Abstract
Continuous increase in population has unbalanced the demand and supply of agricultural produce. In this scenario, food security in a sustainable manner is being challenged due to several factors. Insect pests are considered as one of the major factors, which accounts for 35–100% crop damage, worldwide. Synthetic insecticides contributed significantly, but several safety concerns are associated with them. Transgenic crops with enhanced biotic or abiotic stress tolerance have shown promising contribution in achieving greater crop productivity. Transgenic cotton expressing Cry toxin of Bacillus thuringiensis has tremendously increased the production as well as the societal status of farmers in our country. However, a concomitant increase in the population of minor pests like aphids, whiteflies and others has demanded certain new approaches. Researchers have isolated several other toxic proteins like lectins, protease inhibitors, amylase inhibitors, chitinases, and tried various novel approaches like gene pyramiding, tissue specific expression and modulation in metabolites expression to combat emerging problems of insect pests. Conversely, the emergence of a new type of crop insect pests demands more specific effort for each insect. Besides this, there are several safety and ethical concerns that are associated with the use of genetically modified crops, which also need to be resolved as per demand. Development of a dedicated scientific forum for the proper demonstration of advantages and disadvantages of genetically modified crops to the citizens at ground level might be useful in resolving the societal and ethical concerns in our country.
Keywords
- Insecticidal proteins
- Cry toxins
- Lectins
- Protease inhibitors
- Chinitase
- Transgenic crops
1. Introduction
World population is projected to increase over 1,000 million in the next four decades. An immediate priority for agriculture industry is to achieve maximum production in an environmentally sustainable and cost-effective manner. Food security is on high agenda at the political and social level [1]. Our progeny can face a severe shortage of food supply due to the over demand of continuously increasing population. Jaques Diouf, the Director General FAO, stated (2011) “
Transgenic crops with enhanced biotic or abiotic stress tolerance have shown promising contribution in achieving greater food security. A milestone was established about 25 years ago with the development of genetically engineered tobacco expressing the entomotoxic Cry protein from the bacterium
2. Insecticidal proteins
2.1. Cry toxins of Bacillus thuringiensis
Introduction of Bt-Cry toxins revolutionized the area of insect control through transgenic technology. These are toxic to the insects of orders Lepidoptera, Diptera, Hymenoptera, Coleoptera and also to nematodes. These are produced as parasporal crystalline inclusions in
Several insect-resistant transgenic crops have been developed by expressing Bt-Cry proteins, among which corn, cotton, soybean and canola are the most important crops. These transgenic crops are mostly expressing the Cry1Ac and Cry2Ab to control the chewing pests like
2.2. Lectins
Lectins are carbohydrate-binding proteins, which possess at least one non-catalytic domain for specific and reversible binding to mono- or oligosaccharides [22, 23]. A typical lectin is multivalent in nature, therefore agglutinate cells. Lectins are extensively distributed in nature from prokaryotes to eukaryotes. The specific interaction with glycoconjugates makes them valuable in biomedical sciences and biotechnology [24]. Carbohydrates present in viruses, microorganisms, fungi, nematodes or phytophagous insects interact with plant lectins [25, 26]. In the past decades, many plant lectins are reported to be toxic to several economically important insect pests of various orders [27–29]. To analyze the insecticidal properties under natural conditions, many transgenic plants expressing lectins have been developed. The toxic effects of different lectins have been demonstrated on several insect species; these effects range from a severe delay in development to high mortality in insects [11].
2.2.1. GNA-related lectins
GNA and related lectins have been successfully expressed for resistance against insect pests into a variety of crops [32]. Transgenic rice expressing ASAL caused significant mortality in nymph of hemipteran insect pests [33]. Onion (
2.2.2. Legume lectins
Legume lectins are purified from seeds and bind to carbohydrate structures like Thomsen-nouveau (Tn) antigen or complex N-glycan with terminal galactose and sialic acid residues. Pea lectin (
2.2.3. Hevein-related lectins
Hevein-related plant lectins exhibit specificity for chitin (chitin forms endo- and exo-arthropods, nematodes and fungi). These are also studied for insecticidal properties [45]. Due to the absence of chitin in mammals, hevein-related lectins are considered safe for the usage in genetically modified crops. Wheat germ agglutinin (WGA) has shown a negative effect on the development of the cowpea weevil (
2.2.4. Other insecticidal lectins
Several other plant lectins have shown insecticidal property. Transgenic tobacco plants expressing tobacco leaf lectin (NICTABA) is detrimental to the cotton leafworm (
2.3. Proteinase inhibitors
Proteinase inhibitors (PIs) are small molecular weight proteins which affect several metabolic pathways. They are the major components in seeds and storage organs of crops. Mickel and Standish [55] demonstrated the role of PIs in plant defense for the first time and noticed the abnormality in the development of larvae of certain insects fed on soybean products. The feature was attributed to trypsin inhibitors, and it was found to be toxic to the larvae of flour beetle (
PIs inhibit the digestion of proteins in midgut and cause mortality of insects due to nutritional imbalance [57, 58]. PIs also interfere with several metabolic processes (like moulting) by blocking the proteolytic activation of enzymes [59]. They affect growth and development, multiplication rate and insect life span [60–62]. PIs have been expressed in several transgenic plants for resistance against insect pests of several classes [63–65]. Pea and soybean trypsin–chymotrypsin inhibitors (PsTI-2, SbBBI) belonging to the Bowman–Birk family [66] and mustard-type trypsin–chymotrypsin variant Chy8 [67] cause significant mortality of pea aphid
The majority of plant PIs originate from three main families, namely Solanaceae, Leguminosae and Gramineae [70]. Plant PIs can be grouped into four classes: serine, thiol, metallo and aspartyl. Most plant PIs are inhibitors of microbial and animal serine proteases, such as chymotrypsin, trypsin, elastase and subtilisin [71]. Specificity of protease inhibitor families is mainly based on the amino acid residues present in the active site [72].
