Insect Resistant Transgenic Crops

Published Date: 02 Nov 2017

Abstract

World population is projected to increase well into the next century reaching 9.3 billion by 2050 and with limited arable land available; meeting the growing population’s needs is a hard task which requires accelerated progress for cost effective, sustainable yield increases. Arthropod pests are responsible for major damage to the world’s important agricultural crops reducing yield and acting as vectors of diseases. Insect attack resistant crops made through genetic engineering offer a different approach to pest control compared to using mostly chemical pesticides. Large varieties of transgenic crops which express proteins which confer insecticidal properties from Bacillus thuringiensis have been commercialised starting from the mid 1990’s and have assisted in increasing yields through their ability to kill phytophagous insects, protecting crops and increasing yields. Recently cases of resistance in insect pests to certain strains of B.thuringiensis toxins expressed in transgenic plants have occurred. Resulting in the need to identify novel resistance genes which can be compiled in plants to delay advances in insect resistance to the insecticidal products and widen the domain of pests affected, overall improving the global state of transgenic crops (Tabashnik et al. 2008).

Introduction

Food Security

The projected increase of the world’s 7 billion population rises well into the next century, reaching 9.3 billion by 2050 and a further 10.1 billion by 2100 (UN Population Division 2011). This increase will be primarily visible in developing regions of the world, with Asia staying the most inhabited major world area during the 21st century whereas Africa’s population will more than triple with it increasing through 1 billion in 2011 to 3.6 billion in 2100 (Fig. 1). With this population increase comes a greater drive and demand for food and the need for significant increase in food production and productivity to be able to achieve food security.

Feeding a world population in 2050 of 9.1 billion people has been projected to require food production increases or around 70% between 2005/07 and 2050. Also developing countries production would be required to almost double (Anon. 2009) (Fig. 2).

Figure 1. Projected changes in relative population growth, from 1950-2095.

Source: United Nations, Department of Economic and Social Affairs, Population Division (2011). World Population 2010, (Wall Chart).

. ST/ESA/SER_A/307

Figure 2. Targets of cereal production (left). Between 1961 and 2007 cereal production on a global scale has risen from 877 million tonnes to 2351 million tonnes. Rises in production will need to occur to figures of over 4000 million tonnes by 2050 if these forecasted demands are to be reached. Yield increase rate must increase by 37% to meet these demands.

Source: [Based on FAO data] – FAO world agriculture: toward 2030/2050. Interim report, global perspective studies unit (FAO Rome, 2006).

The question is how to tackle this much needed increase. The majority of production increases will arise from large cropping intensity increases and major yield advances with developing countries having to increase these 80 percent in comparison to the production increases from expansion of arable land at around 20 percent in developing countries (Anon 2009).

Closing the Yield Gap – The use of Biotechnology

Major Constraints on productivity arrive from factors such as plant disease, nutrient and land availability, and damage from pests. With the best locally obtained yields depending on the extent of farmers to use and access, things such as water for irrigation, the right seeds and nutrients, and pest management measures (Charles et al. 2010). This review will focus specifically on strategies’ incorporating transgenic plants to prevent damage by insect pests.

Of the minimal amount of arthropods which are crop pests, many of these induce harmfull implications on crops with destruction of world crop production being 14% causeing around "$100 billion of damage each year" (Ferry and Gatehouse, 2010). Current measures in place to reduce losses in crop yield due to insect pests is the use of synthetic insecticides which without use, would cause drastic losses in global crop yeild. However the major limiting factor on the use of insecticides to tackle crop pests is the increasing resistance of insects to insecticides with reports of resistance rising. There are also environmental and health concerns around insecticides with improper and unreasonable use of pesticides leading to outbreaks of pests due to the unintentional destruction of the pests natural enemies (Pimentel 2009). Thus other strategies are being investigated to address the global crop pest problem with one of these being the use of recombinant DNA technology.

Recombinant DNA technology is used to produce transgenic crops which have increased stress tolerance, biotic or abiotic (Blackburn, et al. 2005). These transgenic crops contribute a notable input into achieving greater food security whether by increasing resistance to disease, insect pests or even by increasing nutrient levels of the plant to enhance the probability of people meeting their dietary needs for a healthy life. Plants containing transgenes are often called transgenic or genetically engineered (GE) crops, however the reality is that all modern crops have been bred and engineered from their wild state by domestication, selection for preferred traits and controlled breeding through time. Currently the major commercialized transgenic crops have undergone simple manipulations to insert genes to benefit the plant for example genes for herbicide tolerance or pest-insect toxin. In the near future developments in combining desirable traits and new novel traits such as resistance to drought in plants will be brought about. However there are issues of public acceptance of biotechnology with differences in acceptance of genetically engineered plants paired to food production in Europe. Currently applications of biotechnology including those of genetic engineering is encouraged but there are some suspicions that application of biotechnological methods towards production of food could jeopardize modern agriculture and the health and safety of our food. However modern molecular biological methods present enormous prospects for expanding production and reducing risks in production of food. Therefore there is a demand for increased acceptance by the public in biotechnology before it can openly assist in improving global food security. This review focuses on insect resistant transgenic crops.

