- freely available
Toxins 2012, 4(6), 455-475; doi:10.3390/toxins4060455
Published: 12 June 2012
Abstract: Transgenic plants expressing combinations of microbial or plant pesticidal proteins represent a promising tool for the efficient, durable control of herbivorous insects. In this review we describe current strategies devised for the heterologous co-expression of pesticidal proteins in planta, some of which have already shown usefulness in plant protection. Emphasis is placed on protein engineering strategies involving the insertion of single DNA constructs within the host plant genome. Multimodal fusion proteins integrating complementary pesticidal functions along a unique polypeptide are first considered, taking into account the structural constraints associated with protein or protein domain grafting to biologically active proteins. Strategies that allow for the co- or post-translational release of two or more pesticidal proteins are then considered, including polyprotein precursors releasing free proteins upon proteolytic cleavage, and multicistronic transcripts for the parallel translation of single protein-encoding mRNA sequences.
Large-scale adoption of plant varieties expressing insecticidal Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) illustrates the growing importance of insect-resistant transgenic plants in agricultural systems worldwide . Cry toxin-expressing plants (or Bt plants) have been grown in more than 25 countries, on a total area of more than 50 million ha annually [1,2]. Their high degree of resistance to economically important insects, along with the adoption of deployment strategies to delay the onset of genetic resistance among target pest populations, have largely contributed to the success and sustained efficacy of these plants since their first introduction on the market in the mid-1990s [3,4]. After more than 15 years of commercial use, transgenic Bt plant lines still show highly toxic effects against target insects, and documented cases of genetic resistance remain scarce considering the extent of plantations .
Resistance monitoring in the field, however, remains challenging [5,6] and genetic adaptation is a threat to the long-term efficacy of any insecticide. The marked lethal effects of Cry toxins exert a strong selection pressure on target pest populations, and genetic resistance to these biopesticides is readily induced among laboratory colonies (e.g., [7,8,9]). Cases of resistance to field-grown Bt plants have also been reported recently in China, India, South Africa and the U.S. [4,10,11,12,13,14,15] despite deployment schemes to prevent resistance, such as the use of Bt lines with high levels of toxin grown along with non-Bt plant refuges for susceptible insects . A reliable strategy to promote long-term effectiveness of transgenic Bt plant lines is to consider these plants as components of much broader, integrated pest management systems involving different approaches for insect control [16,17,18]. A complementary—and conceptually similar—strategy consists of expressing two or more pesticidal proteins in the plants in such a way as to implement an integrated pesticidal system [19,20]. Transgenic plants expressing combinations of Cry toxins interacting with different receptors in the insect midgut were shown to delay the onset of genetic resistance compared to single toxin-plant varieties used alone, sequentially or in mosaics [21,22,23]. Insect adaptation to plant varieties expressing more than one Cry toxin will always remain an issue [3,24,25], but recent surveys for resistance to field-grown Bt plant lines expressing two or three Cry toxins confirm a very low joint resistance allele frequency for the toxins among target populations .
The stacking (or “pyramiding”) of Cry toxins in planta may also provide improved protection against insects partially susceptible to single toxins [27,28], or help broadening pesticidal effects against different insects to minimize secondary pest infestations upon primary pest control [29,30,31]. From a larger perspective, the pesticidal effects of Cry toxins can be extended by the co-expression of complementary resistance factors with different modes of action [16,32]. For instance, Cry toxins have been expressed in combination with the Bt vegetative insecticidal protein Vip3A [33,34,35], or with plant proteins involved in defense responses to biotic stress agents [36,37,38,39,40]. Pyramiding approaches exclusive of Cry toxins have also been devised, most notably involving alternative Bt endotoxins  and plant defense proteins with complementary or synergistic effects such as lectins and protease inhibitors [42,43,44,45,46]. Different strategies have been proposed to co-express several recombinant proteins in plants. Here we review these strategies, with particular emphasis on genetic and protein engineering approaches enabling the coordinated expression of multiple pesticidal proteins under the control of single promoters.
