Bacillus thuringiensis crystal proteins for insect management
Rohini Sreevathsa*, Monika Dalal, Basavaprabhu L. Patil and Subodh Kumar Sinha
National Research Centre on Plant Biotechnology, Pusa, New Delhi-110012
*Corresponding Author E-mail: rohinisreevathsa@rediffmail.com

Introduction
Bacillus thuringiensis is a gram-positive soil bacterium and produces toxins during sporulation that are proteinaceuos crystalline inclusion bodies. A range of well characterized insecticidal proteins or Bt toxins have been identified that belong to many subspecies and serotypes of Bt. Over 60,000 isolates of Bt are being maintained in culture collections worldwide. Known Bt toxins kill insects belonging to the orders Lepidoptera, Coleoptera, Diptera (Hofte and Whiteley, 1989) and nematodes (Feitelson et al., 1992). Insecticidal δ-endotoxins of B. thuringiensis (Bt) have acquired considerable importance because of their specificity to target insects, toxicity at very low concentrations and environment friendly nature (Kumar et al., 1998). Primarily, Bt toxins are classified based on homology of toxin gene sequences and the spectrum of insecticidal activity (Hofte and Whiteley, 1989). Crickmore et al. (1998) introduced a systematic nomenclature for classifying the cry genes and their products based on the homology of amino acids of full length gene products. Eleven Lepidoptera-specific δ-endotoxins have been reported towards H. armigera, an important polyphagous pest on cotton, chickpea, pigeonpea, tomato, sunflower, sorghum, etc. (Chakrabarti et al., 1998).

Mode of action of the Bt toxin
The crystalline protoxins are inactive, until they are solubilized by the gut proteases (Tojo and Aizawa, 1983; Milne and Kaplan, 1993). The protoxins are cleaved in to toxins by trypsin like proteases in the alkaline pH of the insect mid gut, In general, 500 amino acids from the C terminus of 130 kDa protoxins and 28 amino acids from the N terminus are cleaved leaving a 65 to 55 kDa protease resistant, toxic active core comprising the N terminal half of the protoxin (Hofte and Whiteley, 1989). The active toxin consists of three distinct structural domains. Domain I (7 α-helices) determines toxicity and pore formation; Domain II (3 β-sheets) determines receptor binding and specificity whereas domain III (2 α- sheets), is involved in receptor binding and protein processing (Schnepf et al., 1998). The active toxin binds to specific receptors located on the apical brush border membrane of the columnar cells in the midgut of target insect. The toxicity of Bt lies in the organization of α-helices derived from domain I.The α -helices penetrate the membrane and lead to formation of pores (ion channels). The toxin induced pores formed in the columnar cells allow rapid fluxes of ions leading to swelling of the cells and osmotic lysis. There is a positive correlation between toxin activity and ability to bind BBMV (Brush Border Membrane Vesicles) (Gill et al., 1992), and the toxicity is correlated with receptor number rather than receptor affinity (Rie et al., 1989). The disruption of gut integrity leads to death of the insect through starvation or septicemia (Sneh and Schuster, 1981; Salama and Sharaby, 1985).

Transgenic crops with Bt genes
In 1995, the US Environmental Protection Agency (EPA) approved the first registration of Bt potato, corn and cotton crops. The first to reach the market was Monsanto's New Leaf potato variety expressing cry3A, swiftly followed by two transgenic corn hybrids expressing cry1Ab to protect against the European corn borer, i.e. KnockOut by Syngenta (Basel, Switzerland) and NatureGard by Mycogen (both containing Event 176). Monsanto also released the cotton varieties Bollgard and Ingard (Events 531, 757 and 1076) expressing a modified cry1Ac toxin. Two additional Bt corn varieties expressing cry1Ab were released shortly thereafter, namely Agrisure CB by Northrup King (Event Bt11) and the widely discussed YieldGard variety (Event MON 810) by Monsanto. This marked the beginning of the technology.
The demand for a technical solution to the bollworm problem was quite intense in India. Bollgard cotton was the first commercial Bt hybrid cotton sold in India for insect control. Indian Bt varieties were developed by a licensing agreement between Monsanto India Ltd. and the Maharashtra Hybrids Seeds Co. (Mahyco), who bred the resistance gene into locally adapted Indian germplasm. These hybrids were released to farmers and planted on approximately 24,000 ha in 2002 (James, 2012). The use of Bt cotton continues to increase in India, with hectarage expanding to nearly 10.8 million hectares in 2012 making it the fourth country to grow large areas of the Bt crop.

