Bacillus Thuringiensis Approach To Gm Crops Biology Essay

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Genetically Modified (GM) crops also known as transgenic crops are produced by genetically altering the plant genome through the integration of the desired gene by the application of biotechnological techniques. The 1st GM crop to be produced was the Flavr Savr tomato in 1994 in the United States followed by GM cotton, corn, soya bean, canola etc and it was approved to be the first commercialized GM food. Crops are genetically modified so as to possess certain desirable traits such as insecticide resistance, herbicide or virus resistance (Morin, 2008). Currently, the insects or pests pose a major threat to most of the crops globally resulting in decrease in the production rate and yield of the crops. Usage of synthetic pesticides has caused severe environmental effects and adverse health effects in human and other organisms (Ranjekar et al, 2003). This has been overcome by the introduction of gene manipulation, one such being the development of insecticidal toxins such as the Cry protein from the bacteria Bacillus thuringiensis (Bt). Generation of transgenic plants with the gene of interest can be done in a short period and significantly contributes to the food industry (Ignacimuthu et al, 2000). Many countries have already adopted the Bt technology. For e.g. the North America have already developed transgenic crops (de Maagd et al, 1999).

The major strategies so far have been employed to genetically engineer pest resistance in crops plants:

The use of Bacillus thuringiensis approach and its based genetic modification in plants (which includes the regulation of cry genes under the control of a promoter and terminator) .

Copy nature strategy.

Bacillus thuringiensis approach:

Bacillus thuringiensis is a gram positive, spore forming bacteria possessing a genome size ranging from 2.4 to 5.7 million base pairs. Different strains of B.thuringiensis produce different cry proteins which will be susceptible to certain insects (Schnepf et al, 1998).

Table 1: Example of important Bt-transgenic crops (Ranjekar et al, 2003)

Most of the Cry proteins structure has 3 domains: Domains I, II and III. Domain I is present at the N-terminal region and consist of a series of α-helices ordered in a cylindrical form (figure 1). This form is accountable for the pore formation in the mid gut of the larvae. Domain II is a triple β - sheet and it functions in identifying the receptors. Domain III is a β sandwich of 2 anti-parallel β-sheets which are involved in playing vital roles such as receptor binding and also ion-channel activity (Schnepf et al, 1998).

Fig 1: (a) Diagrammatic representation of primary structure of B.thuringiensis cry protein. The full length of the protein with the number of amino acids indicated below. Grey pattern- cleavage of the N and C terminus end by protease in the mid gut of insects.(b) 3 dimensional structure of cry protein. Blue color- Domain I which is involved in pore formation. Green color- represents Domain II involved in receptor identification. Red color- Domain III which is involved in receptor binding (de Maagd et al, 1999).

Mechanism of action:

The cry toxins are generally produced as pro-toxins. When leaves expressing cry protein is ingested in by the larvae, the pro-toxin gets solubilised and activates the toxin which is mediated by the protease present in the gut epithelium (figure 2). The toxin then interacts with the cadherin receptor causing oligomerization of the toxin and whicn further interacts with AminoPeptidaseN (APN) receptor resulting in pore formation. This causes osmotic lysis of mid gut epithelium cells leading to death of the larvae (Schnepf et al, 1998).

Fig 2: Mechanism of action of cry protein in the mid gut of the larvae (This is the work of Dr. Juan Luis Jurat-Fuentes from the university of Tennessee titled 'Characterisation of cry toxin mode of action',

For a transgenic plant to express high level of the cry protein, a cry gene construct is required which requires plant promoter and terminator sequence.

Rice is the most important global food crop consumed by most of the people around the world. As stated by Cheng et al, 1998, infestation of insects on rice, leads to global loss in the production of rice with an estimate of 10 million tonnes. Engineering the cry protein into have shown to be toxic to leptidopteran, dipteran and colopteran insects. The major pest which destroys the crop are striped stem borer and yellow stem borer. Studies have shown that the production of transgenic rice expressing cryIA(b) or cryIA(c) by the method of particle bombardment were very low (Nayak et al, 1997). Hence, application of the modified method of Agrobacterium - mediated transformation has resulted in large scale (approx 2600) regeneration of transgenic rice with the modified cry genes. Such plants showed high level of expression of cryIA(b) and cryIA(c) rendering itself a potent candidate as transgenic plant. Chemical re-synthesis of the sequence coding for the cry toxin by optimization of codon usage, was placed along with maize ubiquitin promoter, CaMV 35S promoter, Bp10 gene promoter (pollen specific promoter) and the nos terminator. 4Bt gene vectors (pKUB, pKUC, pKSB and pKBB) were generated as a result of insertation of the chimeric gene into the Hind III region of the pKHG4 vector (figure 3).

