Transgenic crop technology - Bt toxin.pptx

About Bt toxin - mechanism, advantages, limitations

1. Pest resistance - Bt toxin V. Dhanush Prabhakar II M.Sc Biotechnology 24BIOB002

2. Introduction Transgenic crop technology involves introducing specific foreign genes (transgenes) into plant genomes to provide beneficial traits such as pest resistance , herbicide tolerance, stress tolerance, or improved nutritional value. One of the earliest and most successful applications of this technology is pest resistance achieved through the expression of Bt toxin genes from Bacillus thuringiensis . Bt toxin is produced by Bacillus thuringiensis , a gram-positive, soil-dwelling bacterium. It forms crystalline inclusions during sporulation, called Cry (crystal) proteins or δ-endotoxins . These Cry proteins are highly specific insecticidal proteins that target certain insect orders such as: Lepidoptera (moths and butterflies) - cotton bollworm, armyworm Coleoptera (beetles) - corn rootworm Diptera (flies and mosquitoes)

3. Various Bacillus thuringiensis (Bt) strains generate over 25 distinct yet related insecticidal crystal proteins (ICPs) . These proteins are lethal to the larvae of numerous insects, including those that spread diseases and many agricultural pests. Cotton bollworms , which belong to the Lepidoptera order, are particularly vulnerable to Bt Cry I and Cry II proteins , which target them specifically. The Bacillus Genetic Stock Centre (BGSC) gene bank catalogs a range of Cry (crystal), Cyt (cytolytic), and Vip (vegetative insecticidal protein) genes , including synthetic and modified variants derived from B. thuringiensis . To date, 22 Cry classes encompassing 126 Cry genes have been documented, alongside one Crt gene and three Vip genes. Among these, Cry1Ac and Cry1Ab are the most widely and effectively applied in various crops. Fig 1. 3D structures of insecticidal toxins produced by Bacillus thurin gie nsis

4. Principle and Mechanism of Bt-Mediated Pest Resistance Gene Identification and Isolation Specific cry genes ( cry1Ac, cry2Ab, cry3Bb) are isolated from B. thuringiensis that encode the desired toxin effective against target pests. Genetic Transformation The cry gene is inserted into plant cells using: Agrobacterium tumefaciens - mediated transformation Gene gun (biolistics) method. These transformed cells are then regenerated into whole plants that express the Bt toxin in their tissues. Fig 2 . General schematic of GM crop production

5. Mechanism of Action of Three-Domain Cry Toxins in Lepidopteran Insects Research on Cry toxins has primarily focused on their effects in lepidopteran species. It is well-established that Cry toxins mainly disrupt the midgut epithelial cells of target insects by creating pores in the microvilli membranes lining the gut . Recent studies propose an additional mechanism where Cry toxins may trigger apoptosis through interactions with G-protein-coupled receptors (GPCRs) after binding to specific receptors. Initially, Cry proteins transition from crystal protoxins to membrane-inserted oligomers that induce ion leakage and subsequent cell destruction. When ingested by susceptible larvae, the crystal inclusions dissolve in the alkaline gut environment (around pH 8.5). The inactive protoxins are then activated by midgut proteases , which cleave them into stable, protease-resistant proteins weighing between 60 and 70 kDa. Fig 3. Mechanism of action of Bt toxin

6. Activation of the toxin occurs through proteolytic cleavage, which removes an N-terminal segment - ranging from 25 to 30 amino acids in Cry1 toxins, 58 residues in Cry3A, and 49 in Cry2Aa- and also trims about half of the protein's C-terminal region in the longer Cry protoxins. Once activated, the toxin specifically attaches to receptors located on the brush border membrane of the midgut's columnar epithelial cells, subsequently embedding itself into the membrane. The insertion of toxins triggers the creation of lytic pores within the microvilli of the apical membranes . This event leads to cell rupture and damage to the midgut epithelium, releasing cellular components that serve as a nutrient-rich environment for spore germination, ultimately causing severe septicemia (a critical bloodstream infection) and resulting in insect mortality. A notable aspect of Cry toxin activation involves the modification of the toxin's N-terminal region. Structural analysis of the Cry2Aa protoxin reveals that two α-helices at the N-terminus block a key area responsible for receptor binding. Additionally, research on a Cry1Ac mutant retaining its N-terminal segment after trypsin digestion showed it binds nonspecifically to membranes of Manduca sexta (Tobacco hornworm) but fails to form pores in the brush border membrane vesicles (BBMV) of M. sexta . Therefore, the cleavage and processing of the N-terminal end of Cry protoxins are crucial steps that enhance the toxin's ability to interact effectively with receptors or membranes. Fig 4. Relative length of Cry protoxins and position of protease digestion