Figure 3. Global Map of Biotech Crop Countries and Mega-Countries in 2011. Source: James, Clive. 2011. Global Status of Commercialized Biotech/GM Crops: 2011. ISAAA Brief No.43. ISAAA: Ithaca, NY.

Insect resistant biotech crops

2011 was the 16th year of commercialisation of biotech crops with a 15 consecutive years of increase and an increase of 12 million ha at a growth rate of 8% from 2010-2011 reaching a record of 160 million ha. Biotech crops have been the fastest adopted crop technology in the history of modern agriculture to date with a 94 times rise in hectarage from 1.7 million in 1996 to 160 million in 2011 (James 2011). Out of all 29 biotech crop planting countries in 2011, 10 were industrial and 19 developing (Fig. 3) with developing countries growing near 50% of the global biotech crops. Adoption of biotech crops by trait sees herbicide resistance having the largest sector with 59% of the global crops compared to 15% of insect resistant trait crops. However it is the stacked gene approach which is the fastest growing area trait wise with 26% of global biotech crop coverage (James 2011). Of insect resistant biotech crops commercialised those expressing Bacillus thuringiensis (Bt) δ- endotoxins remain the leading and most successful insecticidal toxins

engineered into plants. Positive yield impacts for the use of biotech IR traits in the corn and cotton sectors have occurred in all countries using them (except genetically modified IR cotton in Australia) (Brookes and Barfoot 2012). The impact that these traits had on yield on average across the area planted from 1996-2010 is +9.96% for corn traits and +14.4% for cotton traits (Brookes and Barfoot 2012) (Fig 4).

Figure 4. Yield impacts on average for the effect of biotech IR traits between 1996-2010 by country and trait. IRCB= resistant to corn boring pests, IRCRW= resistant to corn rootworm. Source: Brookes, G. and P. Barfoot (2012). "The income and production effects of biotech crops globally 1996–2010." GM Crops and Food: Biotechnology in Agriculture and the Food Chain 3(4): 265-272.

The Bt Odyssey

Bacillus thuringiensis is a ubiquitous soil bacterium. The protein crystals it secretes are called Bt-toxins, δ- endotoxins or crystal (cry) proteins which are insecticidal in nature and are produced within its cells during sporulation. Most strains of the bacterium produce several cry-proteins, each of which shows a rather specific host range (Bravo et al. 2007). An example of this comes from the Cry1A, Cry1Ab and Cry1C genes which code for proteins of the same name which have a specific insecticidal spectrum to larval forms of lepidopteran insect pests for example the codling moth (Cydia pomonella), European corn borer (Ostrinia nubilalis)(Cry1A) or African stem borer (Busseola fusca)(Cry1Ab). Differently the CryA3 protein has an insecticidal spectrum to coleopteran pests an example of which is the Colorado potato beetle (Leptinotarsa decemlineata) (George et al. 2012; Bravo et al. 2007). Plants that expressed Bacillus thuringiensis insecticidal proteins were initially commercialized in the 1996 growing season (Bates 2005), and since then a large variety of crop plants have been genetically engineered so that they exhibit the δ- endotoxin gene. Engineering of these plants has been undergone to exhibit the active toxin in the plants tissues to the result that insects which feed on the crops are killed by the toxin.

There are two proposed models for the mechanism of action of cry proteins. The first being the pore formation model described in detail by Bravo et al (2007) whereby the cry proteins lead to the forming of lytic pores in apical membranes subsequently instigating cell lysis and causing insect death. The second is a more recently proposed alternative model which is called the signal transduction model. In this model cell death of the insect comes about without the formation of pores. The detail of the model proposed cited by Soberon et al (2009) "the toxicity of Cry proteins is due to activation of Mg+2-dependant signal cascade pathway which is triggered by interaction of monomeric 3d-Cry toxin with the cadherin protein receptor. This activates a guanine nucleotide-binding protein which in turn activates an adenylyl cyclase promoting the production of intracellular camp. Increase in camp levels causes protein kinase A activation which in turn activates an intracellular pathway resulting in cell death".

Transgenic crops with individual cry proteins expressed were the first of many commercial varieties available which had precise activity against insect pests of Lepidopterans pests, such as ‘MON 810’, a maize crop plant that has been modified developed by Monsanto Company with the trade name YieldGard. This MON810 variety of crop contains a Cry1Ac gene which when expressed is toxic to Lepidopteran insects such as the European Corn Borer.