Bt GM crops are protected specifically against European corn borer, south western corn borer, tobacco budworm, cotton bollworm, pink bollworm and the Colorado potato beetle. Other benefits attributed to using Bt include:
• Reduced environmental impacts from pesticides - When the plants are producing the toxins in their tissues there is no need to spray synthetic pesticides or apply Bt mixtures topically.
• Increased opportunity for beneficial insects - Bt proteins will not kill beneficial insects.
• Reduced pesticide exposure to farm workers and non-target organisms.

Increasing the efficiency of Bt toxins by novel approaches
Various strategies have been developed to increase the efficiency of the toxins as well as for resistance management (Gatehouse 2008; Pardo-Lopez et al. 2013). These include:
1. Using multiple cry toxins to generate transgenic crops, i.e., pyramiding toxins.
2. Combining domains from different Cry toxins (Domain swap) and developing chimeric Cry proteins with novel specificities.
3. Mutagenesis of three-domain Cry toxins to increase toxicity toward target pests.
4. Development of fusion proteins, that is a gene construct containing a single translationally fused coding sequence encoding two Cry proteins.

However, major impetus is on the insect management strategies outlined by the regulatory authorities. Proper implementation of these strategies will help farmers exploit the utility of the toxins to the fullest for crop improvement.

References
1. Chakrabarti, S. K., Mandaokar, A. D., Kumar, P. A. and Sharma, R. P. 1998, Synergistic effect of cry1Ac and cry1F delta endotoxins of Bacillus thuringiensis on cotton bollworm, Helicoverpa armigera. Curr. Sci.,75: 663-664.
2. Crickmore, N., Zeigler, D. R. Feitelson, J., Schnepf, E., Rie, J. V., Lereclus, D., Baum, J. and Dean, D. H., 1998, Revision of the nomenclature for Bacillus thuringiensis cry genes. Microbiol. Mol. Biol. Rev., 62 (3): 807-813.
3. Gatehouse, J. A. 2008. Biotechnological prospects for engineering insect-resistant plants. Plant Physiol. 146: 881-887.
4. Gill, S. S., Cowles, E. A. and Pietrantonio, F. V., 1992, The mode of action of Bacillus thuringiensis endotoxins. Ann. Rev. Ent., 37: 615-636.
5. Hofte, H. and Whiteley, H. R., 1989, Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev., 53:242-255.Feitelson et al., 1992
6. James, C. (2012) Global Status of Commercialized Biotech � GM Crops: 2012. ISAAA Brief 44. Ithaca, NY: ISAAA
7. Kumar, P. A., Mandaokar, A., Sreenivasu, K., Chakrabarti, S. K., Bisaria, S., Sharma, S. R., Kaur, S. and Sharma, R. P., 1998, Insect-resistant transgenic brinjal plants. Mol. Breed., 4:33-37.
8. Milne, R. and Kaplan, H. 1993, Purification and characterisation of a trypsin like digestive enzyme from spruce budworm (Christoneura fumiferana) responsible for the activation of delta-endotoxin from Bacillus thuringiensis. Insect Biochem. Mol. Biol., 23: 663-673.
9. Pardo-López, L., MunËÅ"oz-Garay, C., Porta, H., Rodrıglmazán, C., Soberón, M. and Bravo, A. 2009. Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis. Peptides 30: 589-595.
10. Rie, J. V., Jansens, S., Hoftey, H., Degheele, D. and Mellaert, H. V, 1989, Specificity of Bacillus thuringiensis -endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects. European J. Biochem., 186: 239-247.
11. Salama, H. S. and Sharaby, A., 1985, Histopathological changes in Heliothis armigera infected with Bacillus thuringiensis as detected by electron microscopy. Insect Sci. Appl., 6: 503-511.
12. Schnepf, E., Crickmore, N., van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R. and Dean, D. H., 1998, Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev., 62: 775-806.
13. Sneh, B. and Schuster, S., 1981, Recovery of Bacillus thuringiensis and other bacteria from larvae of Spodoptera littoralis Boisduval previously fed on B. Thuringiensis treated leaves. J. Invert. Pathol., 37:295-303.
14. Tojo, A. and Aizawa, K., 1983, Dissolution and degradation of delta-endotoxin by gut juice protease of silkworm, Bombyx mori. Appl. Environ. Microbiol., 45: 576-580.

About Author / Additional Info: