Oxidase

• Present in many bacteria.

• Catalyzes the oxidation of 3- hydroxysteroids to ketosteroids and H2O2.

• Used to determine cholesterol levels.

• Low levels exhibit high insecticide activity against boll weevil larvae.

• Acts in insect midgut epithelial membrane.

RNA Interference (RNAi)

• Used to develop insect-resistant corn.

• Involves:

  • Creating a larval cDNA library.

  • Testing double-stranded RNAs (dsRNAs).

  • Transforming corn plants and testing for resistance.

Cotton and Gossypol

• Cotton produces gossypol which inhibits dehydrogenase enzymes.

• Insects produce cytochrome P450 monooxygenase to inactivate gossypol.

• RNAi used to inactivate cytochrome P450 monooxygenase.

Herbicide-Resistant Plants

• 10% of global crops lost to weeds.

• $10 billion spent on herbicides.

• Herbicides don’t discriminate and can persist in the environment.

• Biological manipulations for herbicide resistance:

  • Inhibit herbicide uptake.

  • Overproduce the herbicide-sensitive target protein.

  • Introduce a bacterial/fungal gene that produces a non-sensitive but functionally similar protein.

  • Reduce binding affinity of herbicide to the target protein.

  • Enable plants to metabolically inactivate the herbicide.

Glyphosate Resistance

• Glyphosate = "environmentally friendly".

• Inhibits EPSPS (5-enolpyruvylshikimate-3-phosphate synthase).

• EPSPS synthesizes aromatic amino acids in bacteria and plants.

• Glyphosate-resistant E. coli EPSPS gene isolated, placed under plant promoter control, and cloned into plants, making "Roundup Ready" crops which produce resistant EPSPS to replace inhibited protein.

Current Status of Roundup Ready Crops

• Glyphosate patent expired so other companies are actively developing glyphosate-resistant plants.

• Worldwide agriculture has become too dependent on a single herbicide; at least 19 different weeds have acquired naturally-occurring resistance to glyphosate.

Round Up Inactivation by Acetylation

• Transgenic plants expressing glyphosate N-acetyltransferase are tolerant to higher doses of glyphosate.

• Transgenic tobacco plants overexpressing both glyphosate N-acetyltransferase and EPSPS are more resistant to glyphosate.

Dicamba (3,6-dichloro-2-methoxybenzoic acid)

• Controls broadleaf weeds since 1960s.

• Mimics high levels of indole-3-acetic acid, a plant hormone.

• Non-persistent in soils, non-toxic to animals/humans however Resistant weeds have not developed.

• Dicamba monooxygenase converts dicamba to 3,6-dichlorosalicylic acid, which has no herbicidal activity.

Fungus and Bacterium Resistance

• Phytopathogenic fungi cause plant damage and crop loss.

• Fungal rice blast costs farmers billions annually.

Systemic Acquired Resistance

• Plants respond to pathogen invasion by converting salicylic acid 2-O-β-D-glycoside to salicylic acid, inducing defense responses.

• This leads to synthesis of pathogenesis-related (PR) proteins such as β-1,3-glucanases and chitinases.

NPR1 Gene

• Encodes a regulatory protein controlling PR protein expression, activated by salicylic acid.

• Overexpression of NPR1 leads to broad-spectrum disease resistance.

• Transformation of strawberry plants with Arabidopsis thaliana NPR1 protects against fungal pathogens.

Chestnut Blight

• Fungal disease caused by Cryphonectria parasitica devastating American chestnut trees in the 20th century.

• Hybrid trees combining American chestnut with resistant Asian species developed.

Transgenic American Chestnut Trees

• Express a wheat oxalate oxidase gene (oxo) that protects trees from oxalic acid secreted by fungus.

• Oxalic acid acidifies infected tissues to toxic levels.

Salt and Drought Stress

• Drought and high salinity severely impair plant growth.

• Irrigation increases soil salinity, rendering land unsuitable for crops.

• Plants synthesize osmoprotectants to survive like sugars, alcohols, proline, and quaternary ammonium compounds.

Trehalose

• Alpha,alpha-1,1-glucoside consisting of two molecules of alpha-glucose.

• Stable, resistant to acid and high temperature, forms a gel phase during dehydration.

• Protects cells from drought and salt damage, increased via genetic manipulation.

Sequestering Sodium in Vacuoles

• Overproduction of A. thaliana Na+/H+ antiport protein transports Na+ into the vacuole.

• The transgenic plants thrive in high salt conditions.

• Concentrating salt in vacuoles drives water into plant cells, improving water efficiency.

Modification of Plant Nutritional Content

• Rice is a staple food but a poor source of nutrients like vitamin A.

• Vitamin A deficiency affects millions, causing deaths and blindness.

