Global challenges faced by engineered Bacillus thuringiensis Cry genes in soybean (Glycine max L.) in the twenty-first century

Abstract

The most important insect pests causing severe economic damages to soybean (Glycine max L.) production worldwide are Chrysodeixis includens (Walker, Noctuidae), Anticarsia gemmatalis (Hübner, Erebidae), Helicoverpa gelotopoeon (Dyar, Noctuidae), Crocidosema aporema (Walsingham; Tortricidae), Spodoptera albula (Walker, Noctuidae), S. cosmiodes (Walker, Noctuidae), S. eridania (Stoll, Noctuidae), S. frugiperda (Smith; Noctuidae), Helicoverpa armigera (Hübner, Noctuidae), H. zea (Boddie; Noctuidae) and Telenomus podisi (Hymenoptera,Platygastidae). Despite the success of biotech Bacillus thuringiensis (Bt)/herbicide tolerance (HT)-soybean in the past decade in terms of output, unforeseen mitigated performances have been observed due to changes in climatic events that favors the emergence of insect resistance. Thus, there is a need to develop hybrids with elaborated gene stacking to avert the upsurge in insect field tolerance to crystal (Cry) toxins in Bt-soybean. This study covers the performance of important commercial transgenic soybean developed to outwit destructive insects. New gene stacking soybean events such as Cry1Ac-, Cry1AF- and PAT-soybean (DAS-81419-2^®, Conkesta™ technology), and MON-87751-7 × MON-87701–2 × MON 87708 × MON 89788 (bearing Cry1A.105 [Cry1Ab, Cry1F, Cry1Ac], Cry2Ab, Cry1Ac) are being approved and deployed in fields. Following this deployment trend, we recommend herein that plant-mediated RNA interference into Bt-soybean, and the application of RNA-based pesticides that is complemented by other best agricultural practices such as refuge compliance, and periodic application of low-level insecticides could maximize trait durability in Bt-soybean production in the twenty-first century.

Introduction

Soybean (Glycine max L.; Merrill; Fabaceae: Phaseoleae; 2n = 40) is a native crop of East Asia which gained importance for its nutritional attributes and was introduced in Africa by the Chinese in the late nineteenth century and is now cultivated worldwide (Shurtleff and Aoyagi 2013). Commercial soybean expressing crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) is thereafter referred to as Bt-soybean. Prior to global commercialization of Bt-soybean, harmful insects caused the destruction of 20–30% of the world’s crops annually (Estruch et al. 1996). Today, farmers have steadily gained interest in growing genetically modified (GM) soybean either because of their herbicide-tolerant potential or insecticidal activities. For instance, biotech soybean occupies 83% (i.e., 92.1 million hectares) of the total 111 million hectares of soybean farmed globally (James 2015; ISAAA 2016; Marques et al. 2017), of which majority (> 80%) is the Bt-soybean, such as the MON 87701 × MON 87788 hybrid (Monsanto 2012). Soybean is endowed with physiologically active metabolites such as tocopherols, lecithins, isoflavones, saponins, and proteins (Sugan 2005; Yamada et al. 2012). It is a cheap source of nutrition and plays an active role in disease prevention in animals (Sugan 2005; Yamada et al. 2012). Daily multipurpose usage of soybean and the increase in oil demand compelled an increase in soybean farming (Hartman et al. 2011).

The steady increase in human population obliges the agricultural sector to diversify the methods of crop production to match global food demand. Given that the world’s population is predicted at 9.2 billion by 2050, farmers are further compelled to produce an extra 200,000 billion calories per annum (Evans 1998; Bebber and Sarah 2015). This means increasing global acceptance of GM-crops is imminent. Intriguingly, farming indigenous varieties via traditional methods failed to match population demand for soybean and profits for farmers due to damages caused by phytopathogens and harmful insects. Efforts to improve indigenous varieties of soybean began mainly via marker-assisted breeding (MAB) and transgenic approaches (Parrot et al. 1994; Dufourmantel et al. 2005; Cregan et al. 1999; Fast et al. 2015; Ortega et al. 2016; Marques et al. 2017). Nevertheless, the latter approach gained prominence over the former because of the ease and short span of time required to release new hybrids with desired agronomic traits. Despite the preference for transgenic approach to improve plant qualities and agronomic characteristics over classical breeding, the recalcitrance of many countries to accept transgenic food due to perceived safety issues limited the frequency at which biotech soybeans enter the market. Key techniques for development of biotech soybean has been the cotyledonary node Agrobacterium-mediated transformation and somatic embryo particle bombardment-mediated transformation (Yamda et al. 2012).

