Green Biotechnology: Genetic Manipulation for Herbicide/Pest Resistance

Green Biotechnology

Session 1: Herbicides and Herbicide Resistance

• Learning Objectives:
  • Understand the importance of herbicides.
  • Define what constitutes a good herbicide.
  • Describe different strategies for herbicide resistance with examples.
  • Explain potential pleiotropic effects of transgenes.
  • List and discuss environmental stresses on plant growth.

Environmental Stresses

• External stresses influence plant growth and are grouped into biotic and abiotic stresses.

• Almost all stresses lead to the production of Reactive Oxygen Species (ROS), causing oxidative stress.

• Abiotic stresses: Light, water, CO_2, oxygen, soil nutrients, temperature, salts, and heavy metals.

• Biotic stresses: Weeds, pests, and diseases.

• Weeds are a significant biotic stress, reducing the amount of crops available for human consumption.

  • Humans consume 63% of available crops, with the remainder affected by weeds, pests, and diseases.

Stress and ROS

• Oxidative stress is a secondary effect of many stresses resulting in free radical production (superoxide, H2O2, hydroxyl-radical species) and a cascade of reactions.
• This leads to:
  • Membrane damage
  • Amino acid modifications
  • Peptide chain fragmentation
  • DNA damage leading to deletions and mutations.
• Host enzymes like SOD, catalase, and peroxidase counteract these effects by converting to less reactive products.

Need and Use of Herbicides

• Weeds are unwanted plants that compete with crops for light and nutrients and harbor pathogens.
• Crop yield is reduced by 10-15% due to weeds.
• Herbicides are weed killers developed to tackle this problem in modern agriculture.
• Herbicide use is a necessity in modern agriculture.
• Most herbicides are broad-spectrum, killing a wide range of weeds.

Qualities of a Good Herbicide

• Selective killing of weeds without harming crop plants.
• Non-toxic to animals and microorganisms.
• Rapid translocation within the target plant.
• Rapid degradation in the soil.
• Limitations: Current herbicides cannot always discriminate between weeds and crops, requiring usage when crops are not vulnerable.
• Herbicide-resistant crops allow spraying at the most effective time for weed control and play a pivotal role in modern agriculture.

Advantages of Herbicide-Resistant Crops

• Excellent weed control, leading to higher crop yields.
• Flexibility in controlling weeds later in the plant's growth cycle.
• Reduces the number of sprays needed, decreasing fuel use and herbicide usage.
• Reduces soil compaction due to less need for spraying.

Herbicide Activity

• More toxic to plants than animals because they target 'plant-specific' biological processes.
• Many herbicides inhibit a single enzyme/protein.
• Herbicides belong to various chemical families with about 15 broad mode-of-activity classes.
• Some enzymes, like acetolactate synthase (ALS), are highly vulnerable to herbicide activity.
• Two common herbicide classes that target ALS are sulphonylureas and imidazolinones.

Herbicide-Resistant Crops

• Glyphosate (Roundup):
  • Transgene/Mechanism: Agrobacterium CP4-resistant gene, maize-resistant gene, oxidoreductase detoxification.
  • Companies: Monsanto.
  • Crops: Soybean, rape, tomato, maize.
• Phosphinothricin (Basta, Liberty):
  • Transgene/Mechanism: bar gene - phosphinothricin acetyltransferase detoxification.
  • Companies: Hoechst/AgrEvo/Aventis.
  • Crops: Maize, rice, wheat, cotton, rape, potato, tomato, sugar beet.
• Sulphonylurea (Glean):
  • Transgene/Mechanism: Mutant plant acetolactate synthase.
  • Companies: Novartis/Syngenta.
  • Crops: Rape, rice, flax, tomato, sugar beet, maize.
• Imidazolinone (Arsenal):
  • Transgene/Mechanism: Mutant plant acetolactate synthase.
  • Companies: DuPont-Pioneer Hi-Bred.
  • Crops: Soybean.
• S-triazines (Atrazine, Lasso):
  • Transgene/Mechanism: Mutant plant chloroplast psbA gene.
  • Companies: American Cyanamid, DuPont, Ciba-Geigy/Novartis.
  • Crops: Maize, rape, soybean.
• Nitriles (Bromoxynil, Buctril):
  • Transgene/Mechanism: Nitrilase detoxification.
  • Companies: Calgene.
  • Crops: Cotton, rape, potato, tomato.
• Phenoxy-carboxylic acids (2,4-D):
  • Transgene/Mechanism: Monooxygenase detoxification.
  • Companies: Schering/AgrEvo.
  • Crops: Maize, cotton.