2. Transgene Stacking and Pesticidal Protein Pyramiding in Plants
Several studies have reported the successful in planta expression of two or three recombinant proteins for pest resistance in plants by “gene stacking” strategies involving sexual crosses between transgenic parental lines bearing distinct transgenes [22,34,38,47,48]. Others have described DNA cloning strategies, transgene cassettes and transformation procedures enabling the co-integration of different resistance transgenes in a single transformation cycle [36,43,45,46,49,50]. Rapid progress has been achieved over the last decade towards the development of multi-transgenic plant lines [51,52,53], but most transgene stacking approaches still present important practical constraints. The most notable are the considerable amount of work and time required for the stable introgression of multiple transgene sequences in plant hybrids, and the onset of gene silencing following the insertion of homologous promoters or multiple T-DNA sequences in recipient genomes [51,54]. Furthermore, the production and identification of multi-transgenic lines that express sufficient and comparable amounts of the different recombinant proteins is not easy, especially given the likelihood of insertional mutagenesis events and position effects that can take place and influence transgene expression in the modified host plants .
A possible way to overcome these limitations is to use fusion proteins or polyprotein constructs for the co-expression of distinct proteins under the control of a single promoter. These approaches based on single transgenes present several practical advantages, including the single-step production of transgenic lines with pyramided recombinant traits, the avoidance of multiple T-DNA and promoter sequence insertions, and, most importantly, coordinated expression of different recombinant proteins in planta. Three general strategies are currently considered for the co-expression of multiple protein traits encoded by a single transgene sequence in plants: (1) the expression of non-cleavable fusion proteins for the accumulation of multimodal proteins conferring two or several new functional traits; (2) the expression of polyprotein precursors with intrinsic cleavage sites for a co- or post-translational separation of the protein components; and (3) the translation of individual proteins from polycistronic mRNA transcripts.
2.1. Multimodal Fusion Proteins
Fusion proteins are composed of at least two covalently attached proteins, protein domains or polypeptides harboring the biological functions of the single components . An interesting example in nature is potato multicystatin, a wound-inducible Cys protease inhibitor expressed in leaves following insect herbivory . This protein is composed of eight protease inhibitory—or cystatin—domains linked within a single polypeptide chain by short ‘linker peptide’ sequences . The eight domains, which presumably were the result of multiple gene duplications in closely related Solanum species , include hypervariable, rapidly evolving amino acid sites giving the protein a broad range of inhibitory specifities towards insect and plant Cys proteases [60,61]. As shown by in vitro inhibitory assays with the model Cys protease papain, the overall stoechiometric ratio of this natural fusion is comparable to the overall ratio of the eight domains taken separately , thereby providing a relevant example of the potential of defense or pesticidal proteins as structural modules for multimodal fusion protein design. In the laboratory, recombinant fusion proteins may be engineered by simply ligating the DNA-encoding sequences of two (or more) polypeptides, protein domains or whole proteins, either directly or separated by a linker peptide.
2.1.1. Protein Fusions for Insect Control
Several protein fusions or hybrids have been devised to control herbivorous insects, including Bt toxins or plant defense proteins as primary fusion partners [29,30,31,37,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87] (Table 1). For instance, a fusion protein integrating the Bt toxins Cry1B and Cry1Ab was engineered to broaden the insecticidal spectrum of Bt toxin-expressing lines derived from tropical maize varieties . Likewise, toxic peptides and enzymes, such as the spider venom neurotoxin HWTX-I or the Beauvaria bassiana protease CDEP2, were fused to Cry1Ac to enhance its pesticidal effects against lepidopteran insects [72,73]. A number of authors have also devised Cry toxin hybrids by protein domain swapping to integrate structural elements of different native toxins within a single chimeric protein sequence. SN19, a fusion protein consisting of specific domains of the Cry1Ba and Cry1Ia Bt toxins, was designed based on this approach  and used to produce transgenic potato lines resistant to both coleopteran and lepidopteran pests . Another Cry toxin hybrid active against insects of different orders was developed recently based on Cry1Ab modified with part of the Cry3A variable region .