Fig 3: Schematic representation of T-DNA of pKBB, pKSB, pKUB and pKUC which was constructed by incorporating various Bt gene in the Hind III site of pKHG4 (binary vector). BR- Right border, BL- Left border, HPH- Hygromycin phosphotransferase, NPTII- Neomycin phosphotransferase, cryIA(b) and cryIA(c) - cry proteins from B.thuringiensis, P35S- CaMV 35S promoter, Pubi- maize ubiquitin promoter, Pnos- promoter of nopaline synthase, pBp- Bp10 pollen gene promoter, NT- 3' terminator of nopaline synthase (Cheng et al, 1998).

Non transgenic plants expressing low detectable levels of toxin was served as negative control. Experiments carried out showed that the larvae feeding on the plants expressing cryIA(b) and cryIA(c) was susceptible to death reaching mortality of 97% within 5 days after infestation. The level of toxins expressed in these plants was found to be 0.23-0.31%.

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Source: Cheng et al, 1998)

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Genetically engineering a plant with a fusion protein is also another alternative strategy in developing resistance against harmful insects. Mehlo et al, 2005 explains the use of fusion protein containing the combination of δ-endotoxin cry1Ac and the galactose binding domain of non toxic ricin B-chain (RB). This fusion protein termed as BtRB provides more binding domains with the Bt gene at the N-terminal and the RB gene at the C-terminal. Studies have shown that N-acetyl galactosamine residue is a vital component of Bt- toxin binding receptors. Ricin B subunit is a galactose and N-acetyl galactosamine specific lectin and binds with high affinity. RB is also capable of delivering highly toxic ricin A chain. This fusion protein allows binding to a wide range of receptors compared to cry1Ac alone. Increasing the number of binding domains reduces the chance of insect resistance toward the toxin. The BtRB construct and the control genes (unmodified cry1Ac and RB genes) were cloned in pAL76 (ubiquitin promoter based transformation vector). The callus of maize and rice was bombarded with vector featuring selectable marker 'bar' coding for phosphinothricin resistance and 'hpt' marker for hygromycin resistance respectively. Stem borer (C. suppressalis) survival was greatly reduced by 60-90% upon feeding leaves expressing BtRB protein whereas in leaves expressing cry1Ac protein alone, survival was reduced by 20-30% (As shown in figure 5) .

Fig 5: Bioassays representing the effect of BtRB fusion protein on the survival and development of Stem borer (C.suppressalis) in (a) transgenic maize and (b) transgenic rice. WT- Wild type plants (control) (Mehlo et al, 2005).

Cotton leaf worm (S. littoralis), which was generally susceptible to Bt protein, showed significant mortality > 78% on feeding the BtRB expressed leaves. In contrast, with regards to the control plants, the larvae death was observed to be less than 20% (Figure 6). Also, BtRB was found to be toxic to the homopteran pest leaf hopper (C. mbila) which is generally not affected by any Bt toxin. Hence the application of fusion protein such as BtRB in development of transgenic crops against the infestation of insects is a very promising strategy to the global agriculture of the future (as shown in figure 7).

Fig 6: Effect of BtRB protein on survival and development of cotton leaf worm (S.littoralis) in transgenic maize. WT- Wild type non transformed plants, BtRB -ve (negative control), BtRB +ve (plants with BtRB protein). Vertical axis- mean survival of larval (Mehlo et al, 2005).

Fig 7: Effect of BtRB protein on survival and development of leaf hopper (C.mbila) on transgenic maize. Significant difference between BtRB and other protein at P< 0.001 (Mehlo et al, 2005).