More recent releases of Bt transgenic crops express cry protein encoding and vegetative insecticidal proteins (VIP) active against Coleoptera and Lepidoptera insects, and also sometimes HT genes via gene stacking. A recent commercialised example of this comes from Sygenta and their ‘Agrisure® Viptera 3111’ trait stack product which includes triple stacked HT traits, protects against 14- above and below ground insects with combination of Both vegetative insecticidal proteins (Vip3A) and cry proteins derived from B.thuringiensis (Sygenta, 2011). By expressing both VIP and Cry proteins, due to the difference in mechanism of action between the two, the durability of the cultivar is effectively extended by decreasing the likelihood of the insects becoming resistant. Xu et al (1996) established that cowpea trypsin inhibitor (CpTI) expression in rice plants improves resistance of the plant to two rice stem borer species. The study showed significant increases in resistance to the striped stem borer (Chilo suppressalis), and pink stem borer (Sesamia inferens) infestation. In 2000 a trait stacked cotton crop expressing Cry1Ac with CpTI was released in China was employed to improve protection representing the sole commercial development of proteinase inhibitors to date (Gatehouse 2011). This is another example of co-expression to reduce likelihood of resistance in insects to the cultivar.

Evolution of the Resistance

The continued progression in resistance to transgenic crops in insects jeopardises the prolonged success of B.thuringiensis toxin producing crops (Tabashnik 2008). The first documented occurance of field evolved resistance to a B.t toxin provoked by a transgenic crop is of Helicoverpa zea to Cry1Ac in transgenic cotton with significantly increased occurance of resistant alleles being found in field populations of H. zea (Tabashnik 2008)(Fig 5). Further examples of resistance to B.thuringiensis toxins produced by a transgenic crop come from western corn rootworm (Diabrotica virgifera virgifera) resistance to Cry3Bb1 maize (Gassman 2011). Furthermore field-evolved resistance in a major target pest, the cottong bollworm (Helicoverpa armigera) to Cry1Ac has been reported in northern China (Zhang, 2011). Despite laboratory bioassays having detected this resistance, resistance of these insects to Cry1Ac and Cry3Bb1 expressing cultivars hasn’t caused any broad pest control failures. These negative effects shown of Cry protein resistance should instigate reductions in the use of crops which produce only single toxins and progress towards crops which incorporate two or more B.thuringiensis toxins and other proteins and proteinase inhibitors such as VIP and CpTI.

Figure 5. Resistance in the field from the collective Bt crops planted worldwide from 1996-2007 (>200 million ha). Detected in lepidopteron species : Helicoverpa zea (bollworm), to Bt cotton producing Cry1Ac), Spodoptera frugiperda (fall armyworm) to Bt corn producing Cry1F, and Busseola fusca (stem borer) to Bt corn producing Cry1Ab. Tabashnik, B. E., et al. (2008). "Insect resistance to Bt crops: evidence versus theory." Nat Biotechnol 26(2): 199-202.

Currently the approaches to tackle resistance include the use of management strategies such as the refuge strategy (involves growth of non-Bt crops near the planted Bt crops). This has been shown to delay insect resistance evolution through heightening of the chance that resistant insects will mate with non-resistant partners, producing non-resistant offspring. Gene stacking of different B.t toxins or proteinase inhibitors is a strategy that confers elevated levels of pest control (Ferry et al. 2004). These proactive countermeasures combined have so far been successful in preventing widespread insect resistance to many insecticidal crops. However from evidence of the evolution of insect resistance to single B.thuringiensis toxin expressing crops in the field it has seemed that the refuge strategy alone, although postpones resistance for many years, is not enough keep evolution of resistance at bay.

By monitoring resistance of insects collected from biotech crop fields and DNA screening and incorporating gene stacking and refuge strategies, it should be possible to stay ahead of the curve within relation to insect resistance. However the investigation and search for genes conferring resistance to pests needs to be maintained to identify different genes which can be incorporated into plants to provide increases in the variety of plant pests that are effected and to postpone and prevent further insect resistance these gene products which confer resistane (Peferoen 1997; Ferry et al. 2004).

Future Strategies And Prospects

Potential of insect evolved resistance to transgenic crops is present and although methods to reduce this currently being implimented, alternative strategies to deal with insect pests are being developed.

i) Novel insecticidal molecules from bacteria

Future possibilities from bacterial origin a high molecular weight protein complex with insecticidal properties synthesised by Photorhabdus luminescens. Its been discovered that this toxin complex (Tca) is orally toxic to the Colorado potato beetle (Leptinotarsa decemlineata), and the sweet potato whitefly (Bemisia tabaci) biotype B (Blackburn et al. 2005). Description of toxins similar to those produced by P. luminescens have been found in Xenorhabdus nematophilus (Morgan et al. 2001). A study by ffrench-Constant et al. (2007) highlighted the fact that both Xenorhabdus and Photorhabdus have been found to be able produce multiple toxins with oral insecticidal activity. Another future prospective insecticide was discovered in several strains of B.thuringiensis which produce insecticidal proteins during the vegetative phase of growth. These proteins are called vegetative insecticidal proteins (Vip) and do not show any similarity to δ-endotoxins of B.thuringiensis. VIP’s possess toxicity of the same magnitude as that of B.thuringiensis δ-endotoxins against susceptible insects, with new Vips still being identified (Bhalla et al. 2005).