Globally farmed Bt-soybean such as “Roundup-Ready” cultivar (MON 87701—expressing Cry1Ac protein for insect resistance × MON 89788—glyphosate tolerance for herbicide tolerance) has helped solve some specific problems but exposes the crop to phytopathogens and new emerging destructive insects. This is a major unforeseen problem for the global production of Bt-soybean prompting rethinking the strategies to better enhance the resistance of Bt-soybean to destructive insects. Currently, the most cultivated Bt-soybean expresses Cry1Ac, Cry1Ab, Cry1AF, and phosphinothricin acetyltransferase (PAT) proteins. In this study, we search the literature for recent performances of Bt-soybean vis-à-vis targeted harmful insects, evaluate the impact of emerging insect resistance to Cry-protein, and provide a blueprint for gene pyramiding aimed at enhancing Bt-soybean production in the twenty-first century.

Global predictions of commercialized soybean by 2030 reveal upsurge in output

In USA alone, studies in Arkansas, Mississippi, North Carolina, and Tennessee showed that losses caused by bean leaf beetle Cerotoma trifurcata (Forster; Coleoptera: Chrysomelidae) ranges from 3 to 20% (Musser et al. 2014, 2015, 2016, 2017). Despite the deployment of glyphosate-resistant soybean in the field to improve weed management, yield losses associated with early-season weed competition was estimated at 9.3 and 3.1% across 30 fields in 2008 and 40 fields in 2009 in Wisconsin, USA, respectively (Fickett et al. 2009) and a net loss in Bt-soybean worth $16 billion between 2007 and 2013 (Dillie et al. 2016). While current statistics on soybean losses is absent, the United States of America (USA) Department of Agriculture estimated that global soybean production in 2017/2018 will be 351 million metric tons (USDA 2017). As of 2017, we found that there are 37 soybean genetically modified events approved by the International Service for Acquisition of Agri-Biotech Applications (ISAAA; http://www.isaaa.org/gmapprovaldatabase/) indicating the resolve to maintain the current production trends of soybean. We found that Bt-soybean will be predominantly produced by 2050, and that global production is anticipated at 311.10 and 371.30 million metric tons in 2020 and 2030, respectively (FAOSTAT2012). Based on predictions, the rate of soybean production in Argentina is likely to surge by 4.5% from 2010 to 2020 and by 2.8% from 2020 to 2030 (Fig. 1a). It is envisaged that Argentina would have an overall output of 108.4 million metric tons by 2030 (Masuda and Goldsmith 2009; FAOSTAT 2012). Thus, Argentina is expected to become the top soybean grower, contributing 29.2% of the world’s soybean output. Based on this prediction, USA would become the second largest producer (with 99.50 million metric tons) and Brazil the third (with 94.80 million metric tons) of soybean by 2030. Consequently, about 90% of the world’s soybean output by 2030 mostly GM-soybean will be supplied by Argentina, USA, Brazil, China, while India will be supplying high-yielding conventional soybean (Fig. 1a). The world’s total farmed land occupied by soybean would increase to 140.90 million hectares by 2030, which is 1.5 times larger than the area harvested between 2005 and 2007. This could trigger a serious unforeseen global challenge to the production of other crops due to insufficient acreage for farming.

Fig. 1.

a World soybean production forecast summary. The production growth rate is disaggregated into area harvested and yield growth rates. We generated the graph based on prediction data generated in Masuda and Goldsmith (2009) and FAOSTAT (2012). At present, India is only producing high-yielding conventional soybean. b Emergence of destructive fall armyworm (Spodoptera frugiperda) observed in Bt-soybean farms in Vanderbijlpark, Gauteng, South Africa in 2017

According to Food and Agricultural Organization (FAOSTAT2012), Argentina and Paraguay by 2030 would have increased GM-soybean cultivation areas by 31.4 million hectares and 5.0 million hectares, respectively. Regardless of the rising output of GM-soybean, global acceptance still tends to be low, although Bt-soybean does not show any substantial damage to animals, environment, and human health (James 2010, 2015). The major obstacle in acceptance of transgenic soybean is the lack of awareness and the fact that government regulations in some African, European and Asian countries do not permit the commercialization of transgenic plants.

Global profile of destructive insect pest hampering soybean production

We found that fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae J.E. Smith) which was mainly restricted to the South American continent has invaded Africa, especially Bt-soybean farms in South Africa (Fig. 1b). Over the years, some insect species belonging to the order Lepidoptera, Coleoptera, Hemiptera and Orthoptera have been discovered to be the major soybean pests (Grossi-de-Sá et al. 2011). Soybean farming suffers from caterpillars and stink bugs contributing to severe yield losses worldwide (Gamundi and Sosa 2008). Even so, the most important stink bug is Euschistus heros (Hemiptera, Pentatomidae) endemic to South American countries causing severe damage to soybean pods, and it is difficult to control because of its resistance to different insecticides (Sosa-Gomez and Silva 2 010). The most common insects affecting soybean production includes Chrysodeixis includens (Lepidoptera, Noctuidae), Anticarsia gemmatalis (Lepidoptera, Erebidae) (Bernardi et al. 2012), Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) (Azambuja et al. 2015) and the black armyworm, Spodoptera cosmioides (Lepidoptera: Noctuidae) (Silva et al. 2016). Ecological concerns, health hazards and increase in the insect’s population have led to the progressive preference of biological control methods such as the farming of Bt-soybean.