Transforming plants with hybrid Bt cry protein can be employed to fight against pest attack. Fusion of cry1Ba and cry1la result in SN19, a hybrid gene, which when expressed in potatoes, was found to be toxic to colopteran ( and lepidopteran insects. SN19 contained domain I and III of cry1Ba and domain II of cry1la and was under the control of chrysanthenum-1,5-bisphosphate carboxylase/ oxygenase small subunit (RubiscoSSU). Cloning of two expression cassette pSN28 (RBC unmodified SN19) and pSN46 (RBC modified SN19) into pBINPLUS vector resulting in two binary vectors namely pSN32 and pSN48 which was incorporated by electroporation into Agrobacterium tumifaciens. Plants with empty pBINPLUS vector were served as negative controls. The transgenic plants (expressing SN19 gene) were tested for resistance from CPB (Colorado potato beetle, Leptinotarsa decemlineata) larvae, CPB adult, PTM (Potato tuber moth, Phthorimaea operculella) larvae and ECB (European corn borer, Ostrinia nubilalis) larvae. Experiments conducted showed that expression level of SN19 of about 0.25% was achieved resulting in 80% mortality which would give complete plant protection from these pests (Naimov et al, 2003).

Fig 8: Insect bioassays between control plants (a-d) and leaves expressing SN19 gene (e-h). a and e- CPB larvae, b and f- CPB adults, c and g- PTM, d and h- ECB (Naimov et al, 2003).

The advantage of implementing Bt into crop plants are: (i) no negative impact on biodiversity, positive impact in reduced dependence of pesticides and (ii) no evidence of allergenic reaction induced in humans (Mehlo et al, 2005).

Copy Nature Strategy:

The Copy Nature Strategy involves the use or exploitation of the plant natural resource which serves the purpose of anti- insecticidal property. In nature, plants possess a wide range of proteins which helps in the defense mechanism againt insect infestation (Ranjekar et al, 2003). It involves the exploitation of various protease inhibitors, plant lectins,

De Leo et al, 1998 demostrated the effectiveness of a trypsin proteinase inhibitor MTI-2 on Spodoptera littoralis larvae in transgenic Tobacco and Arabidopsis plant. Mustard trypsin PI2 (MTI-2) which was extracted from the white mustard seeds, exhibited similar pattern of expression comparing to the Proteinase Inhibitor (PI) in potato and tomato, which was known to play an important role in plant protection against insects infestation. The gene construct consisted of a 35S Promoter, the Mti-2 gene, Pnos and Tnos gene encoding the promoter and the terminator of Nopaline synthase which was cloned into a pKY-MTI-2 plasmid carrying the kanamycin resistance gene for the selection of the transformant.

Fig 9: Diagrammatic representation of T-DNA of pKY-MTI-2 vector. Pnos- Nopaline synthase promoter, Tnos- Nopaline synthase terminator, mti-2 - sequence coding MTI-2, P35S2- 35S RNA promoter and double enhancer, trbcS- pea RUbisco terminator, LB- left border, RB- right border, nptII- neomycin phosphotransferase. Arrows- direction of transcription of nptII and mti-2 genes (De Leo et al, 1998).

On transferring the gene via Agrobacterium tumifacienes mediated transformation, high level of expression of the MTI-2 inhibitor had adverse effect causing high mortality rate of the larvae and less damage to the leaves. Lower expression of MTI-2 in the leaves resulted in rapid increase in body mass of the larvae and more damage to the leaves due to increase in proteolytic activity whose actual cause is still unknown but could be due to induction of new proteinases in the gut epithelium of the larvae. Significant differences are seen between the proteolytic activity in larvae fed leaves of high levels of MTI-2 and low levels of MTI-2 indicating presence of sensitivity threshold. Below threshold, the larvae develops resistance to inhibitors due to over-expression of proteinases and above threshold, ingestion of PI bring about a tolerable environment leading to induction of new proteinases.

Table 2: Common transgenic plants expressing plant protease inhibitor (Lawrence and Koundal, 2002).