Classification and Properties of Herbicides

• Herbicides vary in properties besides their mode of action due to their wide range of chemical families.
• Classification:
  1. Site of uptake (root vs. shoot).
  2. Degree of translocation (systematic vs. contact).
  3. Time of application (pre-planting, pre-emergence, post-emergence, or pre-harvesting).
• Herbicides differ greatly in toxicity, environmental persistence, and biodegradability.

Strategies for Engineering Herbicide Resistance

1. Overexpression of the target protein:
2. Mutation of the target protein:
   • Resistance to glyphosate and sulfonylurea herbicides is achieved using genes coding for mutant target enzymes 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS), respectively.
3. Detoxification of the herbicide:
   • Resistance to glyphosate has been achieved using the gox gene (glyphosate oxidase) isolated from Achromobacter, which detoxifies the herbicide.
4. Enhanced plant detoxification:

Herbicide Resistance Genes and Their Actions

• EPSPS - 5-enolpyruvyl shikimate 3-phosphate synthase
• ALS - Acetolactate synthase
• PAT - Phosphinothricin acetyltransferase

Glyphosate

• Glyphosate is a broad-spectrum herbicide marketed as ‘Roundup.'
• It is a glycine derivative, less toxic to humans and rapidly degraded by microbes.
• Rapidly transported to the growing points of plants.
• Inhibits the EPSPS enzyme and the biosynthesis of tryptophan and phenylalanine.
• Has a short half-life.
• Marketed by Monsanto (now acquired by Bayer).
• Used in glyphosate-resistant tobacco, tomato, corn, etc.

Glyphosate Resistance: EPSPS Overexpression

• An early approach to engineer glyphosate resistance was to overexpress the EPSPS gene.
• cDNA was isolated from a petunia glyphosate-resistant tissue culture.
• Selection of petunia cells capable of growing in increasing glyphosate concentrations led to cultures with much higher EPSPS levels.
• The EPSPS gene remained wild type.
• Resistance was due to increased enzyme amounts.

Glyphosate Resistance: Mutant/Insensitive EPSPS

• The CP4-EPSPS gene was isolated from Agrobacterium sp. strain CP4.
• Encodes a 47.6 kD EPSPS protein of 455 amino acids.
• CP4 EPSPS is functionally equivalent to endogenous plant EPSPS but displays reduced affinity for glyphosate.

Phosphinothricin (PPT)

• The closest rival to glyphosate in terms of the number and acreage of resistant crops.
• Glyphosate is particularly effective against grasses, while PPT is more effective against broad-leafed weeds.
• PPT is derived from bialaphos, a natural product produced by certain Streptomyces species.
• PPT can be applied directly as a herbicide or converted to an active form (L-PPT) by proteolytic removal of Ala residues.

Herbicidal Action of PPT

• PPT inhibits glutamine synthetase (GS) competitively.
• The immediate effect is the accumulation of ammonia to toxic levels.
• Disrupts photosynthesis.
• Uptake is via the leaf, depending on plant species, growth stage, air humidity, and temperature.
• Translocation within the plant is limited and species-dependent.

Strategy for Resistance to PPT

• PPT is synthesized as an inactive precursor to prevent toxicity to the host Streptomyces.
• The bacteria contain a detoxification gene that protects from the toxic effects of PPT.
• The bar gene of Streptomyces hygroscopicus and the related pat gene of Streptomyces viridochromogenes code for phosphinothricin acetyltransferase (PAT).
• PAT adds an acetyl group to PPT, inactivating it.
• Transferring this gene to a plant provides resistance against PPT.
• The bar gene has been integrated into many plants under the control of the 35S promoter.

Plant Detoxification Enhancement

• Plants possess a natural defense system against toxic compounds.

• Detoxification involves converting a toxic herbicide to a non-toxic/less toxic compound.

• This can involve hydroxylation, conjugation, and transport stages via P450 monoxygenases and other enzymes.

• By enhancing plant detoxification, the impact of the herbicide can be reduced.