7. Development of B. thuringiensis transgenic crops for insect resistance

8. Advantages of Bt Transgenic Crops Effective pest control : Provides season-long, built-in protection without external pesticide application. Reduction in chemical pesticide use : Minimizes environmental pollution and health hazards. Higher yield : Reduces crop losses caused by pests. Eco-friendly and target-specific: Does not harm beneficial insects like bees and ladybirds. Cost-effective: Lowers input costs for farmers due to reduced pesticide need.

9. Limitations and Concerns Insect resistance development: Continuous exposure can lead to evolution of resistant insect populations. Non-target effects: Possible impact on non-target organisms in the ecosystem. Gene flow : Risk of transgene transfer to wild relatives. Public perception and regulation: GMO concerns and strict biosafety laws in some countries. Resistance Management Strategies To delay resistance development in pests: Refuge strategy: Farmers plant non-Bt crops nearby to maintain a susceptible insect population. Gene stacking (pyramiding) : Combine multiple cry genes (cry1Ac + cry2Ab) for broad and durable resistance. Crop rotation: Helps reduce pest buildup over seasons.

10. Global Impact Bt crops have been widely adopted across the world, especially in: India (Bt cotton) USA (Bt maize and cotton) China, Brazil, Argentina, Philippines Studies show: Up to 50–80% reduction in chemical pesticide use. Higher yields and economic benefits for smallholder farmers. Environmental safety due to lower insecticide runoff.

11. Case study

12. Findings: The study assesses the field performance and resistance status after large-scale adoption of MON 87701×MON 89788 soybean (Intacta® RR2 PRO), which expresses the Bt Cry1Ac protein and glyphosate tolerance. Brazil adopted this technology in 2013/14 and by 2020/21 it covered ≈30 million ha (~80% of national soybean area). Primary pests: MON 87701×MON 89788 soybean remains highly effective against primary lepidopteran pests ( Chrysodeixis includens, Anticarsia gemmatalis, Helicoverpa spp., Chloridea virescens ). Phenotypic bioassays showed very high mortality (96–100%) of C. includens at the diagnostic Cry1Ac concentration. Genotypic F₂ screens found low and broadly stable Cry1Ac resistance-allele frequencies in C. includens (values ~0.0024–0.05 across seasons, with pooled estimates ≈0.003–0.01 by year). During 2020/21 the authors detected unexpected injury on Bt soybean from the “loopers” Rachiplusia nu and Crocidosema aporema . Bioassays showed that Brazilian R. nu populations had very high tolerance to Cry1Ac (LC₅₀ > maximum tested; estimated tolerance ratio >2,700 relative to C. includens ), and C. aporema populations also showed decreased susceptibility — evidence consistent with field-evolved resistance in these secondary pests. Bt soybean (MON 87701×MON 89788) continues to control the main soybean lepidopteran pests in Brazil after eight years, but localized field-evolved resistance to Cry1Ac has been documented in two secondary pests ( R. nu, C. aporema ). Continued IRM (refuges, IPM) is essential to preserve Bt efficacy.

13. References Abbas, M. S. T. (2018). Genetically engineered (modified) crops ( Bacillus thuringiensis crops) and the world controversy on their safety. Egyptian Journal of Biological Pest Control, 28(1), 1-12. Sanahuja, G., Banakar, R., Twyman, R. M., Capell, T., & Christou, P. (2011). Bacillus thuringiensis : a century of research, development and commercial applications. Plant biotechnology journal, 9(3), 283-300. https://www.jeb.co.in/journal_issues/200809_sep08/paper_01.pdf Thuringiensis toxin as a natural pesticide - Slideshare Bt Toxin as microbial insecticide - Slideshare Countries Approving GM Crop Cultivation - https://www.isaaa.org/blog/entry/default.asp?BlogDate=10/31/2024 Horikoshi, R. J., Bernardi, O., Godoy, D. N., Semeão, A. A., Willse, A., Corazza, G. O., ... & Head, G. (2021). Resistance status of lepidopteran soybean pests following large-scale use of MON 87701× MON 89788 soybean in Brazil. Scientific Reports, 11(1), 21323.

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