Looking at India's agricultural production, cotton is the most important cash crop and also provides income to the Indian farmers. In the current scenario, to protect their crops from pest infestation, continous application of pesticides has resulted in emergence of pest resistance to pesticides and causing a major impact on the plant. Though most of the insects so far seen in the previous literature are susceptible to Bt plants, the sucking type homopteran pest unfortunately doesn't get affected by the toxin. Moreover, due to their regular habit of sucking the sugars and amino acids from the plant sap, they cannot be controlled by protease or amylase inhibitors. The Red Cotton Bug, upon feeding the leaves and the green bolls of the cotton plant, transmit a bacteria Nematospora gosypii and the affected bolls result in decreased quality of lint, low oil content from the seeds and impaired germination of the seeds. The lectin agglutins from 3 plants namely the bulbs of Allium sativum (ASA), the tubers of Colocasia esculenta (CEA) and the leaves of Dieffenbachia sequina (DEA) was isolated and purified using affinity chromatography techniques. An artificial diet supplemented with ASA, CEA and DEA of varying concentration was given to the bugs and after 48 hours, it was observed that ASA showed high mortality of 77.8% followed by CEA (51%) and DEA (43%) as shown in the figure 10 (Roy et al, 2002).

Fig 10: Insect bioassays between 3 lectins of 20 µg/ml supplied into artificial diet (Roy et al, 2002).

Fig 11: Percentage insect mortality versus time in hours. Artificial diet supplemented with 3 lectins at concentrations 5, 10, 15 and 20 µg/ml (Roy et al, 2002).

Even at varying concentrations, ASA has proved to be an efficient inhibitors in integrated pest management.

Although application of Bt technology not only provide unique advantages in reduction of insect infestation on plant crops, it has its own disadvantages. Especially with regards to evolution of pest developing resistance to transgenic crops which at present, is a major challenge to the agricultural sector. Implementation of integrated pest management has been developed which has been helpful in combating the pest.

4 possible strategies for managing pest resistance towards the plant crops are:

Expression of toxins to a level not affecting all the non- target susceptible individuals.

Expressing different toxins in different plants.

High-dose refugia strategy.

Gene pyramiding strategy (expressing multiple toxins in transgenic plant).

Of the above, refugia and gene pyramiding strategy can provide a ray of hope in overcoming pest adaptation towards transgenic plants (Zhao et al, 2003).

Refugia involve growing transgenic plants expressing high dose of Bt protein adjacent to a refuge of non-transgenic crops that generate susceptible insects upon feeding. Cross breeding between homozygous dominant genotype (resistant) with homozygous recessive genotype (non resistant) result in generation of heterozygous resistant progeny dependable only on non Bt crops. Adoption of Refugia allows high expression of Bt protein thereby eliminating susceptible and heterozygous resistant insects (Manyangarirwa et al, 2006).

Gene pyramiding adopts the concept of using more than two toxins such than multiple insects can be targeted and it serves useful purpose rather than a single toxin gene. E.g. Bollgard II cotton contains two toxin namely cry1Ac and cry2Ac. Cry1Ac is toxic to tobacco budworm and pink bollworm whereas cry2Ac is toxic to corn earworm (Manyangarirwa et al, 2006).


In the earlier decades, farmers were majorly dependent on synthetic pesticides which on continous application, created serious adverse effects such as interference of chemicals with the plant metabolism process, soil infertility, increased insect infestation due to evolution of resistance etc. Currently, special concerns go into the agricultural sector. With increase in the global population, there are large numbers of mouths to be fed thereby causing strain to the natural resources. With the introduction of technology, development of various molecular biology techniques has rendered good service in the field of biotechnology. The introduction of Bt technology have helped to eliminate many insect infestation thereby providing the plant with complete protection. This was only possible due to the different molecular biology techniques currently available. As seen above, different strategies were implemented to genetically engineer insect resistance into plant crops. To date, Bt toxin (cry protein) have proved to be a potent candidate in controlling insect infestation. Though they are beneficial in their way, certain challenges were posed to the agricultural sector such as evolution of insects resistant to Bt toxin. Different integrated pest management strategies have evolved which have been incorporated into plants so as to delay the resistance in insects. Adoption of Bt technology in the current agricultural practices can definitely help in improving the production of major food crops thereby assuring the future generation a better world, a better tomorrow.