• By introducing a foreign gene the herbicide can effectively be detoxified by:

  • Glyphosate oxidoreductase detoxifying glyphosate.
  • Nitrilase detoxifying Bromoxynil.
  • Monooxygenase detoxifying 2,4-D.
  • N-acetyl transferase detoxifying Glufosinate

Pleiotropic Effects of Transgenes

• Insertion of a transgene may result in unforeseen and undesirable effects.
• Roundup Ready crops (soybean, cotton, and oilseed rape) have had some issues.
• Roundup Ready soybeans during hot weather have suffered from stem splitting, possibly due to 20% higher lignin content.
• This may be due to the heightened expression of the EPSPS enzyme under all conditions.

Super Weeds

• A major concern about herbicide-resistant crops is the appearance of super weeds that could evade control by herbicides.
• This is in addition to natural resistance arising from selective pressure due to repeated herbicide usage.
• The use of GM crops could affect the number, rate of appearance, and type of weeds.
• This could occur by 'gene flow.'

Gene Flow: Three Potential Mechanisms

1. The herbicide-resistant crop itself as a 'volunteer' weed in fields where rotational crops are grown; it could reproduce outside of cultivation and form a self-sustaining weed population.
2. Pollen from the herbicide-resistant crop fertilizes weedy relatives, producing herbicide-resistant hybrids.
3. HGT (e.g., viruses) transfers the herbicide-resistant trait into a much wider range of plant species.

• Studies to date tend to indicate no detectable gene flow from crops to weed species.
• Predicting the distance pollen can travel from each GM crop is necessary to minimize potential gene flow.

Abiotic Stress - Salt

• Enhanced tolerance to salt stress during germination and development of young seedlings of transgenic Arabidopsis that harbored a gene for choline oxidase (COD) from Arthrobacter.
• Seeds were placed on solid medium that contained 0, 100, or 200 mM NaCl and were incubated at 22 °C for 7 d for germination and growth of seedlings.
• It appeared that transgenically synthesized betaine enhanced the salt tolerance of transgenic plants that expressed COD.
• Addition of 5 mM betaine to the medium significantly improved the salt tolerance of wild-type Arabidopsis.

Session 2: Genetic Modification for Pest and Virus Resistance

• Learning Objectives:
  • Understand how cry toxins can be used for pest resistance.
  • Describe different steps in the copy nature approach for the development of pest-resistant crops.
  • List how a viral coat protein can be used for viral resistance.
  • Explain.

Nature and Scale of Insect Pest Damage to Crops

• Most crop damage is caused by insect larvae.
• Major classes of insect that cause crop damage:
  • Lepidoptera (butterflies and moths)
  • Diptera (flies and mosquitoes)
  • Orthoptera (grasshoppers, crickets)
  • Homoptera (aphids)
  • Coleoptera (beetles)

GM Strategies: B. thuringiensis Approach

• The bacterium produces insecticidal protein (ICP) which forms inclusion bodies during sporulation.
• One of several classes of endotoxins produced by sporulating bacteria; d endotoxins produced by cry genes carried on plasmids.
• Large difference in size but share a common active core comprising three domains.
• Mode of action of d endotoxins involves a specific interaction between the protein and the insect larva midgut.
• Extremely toxic and can be lethal at low concentrations, but toxicity to other animals is very low.

Genetic Modification with Cry

• First attempts to express Cry1A and Cry3A proteins under the CaMV35S or Agrobacterium T-DNA promoters resulted in very low levels of expression in tobacco, tomato and potato plants.
• The prokaryotic gene sequence itself would need to be extensively modified in order to obtain high levels of expression.
• This was achieved by altering codon usage and overall G +C content, and several potential plant polyadenylation signals and ATTTA sequences were removed.
• Expression was enhanced 100-fold to give levels of expression of the order 100 ng Btprotein per mg total protein.

Commercialization of Bt Technology

Advantages of Bt Crops

• The level of toxin expression can be very high, delivering a sufficient dosage to the pest.
• Toxin expression is contained within the plant system, so only insects that feed on the crop perish.
• Crops can kill insects even after they have invaded the plant tissues.
• The toxin is washed away from plants with water or rain and is broken down by sunlight, providing an environmental benefit.
• Toxin expression can be modulated using tissue-specific promoters, replacing the use of synthetic pesticides.