Types of δ -endotoxins engineered in commercialized soybean

It is observed that since the first isolation of Bacillus thuringiensis (Bt) in Japan by Ishiwata (1901) from silkworm larvae and the subsequent discovery of insecticidal δ-endotoxins crystal proteins (Cry), Bt has been applied as oil-based formulations, granules, and powders to curb insect grazing. This is because Cry-protein kills insect species at the larval stages such as those of the order Lepidoptera (specifically targeted by Cry1Aa, Cry1Ab, Cry1Ac, Cry2Ab), Diptera (specifically targeted by Cry4Aa, Cry4Ba), and Coleoptera (specifically targeted by Cry3A, Cry4b and Cry8Ea) (Ferre and van-Rie 2002). This eco-friendly strategy offers effectiveness in selective elimination of insect chewing pest and acts within a narrow range, but rapidly breakdowns in nature and thus, reduces dependence on chemical insecticides. Advances in genetic engineering led to the development of Bt-soybean to overcome the problem of conventional Bt-insecticide, which is exposed to biodegradation and wash-off. Moreover, specificity of Cry-protein in Bt-soybean greatly reduces insecticide use (Palma et al. 2014), lower production cost, minimizes exposure of lower hazardous chemicals into the environment as well as to humans and animals (Loguercio et al. 2001). The Bt Cry-proteins specifically bind to receptors of the gut epithelial cells by inserting into the target membrane and forming pores (Raymond et al. 2010) and this triggers rapid fluid loss from insect gut, prolongs developmental time, decreases growth rate, reduces the body mass and size of insects, and causes mortality (Lang and Otto 2010). To date, Cry1Ac, Cry1AF, Cry1F, Cry2Ab2, Cry1A.105, and Cry1Ab (Tables 1, 2; Fig. 2) are the most used Cry proteins in Bt-soybean.

Table 1.

List of GM-soybean engineered with Cry-protoxin either by particle bombardment (PB) or Agrobacterium (AG)-mediated transformation

Introgressed gene	Target tissue	Promotors	Transformation method	Effect	References
Insecticidal crystal protein (Cry1Ab)	Whole plant	CaMV35S with an alfalfa mosaic virus leader sequence	PB	Resistance to velvetbean caterpillar (Anticarsia gemmatalis)	Parrot et al. (1994)
Insecticidal crystal protein (Cry1Ac)	Whole plant	CaMV35S	PB	Resistance to A. gemmatalis, corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens)	Stewart et al. (1996)
Insecticidal crystal protein (Cry1Ab)	Plastid	16SrRNA promoter, Nicotiana tabacum Prrn (np)	PB	Resistance to A. gemmatalis	Dufourmantel et al. (2005)
Insecticidal crystal protein (Cry1Ab)	Whole plant	Nicotiana tabacum Prrn (np) + bacteriophage T7 of gene 10L	PB	Resistance to A. gemmatalis	Dufourmantel et al. (2007)
Insecticidal crystal protein gene (Cry1Ac), Agglutinins gene from Pinellia ternata (PTA)	Whole plant	CaMV35S	PB	Resistance to cotton bollworm (Helicoverpa armigera)	Dang and Wei (2007)
Insecticidal crystal protein (Cry1Ac; MON 87701), glyphosate (MON 89788)	Whole plant	35S RNA from figwort mosaic virus (FMV) for Cry1Ac, CaMV35S for EPSP	AG, then traditional breeding of two genetically modified parental lines	Chrysoperla spp., H. zea, tobacco burworm (Heliothis virescens), Hypena scabra	(Monsanto, 2012)
Insecticidal crystal protein gene Cry1AC, Cry1F and PAT proteins, and PAT confers tolerance to the herbicide glufosinate	Whole plant	DAS-81419-2 (Conkesta™ technology) powered by polyubiquitin 10 (UBQ10) gene promotor	AG	Target Elasmopalpus lignosellus, Agrotis ipsilon and Helicoverpa armigera and specific for lepidopteran pests	Fast et al. (2015)

Table 2.

List of Bt-soybean engineered by stacking Cry genes to counter insect resistance and approved in different countries as from November 2017

