Insecticide Resistant Transgenic Plants

Insecticide resistant transgenic plants refer to genetically modified plants that have been engineered to possess resistance to specific insecticides. This resistance is typically achieved through the introduction of genes that encode proteins capable of detoxifying or neutralizing the insecticide, or that modify the plant’s physiology to reduce the insecticide’s efficacy

Introduction to Insecticide Resistant Transgenic Plants

Insect pests pose a significant threat to global agriculture, causing substantial crop losses each year despite the widespread use of insecticides. Modern farming practices, aimed at maximizing yield, have increasingly relied on chemical insecticides for pest control. However, this heavy dependence on agrochemicals has led to the rapid development of resistance among insect pests, such as cotton bollworms becoming resistant to organochlorine insecticides. Additionally, indiscriminate pesticide use often disrupts ecosystems, eliminating beneficial predatory species, which can result in secondary pests becoming primary threats, further compounding crop damage.

Given these challenges, there is a pressing need to develop more sustainable methods for crop protection. Integrated pest management (IPM) offers a balanced approach, combining practices such as selective pesticide use, crop rotation, and field sanitation. Central to IPM is the exploitation of inherently resistant plant varieties. One of the most promising advancements in this area is the development of insecticide-resistant transgenic plants through genetic engineering.

By incorporating insect-resistant genes from a wide range of sources, including the bacterium Bacillus thuringiensis (Bt) and resistance genes from plants themselves, scientists have created crops that can effectively defend against pests. This genetic approach provides an alternative to chemical insecticides, potentially reducing pesticide use, slowing the development of resistance, and increasing crop protection.

Strategies for insecticide resistant transgenic plants

• One approach is to use the entomicidal bacterium Bacillus thuringiensis Berliner as a source of resistance genes
• to identify and use the insect resistance genes present in plants themselves e.g. proteinase inhibitor, cowpea trypsin  inhibitor gene, α amylase inhibitor, lectins.

Plants produce a variety of secondary chemicals, such as pyrethrins from chrysanthemums and nicotine from tobacco, which are toxic or deterrent to potential predators.

USE OF PLANT-DERIVED INSECTICIDAL GENES  (non BT protein)

Proteinase inhibitor

• Interest in protease inhibitors’ effects on insect development began in 1947.
• In 1972, Ryan and colleagues proposed that protease inhibitors play a protective role in plants.
• Damage to solanaceous plant leaves induces the synthesis of protease inhibitors throughout the plant.
• This induction is triggered by a wound hormone called proteinase inhibitor-inducing factor (PIIF), which is released from damaged leaves.
• Gatehouse et al. demonstrated that elevated protease inhibitor levels in cowpea contribute to resistance against the bruchid pest Callosobruchus maculatus.
• This finding led to the use of protease inhibitors in crop protection through genetic manipulation and conventional breeding.
• Transgenic plants expressing protease inhibitors offer an innovative approach to enhancing insect resistance in crops.
• Protease inhibitors target the digestive enzymes of insects, disrupting their ability to process food and causing significant damage to pest populations.

Several protease inhibitors have been identified through studies that assess their activity both in vitro and in vivo in insect gut protease assays. These inhibitors show promise for use in crop protection, although some insects may develop adaptive proteases that bypass inhibition, indicating the need for caution in selecting the most suitable inhibitors for each pest species.

• Serine Protease Inhibitors in Insect-Resistant Plants One of the early successes in this field was the transfer of a serine protease inhibitor gene from cowpea, encoding a double-headed trypsin inhibitor (CpTI), into tobacco plants. The transformed plants exhibited significant resistance against tobacco budworm (Heliothis virescens), a major pest affecting economically important crops such as tobacco, cotton, and maize. Bioassays showed that larvae feeding on CpTI-expressing plants either died or experienced stunted development. This protection has been demonstrated against several other lepidopteran pests, and CpTI has since been engineered into various crops, including potatoes, rice, and strawberries, with field trials showing consistent results.

• Cysteine Protease Inhibitors in Insect-Resistant Plants In addition to serine protease inhibitors, cysteine protease inhibitors have been incorporated into transgenic plants for pest control, particularly for targeting coleopteran insects. While in-vitro studies have demonstrated their efficacy against insect digestive proteases, fewer in planta examples have been published. One notable case involves the engineering of oryzacystatin into poplar trees, providing resistance against the leaf beetle (Chrysomela tremulae)

Table – List of Plant insecticidal gene (Non-bt) of different insect order

Plant Gene	Transgenic plant(s)	Encoded protein	Resistance to insets
CpTi	Potato, apple, rice, sunflower, wheat, tomato	Trypsin	Coleoptera, Lepidoptera
CII	Tobacco, Potato	Serine protease	Coleoptera, Lepidoptera
PI-IV	Potato, Tobacco	Serine protease	Lebidoptera
OC-1	Tobacco	Cystein Protease	Coleoptera, Homoptera

α-Amylase Inhibitors

• Unlike protease inhibitors, α-amylase inhibitors are not synthesized in response to insect attacks. Their precise physiological role in plants remains unclear.
• α-amylase inhibitors from wheat and Phaseolus vulgaris (common bean) have shown insecticidal activity against non-pest species in artificial diet bioassays.
• A specific α-amylase inhibitor isolated from a wild line of P. vulgaris (G12953) is highly effective against the major storage pest Zabrotes subfasciatus.
• Different types of α-amylase inhibitors in wheat show varied activity against lepidopteran pests.
• Genes encoding α-amylase inhibitors have been introduced into tobacco and other plants, showing resistance to insect pests, with strong activity against pests like Z. subfasciatus.