• Engineering rice to produce provitamin A (β-carotene) addresses this deficiency.

Vitamin A – Golden Rice

• Mammals synthesize vitamin A from β-carotene.

• Agrobacterium-mediated transformation introduces the β-carotene biosynthetic pathway.

• Golden Rice 2 uses a corn phytoene synthase gene with higher activity, increasing β-carotene production.

Edible Vaccines

• Limitations to widespread vaccination persist due to cost and infrastructure.

• Edible vaccines offer a solution by reducing production and delivery costs, and eliminating the need for trained personnel.

• Plants glycosylate proteins, potentially enhancing immunogenicity and stability.

Mechanism of Edible Vaccines

• Ingested antigen binds to and is taken up by M cells in the intestine.

• Passed to immune cells, including macrophages and B cells.

• Macrophages display antigen portions to helper T cells, activating B cells to synthesize neutralizing antibodies.

Plant Yield: Altering Lignin Content

• Lignin is the second most abundant organic compound on Earth.

• Lignin is important in mechanical support and pathogen defense.

• High lignin levels decrease nutritional value of forage crops and cause cellulose to require harsh treatments, which causes energy and chemical consumption.

• Low lignin increases accessibility to cellulose- and hemicellulose-degrading enzymes.

• Biochemical steps leading to lignin synthesis are conserved.

• Corn engineered to be a food source and biofuel via lignin modification.

Microbial Insecticides: Benefits and Drawbacks

Benefits:

• Specificity: Often target specific pests, reducing harm to non-target organisms.

• Environmental Safety: Generally considered more environmentally friendly than synthetic pesticides.

• Reduced Chemical Residue: Microbes degrade, leaving minimal chemical residue.

• Development of Resistance: Can reduce the speed at which future generations of insects that develop resistance to standard pesticides.

Drawbacks:

• Narrow Host Range: May only be effective against a limited number of pests.

• Environmental Sensitivity: Efficacy can be affected by environmental conditions (UV radiation, temperature).

• Slower Action: Can take longer to kill pests compared to synthetic insecticides.

• Storage and Application: May require specific storage conditions and application methods.

• Cost: Sometimes more expensive than traditional chemical pesticides

Bacillus thuringiensis (Bt)

• Basic Biology:

  • Soil-dwelling bacterium.

  • Produces crystal proteins (Cry toxins) during sporulation.

• Cry Proteins and Crystals:

  • Cry toxins are protoxins that are activated in the insect gut, and are the the protein crystal inclusions within the bacteria.

  • Different Cry proteins have different insecticidal specificities.

  • Coded for by different genes.

Mechanism of Action of Cry Proteins

1. Ingestion: Insect consumes Bt crystals.

2. Activation: Cry protoxins are cleaved into active toxins by gut proteases.

3. Binding: Activated toxins bind to specific receptors on the midgut epithelial cells.

4. Pore Formation: Toxin insterts into membrane, forming pores. This disrupts ion flow and causes cell lysis.

5. Paralysis and Death: Disruption of digetsive system leads to paralysis, starvation and insect death

Increasing Bt Toxin Expression in Plants

• Genetic Modification of Wild-Type Bt Toxin:

  • Codon Optimization: Modifying the gene sequence to use codons preferred by the plant to enhance translation efficiency.

  • Reasons: Increases the amount of toxin produced leading to better pest control.

• Expression of Modified Bt Toxin in Chloroplasts:

  • Chloroplast Transformation: Inserting the Bt gene into the chloroplast genome.

  • Reasons:

    • High levels of protein expression.

    • Reduced gene silencing.

    • Containment of the transgene through maternal inheritance.

Protease and Alpha Amylase Inhibitors, and Cholesterol Oxidase

• Protease Inhibitors: Interfere with insect digestion by inhibiting proteases, reducing nutrient uptake.

• Alpha Amylase Inhibitors: Disrupt starch digestion by inhibiting amylases, limiting available sugars.

• Cholesterol Oxidase: Disrupts insect development by interfering with sterol metabolism.

RNA Interference (RNAi) in Insect Control

• Mechanism: Introduce dsRNA that targets essential insect genes to silence gene expression.

• Examples:

  • Corn: Targeting genes involved in insect development or immunity.

  • Cotton: Targeting genes essential for cotton bollworm survival and growth.

Modification of Plants for Herbicide Resistance

• Roundup (Glyphosate) Resistance:

  • Mechanism: Introducing a modified EPSPS gene that is resistant to glyphosate.

  • Benefits: Selective weed control, reduced tillage.

  • Drawbacks: Development of glyphosate-resistant weeds, potential environmental impacts.

• Dicamba Resistance:

  • Mechanism: Introducing a dicamba monooxygenase (DMO) gene that degrades dicamba.

  • Benefits: Control of broadleaf weeds, especially those resistant to glyphosate.