Event code and developer	Stacked genes introduced	Product of engineered genes	Function-engineered genes	Method of trait introduction	Country approved as from 2017	Targeted insects
MON 87701 × MON 89788 Monsanto Company	Cry1Ac	Cry1Ac delta-endotoxin	Confers resistance to lepidopteran insects by selectively damaging their midgut lining	Conventional breeding—cross hybridization and selection involving transgenic donor(s)	Argentina, Brazil, China, Colombia, European Union, India, Japan, Mexico, Paraguay, Philippines, South Africa, South Korea, Taiwan, Turkey, Uruguay	Lepidopteran insect resistance
	Cp4 EPSPS (aroA: CP4)	Herbicide-tolerant form of 5-enolpyruvulshikimate-3-phosphate synthase (EPSPS) enzyme	Confer increase tolerance to glyphosate herbicide
DAS81419 Dow AgroSciences LLC	Cry1Ac	Cry1Ac delta-endotoxin	Confers resistance to lepidopteran insects by selectively damaging their midgut lining	Agrobacterium tumefaciens-mediated plant transformation	Argentina, Australia, Brazil, Canada, Japan, Mexico, New Zealand, South Korea, Taiwan, USA	Lepidopteran insect resistance
	Cry1F	Cry1F delta-endotoxin	Confers resistance to lepidopteran insects by selectively damaging their midgut lining
	PAT	Phosphinothricin N-acetyltransferase (PAT) enzyme	Eliminates herbicidal activity of glufosinate (phosphinothricin) herbicides by acetylation
MON 87751 Monsanto Company	Cry1A.105	Cry1A.105 protein which comprises the Cry1Ab, Cry1F and Cry1Ac proteins	Confers resistance to lepidopteran insects by selectively damaging their midgut lining	Agrobacterium tumefaciens-mediated plant transformation	Australia, Canada, Colombia, Japan, Mexico, New Zealand, South Korea, USA, Taiwan	Lepidopteran insect resistance
	Cry2Ab2	Cry2Ab delta-endotoxin	Confers resistance to lepidopteran insects by selectively damaging their midgut lining
MON 87751 × MON 87701 × MON 87708 × MON 89788 Monsanto Company	Cry1A.105	Cry1A.105 protein which comprises the Cry1Ab, Cry1F and Cry1Ac proteins	Confers resistance to lepidopteran insects by selectively damaging their midgut lining	Conventional breeding—cross hybridization and selection involving transgenic donor(s)	Taiwan, South Korea	Lepidopteran insect resistance
	Cry2Ab2	Cry2Ab delta-endotoxin	Confers resistance to lepidopteran insects by selectively damaging their midgut lining
	Cry1Ac	Cry1Ac delta-endotoxin	Confers resistance to lepidopteran insects by selectively damaging their midgut lining
	Dmo	Dicamba monooxygenase (Dmo) enzyme	Confers tolerance to the herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) by using dicamba as substrate in an enzymatic reaction
	Cp4 EPSPS (aroA:CP4)	Herbicide tolerant form of 5-enolpyruvulshikimate-3-phosphate synthase (EPSPS) enzyme	Confer increase tolerance to glyphosate herbicide

Fig. 2.

Molecular analysis of two Cry-protoxins (Cry1Ac and Cry1Ab) commonly introgressed in Bt-soybean. A Molecular phylogenetic analysis by maximum likelihood (ML) method and bootstrap values ≥ 50% from 1000 iterations are shown and the ML tree was rooted as previously described (Louis et al. 2014). b Three-dimensional structure of Cry1Ac (GenBank accession KKB28329.1) and CryAb (GenBank accession AEV45790.1) protoxins modeled in Phyre2 server at 90% accuracy at intensive mode. c Overall view of the functional domain of Cry1Ac or Cry1Ab

Current field performance of Cry genes in Bt-soybean and insect tolerance

We found reports that when truncated Cry1A(b) was introduced into a soybean line that is partially resistant to defoliating insects, the T₁ plants deterred feeding, development, and survival of velvetbean caterpillar Anticarsia gemmatalis Hübner (Parrott et al. 1994), but unfortunately it never entered the regulatory and commercial pipeline. Developed in China, the first Bt-soybean expressing Cry-protein with resistant insect trait was introduced into the commercial and regulatory pipeline in January 2009 (Stein and Rodríguezo 2009). By 2013, Monsanto stacked MON 87701 (expressing Cry1Ac for insect resistance) and MON 89788 [glyphosate tolerance for herbicide tolerance (HT)] in the same soybean (codename: MON 89788 × MON 87701) which were then entered in phase III trials in Brazil (Stein and Rodríguezo 2009) and eventually became the first commercial Bt-soybean in Brazil in 2010 and Argentina in 2012 (Bortolotto et al. 2014). Recently, the deployment of Bt-soybean DAS-81419-2 (Conkesta™ technology) expressing Cry1Ac, Cry1F, and phosphinothricin acetyltransferase (PAT) proteins in Brazil have shown high efficiency in resistance against Lepidoptera insects (Fast et al. 2015; Marques et al. 2017).