|Table 1. Hybrid and fusion proteins devised for herbivorous insect control—Selected examples from the current literature|
|Fusion partners||Intended uses / Improved effects||Refs.|
|Cry1Ab toxin modified with domain III of Cry1C||Improved efficacy against Spodoptera exigua|||
|Cry1Ab toxin modified with C-terminal region of Cry1Ac||Improved efficacy and range against Lepidoptera|||
|Cry1Ab toxin modified with part of Cry3A variable region||Resistance to insects of different orders|||
|Cry1Ac or Cry1E modified with domain III of Cry1C||Improved efficacy against Spodoptera exigua|||
|Cry1Ba toxin modified with domain II of Cry1Ia||Resistance to insects of different orders||[31,66]|
|Cry1Ca, Cry1Fb and Cry1Ba modified with Cry1Ac domain III||Improved efficacy against Heliothis virescens|||
|Cry1Ea toxin modified with part of Cry1Ca toxin domain III||Improved efficacy against Spodoptera litura|||
|Cry hybrid SN19 modified with domain II of Cry1Ba||Resistance to insects of different orders|||
|Sunflower multicystatin integrating gourd trypsin inhibitor||Broader inhibition of Lepidoptera midgut proteases|||
|Bi- or multimodal translational fusions|
|Cry1B and Cry1Ab toxins||Durability and broader range against Lepidoptera|||
|Cry1Ac toxin and galactose-binding domain of ricin B chain||Improved efficacy and broader insecticidal range|||
|Cry1Ac toxin and cowpea trypsin inhibitor CpTI||Dual effect against cabbage worms and durability|||
|Cry1Ac toxin and spider venom neurotoxin HWTX-I||Dual effect against Plutella xylostella|||
|Cry1Ac toxin and Beauveria bassiana subtilisin CDEP2||Dual effect against Helicoverpa armigera|||
|Cry1Ac toxin and baculoviral polyhedrin||Increased stability and expression in E. coli|||
|Cry1Ab toxin C-ter peptide and spider toxin ACTX-Ar1||Improved efficacy and range against Lepidoptera||[75,76,77,78]|
|Snowdrop lectin and arthropod peptide toxins||Delivery of toxic peptides to the haemolymph||[79,80,81,82,83]|
|Soybean cystatin N2 and GSII lectin||Dual effect against Callosobruchus maculatus|||
|Various plant and animal cysteine protease inhibitors||Broader inhibition of Thysanoptera midgut proteases|||
|Tomato cathepsin D inhibitor and corn cystatin II||Broader inhibition of Coleoptera midgut proteases|||
|Oryzacystatin I and potato carboxypeptidase inhibitor domains||Broader inhibition of Coleoptera midgut proteases|||
Pesticidal effects for fusion proteins employing plant proteins or protein domains have also been reported. For instance, Mehlo et al.  fused a galactose-binding domain from ricin B to Cry1Ac to improve its binding properties in the insect midgut epithelium and to broaden its pesticidal effects against different lepidopteran insects. Translational fusions with different plant lectins and/or protease inhibitors have been devised as a way to improve the biological effects of the individual components [70,84,86,87] or to stabilize protein partners prone to degradation along the plant–insect continuum [84,85,88]. Plant lectins have also been used as fusion partners to deliver toxic polypeptides, such as spider or scorpion venom neurotoxins, into the haemolymph of various insects [79,80,81,82,83]. These promising developments, along with rapid advances in protein structural biology and molecular modeling allowing for the design of potentially effective fusions in silico , confirm the potential of fusion protein design as a realistic avenue for the production of recombinant proteins integrating multiple pesticidal functions.
2.1.2. Non-Cleavable Linker Peptides
On the other hand, protein domain grafting may alter the structure and function of some proteins, with possible unexpected effects on the resulting protein fusions. For instance, the Allium sativum lectin “ASAL” showed an altered glycosylation pattern, decreased thermal stability and no bacteriocidal activity when fused to the small ubiquitin related modifier (SUMO) peptide . Similarly, the inhibitory activity of rice cystatin I against papain and herbivorous pest Cys proteases was substantially lowered when the 39-amino acids inhibitor of carboxypeptidase A, potato carboxypeptidase inhibitor, was grafted at the C-terminus . A strategy to avoid such effects consists of adding a linker peptide to seperate the protein partners in such a way as to avoid detrimental non-specific physicochemical interactions between the non-cognate polypeptides [91,92]. Computational approaches have been developed to devise linker peptides, such as the program LINKER, which generates synthetic linker sequences from known protein structures based on user-defined requirements [93,94]. Another approach takes into account the important issue of proteolytic lability by a systematic screening of candidate linker sequences against the substrate specificities of proteases listed in the MEROPS peptidase database [95,96].