RNA interference (RNAi) has been considered as a technique for considerable potential for the regulation insect crop pests. Baum et al. (2007) and Mao et al. (2007) showed from bioassays of transgenic plants insects development can be inhibited and reduced damage to plants can be obtained by feeding of dsRNA produced by the plant, providing encouraging applications of this technology in the field of RNAi for regulation of insect pests.

ii) Plant-derived insecticidal molecules

At present, two main groups of genes giving rise to insect resistance have been established from higher plants which provide crops with insect resistance. These are lectins and proteinase and amylase inhibitors. The enzyme inhibitors inhibit digestive enzymes hindering digestion due to the inhibitory effects on insect gut digestive a-amylases and proteinases. Proteinase inhibitors provide plants with a natural endogenous defence system against insects by effecting their growth and development (Murdock and Shade 2002). Multiple insects have trypsin and chymotrypsin for use in digestion, and proteinase inhibitors mostly inhibit these enzymes by mimicking substrates (Haq et al. 2004). There are different classes of proteinase inhibitors. The four discovered are aspartic, cysteine, serine and metallo-protease inhibitors, with cysteine and serine proteinase inhibitors showing the most promising results in combat against Lepidoptera and Coleoptera. Proteinase inhibitors have already been expressed in plants and CpTI has been commercialised but further research could assist in improving crop resistance. A protective role by α-amylase inhibitors in seeds have also been found to enhance resistance against insect attack (Ishimoto and Chrispeels 1996). A recent promising study by Dias et al. (2005) revealed that an α-amylase inhibitor gene from rye seeds (Secale cereal) expressed in tobacco plants managed to induce development of resistance against cotton boll weevil (Anthonomus grandis), providing the grounds for rye inhibitor to be used in creation of biotech cotton plants to provide increased resistance to cotton boll weevil.

Lectins are reversible carbohydrate-binding proteins, which can bind to complex glycans or simple monosaccharides. (Peumans and Van Damme 1995) A few lectins have been displayed toxic activity against species In orders Homoptera, Coleoptera, Lepidoptera and Diptera (Schuler 1998). However most interest is mainly concentrated on snowdrop lectin, Galanthus nivalis agglutinin (GNA) due to being the first plant lectin demonstrating insecticidal activity against Hemiptera. It to high-mannose glycans specifically and it has been shown to have a limited detrimental effects on many features of insect life when expressed in plants such as reduced survival, reduced feeding and delayed larval development (Nagadhara et al. 2004; Mehlo et al. 2005). However it is the ability of GNA to transverse the insect gut epithelium and remain stable and active, as it is resistant to proteolytic activity, which allows use of it as a ‘carrier’ via creation of fusion protein molecules to deliver other peptides to the haemolymph by transporting them across the insect gut (Fitches et al. 2002).

iii) Fusion proteins as insecticidal molecules

Fitches et al. (2004) also demonstrated that GNA can be used to as a carrier protein, delivering insecticidal spider venom neurotoxin (Segestria florentina toxin 1:SFI1) to the haemolymph of lepidopteran larvae, causing 100% mortality to first stage larvae of tomato moth (Lacanobia oleracea) after six days. Fitches et al. (2012) also demonstrated the ability of GNA to mediate transport of ω-Hexatoxin-Hv1a peptide across the gut epithelium in lepidopteran larva but also the capacity for GNA to deliver HV1a to action sites within the insect central nervous system, significantly enhancing oral activity of insecticidal proteins. This outlines a potent way to deliver many natural insect selective toxins as novel biopesticides using the huge diversity of toxins from venom of organisms such as spiders, scorpions (Fitches et al. 2010) and sea anemones (Bosmans and Tytgat 2007), possibly providing a whole range of insect specific insecticidal crops.

Conclusion

A revolution in modern agriculture has come about. The increasing moving away from pesticides and the recent advances in genetic engineering has resulted in successful control of many pests of important food crops. The transgenic approach to tackling crop pests should allow increases in yield quality and productivity in an environmentally friendly manner. With the large variety of different novel insecticides possible due to the remarkable diversity of compounds with potential insecticidal activity there should be many different ways to prevent resistance, and if it does occur, the numerous number of compounds with insecticidal activity can be used to engineer new insect resistant crops.