Prior to commercialization of MON 8788 × MON 87701, fertile Bt-soybean engineered with synthetic Cry1Ac was developed (Cregan et al. 1999). Here, Cregan et al. (1999) identified two quantitative trait loci (QTLs; 229-H and 229-M) from native Japanese soybean lines conferring antibiosis and antixenosis resistance against Lepidopteran insects. By pyramiding these QLTs (229-H and 229-M) with synthetic Cry1Ac enhanced the plant defense against destructive insects (Cregan et al. 1999). Antibiosis is a resistance phenomenon whereby a plant has a detrimental effect on insect growth, development, and reproduction (Painter 2005). On the other hand, antixenosis is a resistance phenomenon whereby plant affects insect behavior by discouraging oviposition, colonization or feeding (Kogan and Ortman 1978). It was observed that Bt-soybean that constitutively expresses stacked genes viz., Cry1Ac and 229-M generates strong insecticidal activities against larvae of Lepidopteran’s—Heliothis virescens, Helicoverpa zea, and Pseudoplusia includens (Walker et al. 2004). Interestingly, Bt-soybean over-expressing synthetic Cry1A significantly reduced Lepidopteran population densities notably, A.gemmatalis, P. includens, and Hypena scabra under field conditions (Mcpherson and Macrae 2009). Using Bt-soybean (MON 87701 × MON 87788 expressing Cry1Ac protein), Bernardi et al. (2012) found high effectiveness against C. includens (Lepidoptera, Noctuidae) and A. gemmatalis (Lepidoptera, Erebidae), yet, this Bt-soybean failed to show activity against stink bugs E. heros or its egg parasitoid Telenomus podisi (Hymenoptera, Platygastidae) under laboratory conditions (Silva et al. 2014). We equally observed that during the outbreak of fall armyworm (S. frugiperda) in South Africa Bt-soybean (MON 87701 × MON 87788; Fig. 1b) was ravaged. Given the polyphagous nature of S. frugiperda and E. heros, this calls for the development of other Bt-soybean with other Cry genes.

In vivo climatic condition alters the performances of Cry genes in Bt-soybean

From the literature search, we identified that a major problem associated with Cry in Planta is the low expression levels that can go below 3 ng/mg of soluble protein in plant generations succeeding T₀ plants. This is because native Bt-proteins contain regulatory sequences and require tRNAs not found in plants, or Cry mRNA form secondary structures that hinder proper translation (Perlak et al. 1990). Also, the expression of transgene and the overall phenotype of the plants are not stable (Parrott et al. 1994). Another key issue affecting the performances of Bt-crops is the variations of Cry-proteins as a function of plant variety, plant age, environmental temperature, relative humidity, light and soil properties (Kranthi et al. 2005; Rochester 2006; Addison and Rogers 2010; Chen et al. 2011; Ranjith-kumar et al. 2010; Hagenbucher et al. 2013). Additionally, it was noted under field conditions that the concentration of Cry1Ac protein was higher in Bt-soybean leaves than in any other tissues but drops during anthesis and gradually rebounds (Yu et al. 2014). This indicated that changes in climatic events can alter Bt-soybean’s global defense against destructive insect and thus, affect farmers’ output.

Though the majority of biotech soybean farmed globally is MON 87701 × MON 87788 expressing Cry1Ac, it failed to affect the development and reproduction of black armyworm S. cosmioides (Silva et al. 2016) as well as fall armyworm (S. frugiperda) (Fig. 1b). Intriguingly, Silva et al. (2016) suggested that MON 87701 × MON 87788 is a good host for the development of S. cosmioides and allows for more than 80% of larvae-to-adult survival. Thus, black armyworm S. cosmioides is a major unforeseen menace for global Bt-soybean industries should there be an outbreak. The control of destructive insects by planting Bt-soybean (MON 87701 × MON 87788) could trigger interspecific competition and prompt an upsurge in the population density of polyphagous insects such as black armyworm and fall armyworm.

Divergence in the interaction dynamics of Cry proteins reduces insecticidal potential

Due to the functional complementarities between Cry1Ab and Cry1Ac and their common use for controlling polyphagous insects, we retrieved their sequences in NCBI database, and check for their phylogenetic and structural differences. The sequence set was filtered at default mode for redundancy using ElimDupes (available at http://hcv.lanl.gov/content/sequence/ELIMDUPES/elimdupes.html). From the unique sequence set, the Akaike Information Criterion, corrected (AICc) and Bayesian Information Criterion (BIC) were determined. A maximum likelihood phylogenetic analysis was performed using MEGA 6.1 software (Tamura et al. 2013). The structure of Cry1Ab and Cry1Ac were model using The Phyre2 web portal at default mode (Kelley et al. 2015).

Based on the comparative phylogenetic placement of the most used Cry proteins (Cry1Ac and Cry1Ab) in soybean, the best substitution method was JJT, AICc was 9252.90, BIC was 9541.13 and the highest log likelihood was− 4587.32. The maximum likelihood tree revolved the independent evolution of Cry1Ac and Cry1Ab (Fig. 2a), yet they function by damaging the midgut lining of targeted insects. Although Cry1Ac and Cry1Ab evolved independently, Cry1Ac (in group “a”) showed more diversity hallmarked by singleton AANO7788.1, AHK23266.1 and ADH94040.1. It is worth noting that independent evolution of three domains (I, II and III) and swapping of domain III between different Cry proteins is responsible for the varieties of specificities (De-Maagd et al. 2001; Kumar and Kumari 2015). Structural analysis revealed strong resemblance at main functional domains (I, II and III; Fig. 2b) for Cry1Ac (Genbank: KKB28329.1) and CryAb (Genbank: AEV45790.1).