2.3.1. Serine (Serpin) protease inhibitors
It is found in almost all kingdoms of organisms [73–76]. Several serine PIs have been purified from plants and characterized [77, 78]. Plant serine PIs show inconsistent and varied specificities towards plant proteases [79].
2.3.2. Cysteine protease inhibitors
An inhibitor of cysteine proteinases was first described in egg white by Sen and Whitaker [86] and was later named cystatin [87]. Cysteine proteinases inhibitors are widely distributed in plants, animals and microorganisms [88]. Their role in defense has been explored by
2.3.3. Aspartyl protease inhibitors
It is relatively less studied class, due to the rare occurrence [91]. Potato tubers possess cathepsin D, an aspartic proteinase inhibitor which showed substantial amino acid sequence similarity with the soybean trypsin inhibitor [96]. Aspartic proteases have been found in coleoptera species, such as
2.3.4. Metallo-proteases inhibitors
The metallo carboxypeptidase inhibitors (MCPIs) have been identified in solanaceaous plants tomato and potato [99]. The MCPIs are 38–39 amino acid residues long polypeptide [100, 101]. Plants have evolved at least two families of metalloproteinase inhibitors, the metallo-carboxypeptidase inhibitor family in potato and tomato [102] and a cathepsin D inhibitor family in potato [103]. The inhibitor is produced in potato tubers and accumulates with potato inhibitor I and II families (serine proteinase inhibitors) during tuber development. The inhibitor also accumulates in potato leaf with inhibitor I and II in response to wounding and have the potential to inhibit all the major digestive enzymes (like trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B) of higher animals and many insects [104].
2.4. α-Amylase inhibitors
α-Amylases (α-1,4-glucan-4-glucanohydrolases) are hydrolytic enzymes, which catalyze the hydrolysis of α-1,4-glycosydic bonds in polysaccharides. They are present in microorganisms, animals and plants [105–107]. They are the most important digestive enzymes of many insects which feed exclusively on seed products. Inhibition of α-amylase impairs the digestion in an organism and causes shortage of free sugar for energy. α-Amylase inhibitors (α-AI) are found in many plants as a part of the defense system and abundant in cereals and legumes [108–111].
α-AI of
They are potential molecules for the development of insect-resistant transgenic plants [114, 115]. Seeds of transgenic pea and azuki, expressing α-AI-1 inhibitor of
2.5. Chitinase
Chitinases are being employed in plant defense in many ways. It has been used in controlling the growth of fungi and insects. Expression of poplar chitinase in tomato leads to growth inhibition in Colorado potato beetle [117]. Secretome analysis of tobacco cell suspension represents chitinase as the major defense protein [118]. A chitinase-like domain containing 56-kDa defense protein (MLX56) provides strong resistance against cabbage armyworm
Chitinases have also been isolated from insects and found to be equally promising in plant defense. Transgenic tobacco plants expressing chitinase of tobacco hornworm (
3. Insect-resistant transgenic crops
Development of many transgenic crops has been reported for insect resistance. Both private and public sector organizations are involved in the process and they used δ-endotoxins of
3.1. First-generation insect-resistant transgenic crops
Insect-resistant transgenic crops have not only increased the economy but also the environmental and health benefits [69, 124]. Six transgenic crops (canola, corn, cotton, papaya, squash and soybean) were planted in 2003 in the USA alone. These crops increased farm income by US$ 1.9 billion by producing an additional 2.4 million tonnes of food and fiber and reduced the use of pesticides by 21,000 tonnes.
In 2009, China government approved the cultivation of Bt-rice (the country has been growing Bt-cotton since 1997). Farm surveys of randomly selected households cultivating Bt-rice varieties have been performed. The benefit of Bt-rice has been acknowledged to the level of small and poor farmers, it is due to the lesser crop damage by the insects and therefore higher crop yields and less use of pesticides. An improved health has also been observed in Bt-rice cultivating farmers compared to non-Bt rice cultivating farmers [126]. Government of India approved the cultivation of Bt-cotton in 2003, which resulted in a 70% reduction in insecticide applications. This saves up to US$ 30 per ha in insecticide costs and results 80–87% increase in cotton yield [127]. A spectacular decrease in pesticide usage in Bt cotton fields has also been reported from China. The pesticide poisoning to the farmers reduced from 22% to 4.7% [128].