Table:  List amylase inhibitor gene resistance to different insect order

Plant Gene	Transgenic plant(s)	Resistance to insets
α-A1-Pv	Pea, tobacco	Coleoptera
WMAI-1	Tobacco	Lepidoptera

Lectins

• Lectins are carbohydrate-binding proteins that can agglutinate (clump together) specific carbohydrates, which can affect insect feeding and growth.
• Lectins have been proposed as defensive proteins in plants, offering protection against insect pests, particularly in seeds.
• Common bean lectin (PHA) and related proteins like arcelin have been shown to be toxic to coleopteran pests such as the cowpea bruchid (Callosobruchus maculatus).
• Lectins like wheat germ agglutinin (WGA) have demonstrated high toxicity to lepidopteran pests like the European corn borer (Ostrinia nubilalis).
• Lectins like GNA (mannose-specific) and WGA have been found toxic to homopteran pests, significantly reducing survival and fecundity.
• Transgenic plants expressing lectin genes, such as pea and snowdrop lectins, have shown strong protection against insect pests, reducing larval biomass and damage in crops like tobacco, potato, and rice.
• Table:  List lactin gene resistance to different insect order

Plant Gene	Transgenic plant(s)	Resistance to insets
GNA	Potato, rice, Sugarcan, Sweet potato, tobacco	Homoptera, Lepidoptera
WGA	Maize	Lepidoptera, Coleoptera

Chitinases

• Though primarily studied for anti-fungal properties, chitinases can also reduce the fecundity of certain insect pests, particularly aphids, by breaking down the chitin in their exoskeletons.
• Transgenic potatoes expressing bean chitinase (BCH) have shown reduced aphid size and fecundity, though the results are not always statistically significant.
• e.g. BCH gene provide resistance to Homoptera, Lepidoptera

Bacillus thuringiensis (BT) Toxin –

BT toxin, or Bacillus thuringiensis toxin (intracellular crystal protein)/δ-endotoxin, is a natural insecticide produced by the bacterium Bacillus thuringiensis, a gram –ve soil bacteria. It was first discovered by Ishiwaki in 1901.  It works by targeting specific insects, usually caterpillars, beetles, and flies, by causing them to stop feeding and eventually die. The toxin binds to receptors in the insect’s gut, forming crystals that are then broken down into toxic proteins. These proteins disrupt the insect’s digestive system, leading to its demise.

Various strains of Bacillus thuringiensis produce a range of endotoxins (crystal proteins/cry genes). The bacterium produces a protoxin (approximately 130 kDa), which becomes an active toxin (about 68 kDa) upon ingestion by insects (Lepidoptera, Diptera, and Coleoptera larvae) in the gut under alkaline conditions (pH 7.5-8.5)

Crop	BT Gene	Resistance to insects
Cotton	Cry1Ac	Cotton bollworm, tobacco budworm
Maize	Cry1Ab, Cry9C, Cry1F,	European corn borer
Potato	Cry3A	Colarado beetle

Steps of BT-toxin activity in larvae

• Ingestion: The larvae of susceptible insects (such as those from the orders Lepidoptera, Diptera, and Coleoptera) consume plant material or soil contaminated with Bacillus thuringiensis spores or toxins.
• Protoxin Release: The ingested Bacillus thuringiensis cells release protoxin proteins in the insect gut. These protoxins are large, inactive precursors of the active toxins.
• Activation in Alkaline Environment: The protoxins are soluble and remain inactive until they encounter the alkaline conditions of the insect gut (pH 7.5-8.5). In this environment, proteolytic enzymes present in the gut cleave the protoxins, converting them into active toxins.
• Toxin Binding: The activated toxins bind specifically to receptors on the surface of gut epithelial cells. This binding is highly specific, as the toxins recognize and attach to certain protein receptors on the cell membranes.
• Toxin Insertion: After binding, the toxins insert themselves into the gut cell membranes. This insertion forms pores or channels in the cell membranes, disrupting the integrity of the gut epithelium.
• Cell Disruption: The formation of pores leads to cell lysis, causing the gut cells to rupture and die. This disrupts the digestive process, leading to leakage of cell contents and further damage to the gut lining.
• Feeding Disruption: As the gut cells are damaged, the larvae experience a loss of appetite and stop feeding. The destruction of the gut lining impairs nutrient absorption and digestion.
• Larval Death: The continued feeding disruption and gut damage lead to starvation, dehydration, and eventually death of the larvae.
• Degradation: After the larvae die, the Bt toxins and bacterial remnants are broken down in the environment, reducing the risk of further impact on non-target organisms.

Steps involved in insecticide resistant transgenic plants

• Identification of lead
• Isolation ad purification of protein
• Bioassay of isolated protein
• Testing of toxicity in mammals
• Gene transfer
• Selection of transgenic plants
• Evaluation of biosafety

Future Directions:

• Integrated Pest Management: Combining different resistance mechanisms to create crops with multi-faceted pest resistance.
• Gene Stacking: Incorporating multiple resistance genes into a single crop variety to broaden the spectrum of pest resistance.
• Environmental Considerations: Monitoring and assessing the ecological impact of transgenic crops to ensure their safety and effectiveness.

Question

1. How do protease inhibitors disrupt insect digestion and lead to insect mortality in transgenic plants?
2. What are the potential challenges in using protease inhibitors for crop protection?
3. How might insects adapt to overcome the effects of protease inhibitors, and how can scientists address this?
4. Why is it important to use both serine and cysteine protease inhibitors in developing insect-resistant plants?