It was shown that Cry1Ac protein in cotton do not have an effect on Spodoptera insects (Greenberg et al. 2010) and could be due to natural high tolerance of these insects to Cry1 protein (de Maagd et al. 2000). Interestingly, Cry-protein domain I creates pore in epithelium midgut, while domains II and III are involved in receptor recognition and binding (de Maagd et al. 2000, 2003) and contribute to osmotic cell death (Zhuang et al. 2002; Pardo-Lopez et al. 2006). Additionally, domain III structurally resembles the carbohydrate-binding domain 6 (CBM6; Fig. 2b). However, the interaction dynamics of Cry-protein with Spodoptera differs. It was shown that domain III of Cry1C can function as a specific determinant for Spodoptera exigua, but not in all pyramid Cry1-Cry1C combinations (de Maagd et al. 2000). Based on this, it would not be unreasonable to hypothesize that an unexpected mutation in domain III of Cry1Ac could be fatal for Bt-soybean susceptibility to destructive insects. Incontrovertibly, the midgut of Cry-resistant mutant insect larvae are able to tolerate very high doses of protein because of the following: (1) down-regulation in the expression of Cry-protein receptors such as aminopeptidases, alkaline phosphatase and cadherin-like proteins, (2) decrease in the quantity of proteases as well as increase in the amount of esterases and glycolipids such that the level of lethal free and active toxin is non-optimal, and (3) suppression in the expression of ATP-binding cassette transporter C2 (Griffitts and Aroian 2005; Kumar and Kumari 2015). Interestingly, instead of mutations in the Cry, four Cry1Ac-resistant mutations were discovered from Pectinophora gossypiella and found to be recessive and were later mapped onto the cadherin locus (Morin et al. 2003; Fabrick and Tabashnik 2012). This indicates that in an event of concomitant mutations in Cry genes and mutations in insect gut epithelial cell receptors (such as aminopeptidase, alkaline phosphatase and cadherin-like proteins) could compromise the effectiveness of Cry-protein even if the Cry-protein is highly expressed in the Bt-crop.

Cohort of factors that favors the emergence of insect tolerance to Bt-soybean

A fundamental problem affecting the performance of Cry1Ac-crops against key destructive insects such as S. frugiperda and S. exiguaare (1) poor binding to the midgut (Aranda et al. 1996), and (2) endogenous secretion of insect proteases to deactivate Cry1Ac (Miranda et al. 2001; Rahman et al. 2012). Often intercropping of Bt-crops such as soybean and maize could exert selective pressure on polyphagous feeders such as H. armigera that is distributed in Asia, Africa, Europe, and Australia (Tay et al. 2013) and accelerates the emergence of resistant insects. This is supported by a report of H. armigera resistance to Bt-cotton expressing Cry1Ac protein in Australia (Gunning et al. 2005).

Critical for Cry-protein optimal insecticidal activity, it should be overexpressed at all stages of plant growth that coincide with the occurrence of insect pests at the vulnerable stage. Equally, abiotic factors may cause a decrease or fluctuation in the total soluble proteins and negatively influences insecticidal activity. For instance, Luo et al. (2008) showed that the combination of waterlogging and salinity stress on Cry1Ac-cotton inhibited nitrogen metabolism which reduces the production of total soluble protein and reduces insecticidal potential. Also, higher temperature is known to degrade total soluble protein by 47–55%, thus, decreasing the insecticidal activity of Cry1Ac expressed in cotton (Chen et al. 2005). This may explain in part why Cry1Ac does not sufficiently protect Bt-soybean from fall armyworm (S. frugiperda), Southern armyworm (S. eridania), and the velvet armyworm (S. latifascia) (Bernardi et al. 2014). Bt-soybean the MON 87701 × MON 87788 bearing the Cry1Ac is the most farmed, indicating a wide range of the target and non-target insects that are continuously exposed to this protein. Thus, there is a high likelihood of insects evolving tolerance to Cry1Ac. This could be the case for H. armigera resistance to Cry1Ac-cotton in Australia (Gunning et al. 2005).

Higher temperatures in growing area at high latitudes lead to a longer growing season and this subsequently leads to an increase in pest and disease pressure (Haverkort and Verhagen 2008). Climatic changes could also trigger unfavorable epigenetic DNA changes. For instance, DNA methylation of promotor led to reduce late-season expression of Cry-protein (Müller-Cohn et al. 1996; Dong and Li 2007) and this phenomenon could increase the likelihood of broad insect resistance in regions prone to heavy and unseasonal rains, increased humidity, drought, cyclones and hurricanes, and warmer monsoon temperatures. At present, Bt-soybean based on Cry1Ac, and Cry1Ab are widely cultivated and hypothetically, extensive farming of these hybrids could accelerate the development of resistance since Cry1Ab and Cry1Ac show high-level structural and functional similarities (Fig. 2).