To assess probable hazards of Bt toxins on non-target insects, field evaluation was performed in Spain [129]. Bt-maize did not show negative impact on non-target pests. Similar numbers of cutworms and wireworms were present in Bt versus non-Bt fields. Surprisingly, higher numbers of aphids and leafhoppers were observed in Bt field.
3.2. Strategies for next-generation insect resistance
3.2.1. Engineering of Cry toxin by domains swapping
Most of the Cry toxins share common three-domain structure in activated form [130]. Domain I gets inserted into the target membrane and forms pore; domain II is associated with receptor binding and thus determines specificity, and domain III is also involved in receptor-binding specificity. It has been demonstrated in a couple of studies that hybrid Cry toxins exhibit higher toxicity. Domain III of Cry1Ac increased the efficacy of various other Cry1 proteins in Cry1–Cry1Ac hybrid [131]. Similarly, Singh et al. (2004) developed a hybrid toxin against
3.2.2. Plant-derived insecticidal lectins and protease inhibitors
Detail about lectins and protease inhibitors have been discussed in earlier section. Some other insecticidal roles are summarized here. Besides insecticidal potential, GNA and ASAL also serve as a carrier protein for other insecticidal peptides and proteins to the haemolymph of lepidopteran larvae. It has been demonstrated by feeding GNA-allatostatin and GNA-SFI1 fusions to the tomato moth
Lectins are reported to be insecticidal towards sap-sucking insects, where Bt-toxins are not effective. Transgenic tobacco expressing garlic (
Protease inhibitors (PIs) expressing transgenic plants are not as effective as Bt and insecticidal lectin expressing plants. This is due to the adaptation in gut proteases in phytophagous insects. High genetic diversity in gut proteases and low potency of protease inhibitors is responsible for such adaptation. The combination of inhibitors (potato PI–II and carboxypeptidase) is not enough to avoid the compensatory adaptation [68]. However, inhibitors like barley trypsin inhibitor [65], equistatin from sea anemone [138], other cystatins [139, 140] or use of multiple inhibitors [141] or combination of inhibitors and lectins [142] might also be useful to provide resistance against insects in transgenic plants.
3.2.3. Multiple insecticidal proteins containing transgenic crop
Second-generation Bt transgenic cotton [Bollgard II (Cry1Ac + Cry 2Ab) and Widestrike (Cry1Ac + Cry1F)] are developed to increase the level of resistance against cotton bollworm [143, 144]. It has also been demonstrated that the expression of three insecticidal proteins (Cry1Ac, Cry2A and GNA) into Indica rice control three major pests, rice leaf folder (
3.2.4. Tissue-specific or regulated expression
Insecticidal proteins are usually expressed under constitutive promoter for higher accumulation of the proteins. Although the constitutive expression has some advantages, tissue-specific or inducible expression is desirable under certain circumstances. Insect attacks epidermal cells first and therefore the expression of insecticidal proteins under epidermal cell-specific promoters can be a useful strategy. For example, CER6 is an epidermal cell-specific promoter responsible for the expression of an enzyme for cuticular wax production [150]. Similarly, phloem-feeding insects can be targeted by using phloem-specific promoter like PP2 promoter of pumpkin [151], rice sucrose synthase Rss promoter [152] and root phloem-specific promoter AAP3 [153]. Tissue-specific expression of several insecticidal proteins has demonstrated as a good potential for insect control in several studies. Phloem-specific expression of ASAL under promoter Asus1 protects tobacco against aphid,
3.2.5. Strategies to over express secondary metabolites
Secondary metabolites synthesized by the plants participate in a number of physiological and biochemical processes. Our group demonstrated that the over-expression of pectin methylesterase of
4. Conclusions and perspectives
Transgenic technology (especially Bt crops) has contributed significantly in increasing the crop production worldwide. The crops are protected from being damaged by insect pests. Certainly, this methodology provides an environmentally safe alternative for the synthetic pesticides. Further, it has also been proven to be useful in enhancing nutritional values of crops, improvement of stress tolerance and production of pharmaceutical proteins. Introduction of Bt cotton varieties in India has tremendously increased the yields of cotton and thereby profits to the farmers. Bt proteins are able to control the damage caused by Lepidopteran and Coleopteran insects, but not effective against sap-sucking Homopteran pests [8, 9]. Therefore, an unusual increase in the population of homopteran pests like whiteflies, aphids and leafhoppers on transgenic cotton has been reported [7]. Further, development of resistance in insects against toxins is also going to be a major point of concern, which might ultimately challenge the future of Bt crops. Some defense-related proteins like plant lectins, PIs and chitinases are reported to be toxic to various homopteran insect pests. However, several safety and societal concerns are raised from time to time. Further, there is non-availability of an effective and safe protein against several important and emerging insects, which need an
Acknowledgments
SKU acknowledges Department of Science and Technology-INSPIRE faculty fellowship.
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