  • Drawbacks: Potential for dicamba drift harming non-target plants, herbicide resistance.

Pathogenesis-Related (PR) Proteins and Salicylic Acid

• PR Proteins: Plant proteins induced upon pathogen attack; have antimicrobial activities.

• Salicylic Acid (SA): Signaling molecule that activates PR protein expression.

• Mechanism: Fungal or bacterial pathogens trigger SA production, leading to expression of PR proteins.

• Genetic Modifications:

  • Overexpressing genes encoding specific PR proteins for enhanced resistance.

  • Modifying signaling pathways to enhance SA production or response.

Chestnut Blight

• Causative Agent: Cryphonectria parasitica, a fungal pathogen.

• Mechanism:

  • Fungus enters through wounds in the bark.

  • Produces oxalic acid, which kills cambial cells, leading to cankers.

  • Girdling of the tree eventually causes death.

Genetically Modified American Chestnut Trees

• Development: Introduction of an oxalate oxidase gene to break down oxalic acid produced by the fungus.

• Goal: Enhance resistance to chestnut blight and rescue the American chestnut from extinction.

Salt and Drought Stress Resistance

• Significance: Increases crop yields in marginal lands affected by salinity and water scarcity.

• Mechanisms:

  • Na+ Sequestration: Expressing genes to sequester sodium ions in vacuoles.

  • Increasing Trehalose Expression: Trehalose acts as a protectant, stabilizing proteins and membranes under stress.

Genetic Modification of Plant Nutritional Content: Golden Rice

• Golden Rice: Genetically engineered rice to produce beta-carotene (provitamin A) in the endosperm.

• Goal: Combat vitamin A deficiency in populations where rice is a staple food.

Genetically Engineered Plants for Edible Vaccines

• Mechanism: Expressing antigenic proteins from pathogens in plant tissues.

• Advantage: Cost-effective and convenient delivery of vaccines, potential for mass immunization.

Modification of Plants to Alter Lignin Production

• Lignin is the second most abundant organic compound on Earth.

• Lignin is important in mechanical support and pathogen defense.

• High lignin levels decrease nutritional value of forage crops and cause cellulose to require harsh treatments, which causes energy and chemical consumption.

• Low lignin increases accessibility to cellulose- and hemicellulose-degrading enzymes.

• Biochemical steps leading to lignin synthesis are conserved.

• Corn engineered to be a food source and biofuel via lignin modification.

Definitions in Outline Format:

1. Bacillus thuringiensis (Bt)

   • Gram-positive soil bacterium producing insecticidal proteins.

2. BT Toxin

   • Crystalline proteins produced by Bacillus thuringiensis that act as effective insecticides.

3. Parasporal Crystal

   • Crystal containing toxic protein produced during sporulation of Bacillus thuringiensis.

4. Cowpea Trypsin Inhibitor (CpTI)

   • A protease inhibitor that prevents insects from hydrolyzing proteins, causing starvation.

5. Gossypol

   • A substance produced by cotton plants that inhibits dehydrogenase enzymes in insects.

6. Herbicide

   • A substance used to kill unwanted plants or weeds.

7. EPSPS (5-enolpyruvylshikimate-3-phosphate synthase)

   • An enzyme inhibited by glyphosate, essential for aromatic amino acid synthesis in plants and bacteria.

8. Glyphosate

   • A broad-spectrum herbicide that inhibits EPSPS.

9. Round-Up Ready

   • Crops genetically modified to be resistant to glyphosate.

10. Dicamba (3,6-dichloro-2-methoxybenzoic acid)

    • A herbicide that mimics high levels of indole-3-acetic acid, used to control broadleaf weeds.

11. Dicamba Monooxygenase

    • An enzyme that converts dicamba into a non-herbicidal substance, providing resistance to plants.

12. Salicylic Acid

    • A plant hormone involved in systemic acquired resistance, inducing defense responses against pathogens.

13. Pathogenesis-Related (PR) Proteins

    • Proteins synthesized by plants in response to pathogen invasion, such as β-1,3-glucanases and chitinases.

14. Glucanase

    • A type of PR protein that breaks down glucans, often found in fungal cell walls.

15. Chitinase

    • A type of PR protein that breaks down chitin, a component of fungal cell walls and insect exoskeletons.

16. Thaumatin-like proteins

    • Function and definition were not specifically mentioned in the provided text

17. NPR1 Gene

    • A gene encoding a regulatory protein that controls PR protein expression, activated by salicylic acid.

18. Trehalose

    • A sugar that protects cells from drought and salt damage, increased via genetic manipulation.

19. Golden Rice

    • Genetically engineered rice to produce beta-carotene (provitamin A) in the endosperm.

20. Lignin

    • Second most abundant organic compound on Earth, important