Blueprint in integrated gene pyramiding strategy to counter insect resistance challenges

Current trends in soybean protection revealed that resistance to destructive insects hugely relies on coupling multiple endogenous quantitative resistance traits in succession to achieve the desired trait as a core part of integrated pest management strategy (Fig. 3; Table 2). To avoid insect evolving resistance to Cry1Ac-soybean commonly farmed, gene pyramiding consisting of coupling more than one Cry in a transgenic plant is encouraged (Perlak et al. 1990; Monsanto 2012; Ortega et al. 2016). For instance, pyramiding in Bt-soybean (MON 89788 × MON 87701) solves at least two problems (Monsanto 2012), but reliance on only one Cry-protein cannot guarantee complete elimination of all soybean destructive insects.

Fig. 3.

a Modification of Ortega et al. (2016) depicting the strategy of pyramiding QTLs resistance with Cry1Ac-protoxin to control various soybean chewing insects. Here, Benning^{M−E−Cry1Ac} was found to be more resistant than Benning^ME and Benning^Cry1Acagainst soybean looper Chrysodeixis includens (Walker) and Southern armyworm Spodoptera eridania (Cramer). b Cry was coupled with artificial microRNAs to specifically target pests

Gene pyramiding in plants such as soybean that are recalcitrant to transform is challenging (Joshi and Nayak 2010; Ortega et al. 2016), although marker-assisted selection (MAS) and single nucleotide polymorphism (SNP) has eased the process of selecting the desire trait (Fig. 3; Table 2). Combining dominant resistance traits offers broad spectrum resistance to multiple destructive insects of soybean. For example, Ortega et al. (2016) used soybean line PI 229358 and PI 227687 that harbors broad range resistance to chewing insects. PI 229358’s resistance is conferred by QTLs: M, G, and H. PI 227687’s resistance is conferred by QTL-E, where the letters indicate linkage groups on which the QTL are located, plus the incorporated Cry1Ac-protein to soybean-Benning^{M−E−Cry1AC} (Ortega et al. 2016; Fig. 3a). It was found that Benning^{M−E−Cry1Ac}was more resistant than Benning^ME and Benning^Cry1Acagainst soybean looper C. includens and Southern armyworm (Spodoptera eridania). In fact, the rationale behind the success of gene pyramiding depends on the genetic background of the host lines to ensure inheritance of the desired traits. For instance, PI-227687 is resistant to defoliating insects and PI-229358 exhibits antibiosis and antixenosis (Rector et al. 2000a, b) and thus, stacking more traits enhances defense against destructive insects. It would be interesting to observe the field performance of Cry1Ac-, Cry1AF- and PAT-soybean (DAS-81419-2^®, Conkesta™ technology; Fast et al. 2015) bearing multiple stack genes. Furthermore, we proposed that engineering a soybean with inherent multiple resistant QTLs with multiple Cry-proteins whose product bind to different receptors in the same insect is an important prospective to effectively counter insect resistance challenges. While Cry is widely accepted in soybean, we proposed that incorporating RNA interference (RNAi) technology into transgenic soybeans could complement the Bt-soybean hybrids already compromised by insect-evolved field tolerance. In this vein, the use of artificial microRNAs designed to be constitutively expressed and target midgut proteases of insects could ensure durability of already developed Bt-soybean (Fig. 4).

Fig. 4.

Important δ-endotoxins crystal proteins such as Cry1 (~ 130 kDa specifically target butterflies and beetles) Cry2 (~ 70 kDa specifically target butterflies and Diptera), Cry3 (~ 70 kDa specifically target beetles), and Cry4 (~ 130 kDa specifically target Diptera) (Palma et al. 2014; Ibrahim and Shawer 2014). Coupling PMRi with stacked Cry genes could better protect Bt-soybean against destructive insects

Currently, QTLs/Cry-protein applied in soybean is expressed in the whole plant (Cregan et al. 1999; Ortega et al. 2016), whereas insects feed mostly on the soybean leaf and pods. It was suggested that Cry-protein should be expressed in the chloroplast genome since it offers the possibility of gene transfer to pollen (Daniell 2000) and high-level toxicity in the leaves. Therefore, transforming chloroplast genome provides high dosage of Cry-protein/mg leaf consumed and offers a more effective tool of controlling the destructive insects. With advances in DNA delivery method, Dufourmantel et al. (2005) transformed soybean plastid with Cry1Ab (Table 1) and found a strong resistance against velvetbean caterpillar (A. gemmatalis) a difficult insect to control. Plastid transformation offers multiple advantages such as (1) specific placement of the transgene, (2) sub-lethal dosage to leaf-chewing insect are avoided, (3) since plastids genes are inherited via the mother ovary only, the likely risk for out-crossing transgenes to other species in the same biota is greatly reduced (Mikkelsen et al. 1996; Ibrahim and Shawer 2014), and (4) high copy number of transgene is achieved since DNA placed in the chloroplast will be copied 5000–10,000 times in a cell, while a typical genomic DNA will produce 1–4 copies per cell. Thus, plastid transformation offers a direct and rapid approach for controlling destructive insects of soybean. In this light, Bt-soybean developers should consider chloroplast transformation for the stacked Cry genes constructs (Table 2). To preserve the effectiveness of the current Bt-technology, a refuge block (or strips) of non-Bt-crops could be included in the field harboring the same Bt-crop as buffer zones. Furthermore, combining the refuge technique with other traditional techniques such as (1) crop rotation, (2) rotating Bt traits, (3) use of pyramided traits, and (4) scout fields for pest can effectively slow insect resistance to Bt-crop.