Insect Resistance to Bt

• The rapid appearance of resistant pests is a problem with accelerated Bt technology use.
• The resistance mechanism relates to the specific binding involved in the mechanism of action of the Cry proteins.
• A small number of significant mutations in the insect gene coding for the receptor protein can greatly reduce the binding of a particular Cry protein.
• Strategies to counter the build-up of insect resistance:
  • Pyramiding
  • Chimeric protein
  • Integrated pest management

Pyramiding

• Uses more than one transgene.
• Transgenes are ‘stacked’ by conventional crosses between different transgenic lines.
• Has been a more effective strategy than single-gene technology for insect and disease resistance.
• To avoid ‘cross-resistance’ to 2 different Btgenes, it is better to pyramid genes with unrelated resistance mechanisms, such as a proteinase inhibitor gene.
• It is highly unlikely that resistance to two completely different genes will arise at the same time.

Integrated Pest Management (IPM)

• Includes selective usage of Bt crops by rotating Bt crops with non-Bt crops and by growing different Bt crops within the migration distance of pests common to both (high dose/refuge approach).
• This helps to prevent the build-up of resistance when there is a continuous selection pressurecreated by continuous use of Bt crops.
• High dose/ refuge:
  • Grow transgenic crops expressing a high dose of Btprotein alongside a smaller refuge of non-transgenic crop, or any other plant that the insect pest feeds on.
  • High dose ensures only homozygous resistant insects would tolerate feeding on GM crop – this is rare in early stages of resistance development.
  • The refuge ensures the presence of a much larger population of susceptible insects in the vicinity of the GM crop.

Copy Nature Strategy

• Involves a rational approach to developing pest-resistant crops.
  1. Identification of leads via literature, world seed collections, or field observations.
  2. Protein purification with insecticidal properties.
  3. Artificial-diet bioassay.
  4. Mammalian toxicity testing.
  5. Genetic engineering with a strong constitutive promoter.
  6. Selection and testing
  7. Biosafety

Promoters Used with Insect Resistance Genes

Cowpea Trypsin Inhibitor (CpTI)

• CpTi as an anti-insect, pest control mechanism came from identifying strains of Cowpea in Africa resistant to insect pests.
• Found to be a Trypsin inhibitor and in artificial-diet bioassays, was shown to be effective against Lepidoptera, Orthoptera and Coleoptera.
• Not toxic to mammals.
• The gene was isolated and inserted into the transformation vector pROK 2.
• Transferred into tobacco for initial trials, and the transgenic plant was visibly more resistant to damage under laboratory conditions versus control plants.
• In field tests, CpTi tobacco showed a significant level of protection from damage by lepidopteran pests and a reduction of feeding larvae.

Viral Resistance

• Plant viruses cause serious reductions in marketable crop yield and, in some cases, even plant death worldwide.
• In most cases, the most effective way to control virus diseases is through genetically controlled resistance.
• Developing virus-resistant (VR) crops through traditional breeding can take many years and is not always possible.
• Approaches to engineer plants for virus resistance include the CP gene, antisense RNA approach, and ribozyme-mediated protection.
• The use of the CP gene has been the most successful.

CP Gene Approach

• Transgenic plants with a virus CP gene linked to a strong promoter have been produced in many crops, such as tobacco, tomato, alfalfa, sugar beet, and potato.
• The first transgenic plant of this type was tobacco containing the CP gene of tobacco mosaic virus (TMV) strain UI.
• When these plants were inoculated with TMV UI, symptoms either failed to develop or were considerably delayed.
• Disease symptoms were delayed when inoculated with related viruses.

How Does This Work?

• Expression of a virus CP gene confers resistance to the concerned virus and related viruses.
• The effectiveness of the CP gene is affected by the amount of CP produced and by the virus inoculum concentration.
• The resistance generated by CP is most likely due to blocking the uncoating of virus particles, which is necessary for viral genome replication and expression.
• Other effects include preventing or delaying the systemic spread of the viruses.

Are Transgenic Plants Safe?

• Antibiotic resistance genes used for selection are expressed in every cell of transgenic plants.
• The protein products of such genes could be toxic to humans/animals.
• Bacteria in the human intestine could acquire the antibiotic resistance gene, becoming resistant to antibiotics.
• The antibiotic resistance gene could also be passed on to other organisms in the environment, damaging the ecosystem.