Enhancing plant defense by coupling Cry traits with plant-mediated RNAi (PMRi) silencing of insect’s genes

Mobile small RNA (sRNA)-mediated RNA interference (RNAi) is a highly conserved regulatory mechanism that governed cellular processes in eukaryotes and ensure communication between kingdoms (Weiberg et al. 2013). Despite the diversity of sRNA, the most used of this non-coding regulatory molecules in transgenics are small interfering RNAs (siRNAs), piwi-associated RNAs (piRNAs) and microRNAs (miRNAs) generated via the Dicer and Argonaute (AGO) protein complexes (Fig. 4). MicroRNAs designed from acetylcholinesterase 2 gene (MpCHE2) to target two different sites on MpCHE2 of aphid Myzus persicae (family Aphidoidea, order Hemiptera) and introgressed in tobacco plants (Nicotiana tabacum cv. Xanti) exhibited resistance against aphid (Guo et al. 2014). This study demonstrated that plants expressing artificial sRNA (asRNA) can effectively control destructive insects. Other genes used successfully in plant-mediated RNAi (PMRi) silencing of insect’s genes where transgenic plants expressed insect double-stranded RNA (dsRNA) include cytochrome P450 genes CYP6AE14 and CYP6B6, arginine kinase (AK) gene, and HMG-CoA reductase (HMG) gene of cotton bollworm Helicoverpa zea (Mao et al. 2007, 2011; Tian et al. 2015), hormone receptor 3 (HR3) molt-regulating transcription factor involved in metamorphosis of Helicoverpa armigera (Han et al. 2017), and induced cytochrome P450 monooxygenase CYP6B46 and β-glucosidase genes of Manduca sexta and M. quinquemaculata (Lepidoptera, Sphingidae) (Poreddy et al. 2017), all leading resistance against the targeted insects. Taking into consideration that insects bites triggers host-induced gene silencing (HIGS) via transgenes in plants and that HIGS sRNAs are effective against destructive insects by translocating into insect cells (Weiberg et al. 2013), associating PMRi with Cry genes would better protect Bt-soybeans than overdependence on Cry genes only (Fig. 4).

The quiet approval and commercialization of Monsanto non-specific DvSnf7 dsRNA expressed in MON 89034 × TC1507 × MON 87411 × DAS-59122-7 cassette in corn (Bolognesi et al. 2012; Vélez et al. 2016; EPA 2016) to counter the Western corn rootworm (Diabrotica virgifera virgifera LeConte) in USA, marks the beginning of RNAi biospesticide into agriculture. Reasonably, it might not long to see DvSnf7 dsRNA incorporated in different soybean hybrids. Regardless of RNAi approach, it could be less effective because of low uptake in the insect, high alkaline gut environment (pH > 9.0) and a strong intestinal nucleolytic activity which degrade dsRNA. To counter alkaline hydrolysis in insect gut, Christiaens et al. (2018) developed a guanidine-rich polymer capable of protecting dsRNA up to 30 h at pH 11. By feeding beet armyworm S. exigua larvae with cabbage leaf disc coated with protected dsRNA guanidine-rich polymer nanoparticle in vivo, 53% and 16% mortality was observed with protected and naked dsRNA (Christiaens et al. 2018), respectively. This approach of protecting dsRNA with guanidine-rich polymer nanoparticle paved the way for RNAi-based pesticides as sprays for the control of invasive insect pests.

Conclusion

In the twenty-first century, Bt-soybean like other GM crops provide useful agronomic traits for crop protection and production, but not a panacea, yet, Bt-soybean helped to curb the problems caused by specific pests, lower farming input and increase the yield. It could be an oversight not to think of new strategies to control destructive insect and ensure the achievement of 2050 forecast of 371.3 metric million tons. We proposed that combining two Cry-protein that binds to different receptors in the same insect and associated RNAi technology could enhance insecticidal potential of Bt-soybean. While gene pyramiding appeared the way forward to counter destructive insects, stringent caution must be taken not to compromise human health, animal health and abrupt collapse of biota due to the elimination of non-target pests.

Abbreviations

PMRi

Plant-mediated RNA interference

Bt

Bacillus thuringiensis

HT

Herbicide tolerance

GM

Genetically modified

MAB

Marker-assisted breeding

PAT

Phosphinothricin acetyltransferase

AICc

Akaike Information Criterion, corrected

BIC

Bayesian Information Criterion

Cry

Crystal

Compliance with ethical standards

Conflict of interest

No potential conflict of interest was reported by the authors.