Empirical assessments with different linker peptides will possibly remain the most robust approach to identify linkers adapted to specific macromolecular contexts, but current knowledge on the properties of natural and synthetic linkers already provides helpful hints for successful outputs [92,97]. Gly-rich peptide motifs, such as the well-characterized pentapeptide motif Gly–Gly–Gly–Gly–Ser, are known to ensure a certain flexibility of protein fusions and are useful in preventing functional disturbance and destabilization of the domain partners . By comparison, Pro-rich motifs confer rigidity to the proteins and are useful in maintaining a minimal distance and preventing potential non-specific interactions between the fusion partners [91,92]. Data are still scarce about the performance of linker peptides in plant biotechnology, but a number of studies suggest the potential of Gly-containing linkers to produce protease-resistant fusions with dual pesticidal activities against insects or pathogens [84,85,86,99,100]. A rigid linker of fungal origin has also been used to produce a bifunctional protein, resulting in a dual inhibitor of herbivorous pest proteases that was partially resistant to proteolytic processing .
Despite promising results, fusion proteins integrating linker peptides might not always be the optimal alternative to linker-free, tail-to-tail translational fusions. The choice and number of amino acids used to devise an effective linker are critical because this structural element may itself impact the overall performance of the fusion partners [102,103], fusion protein yield , tendency to aggregate , or robustness of the fusion product in terms of thermal stability [95,104] and resistance to proteolysis [95,103,105]. Biological context-dependent effects, including the host organism used for expression, further add to the complexity of linker sequence design and stress the need for more empirical data on natural and synthetic linkers. For example, a helical linker that allows for more activity of fusion partners than a flexible linker was also more sensitive to proteolysis in yeast  and prone to autocatalytic cleavage under certain pH conditions . A trade-off might be unavoidable, in some cases, between the possible gains obtained with linker peptides in terms of yield and activity of the fusion partners, and different constraints impacting their structural stability in the host environment selected for expression.
2.2. Polyprotein Precursors
One possible way to avoid such trade-offs is to express polyprotein precursors with cleavable linkers between the proteins of interest. This approach is based on the gene/protein expression strategy adopted by some viruses to ensure a quantitative balance of protein components in vivo . The protein expression pattern of these viruses first involves the synthesis of a large polypeptide precursor, which is then processed to release the mature proteins [107,108]. Recombinant polyproteins may be engineered to include the coding sequences of multiple proteins separated by accessory DNA sequences encoding a proteolysis-susceptible peptide for processing by either host endogenous proteases or an exogenously supplied recombinant protease (Figure 1).
2.2.1. Exogenous Protease Cleavage
An exogenous protease with high specificity towards linker cleavage sites may be supplied as a self-processing component of the same polyprotein as the proteins of interest for cleavage in cis, or from a co-expressed DNA sequence as a viral accessory endoprotease for cleavage in trans (Figure 1a). Examples of viral proteases used in plant biotechnology are the well-characterized tobacco etch virus nuclear inclusion NIa protease  and the cowpea mosaic virus 24 K proteinase . These proteases excise themselves from viral polyproteins during natural infections, where they then go on to process the remaining polyprotein fragments to release the individual proteins [111,112]. For instance, the NIa protease specifically recognizes and cleaves the heptapeptidic sequence “Glu–X–X–Tyr–X–Gln–↓[Gly or Ser]”, with the scissile bond located between the Gln–Gly (or Gln-Ser) dipeptide motif [113,114]. In practice, the NIa protease may be co-expressed in planta along with an engineered polyprotein bearing the corresponding protease-cleavable motifs between the C- and N-termini of the proteins to be produced. NIa protease/polyprotein precursors have been expressed in several plant systems, notably allowing for the ectopic implementation of short metabolic pathways in transgenic plant cells , the coordinate expression of interacting transcription factors to activate endogenous metabolic pathways , and the differential subcellular translocation of recombinant proteins harboring distinct targeting signals after polyprotein processing . NIa/polyproteins have also been used to co-express defense proteins in plants, such as the coat proteins of different plant viruses [118,119] and a number of antimicrobial peptides and proteins [120,121].
2.2.2. Endogenous Protease Cleavage
An alternative to supplying exogenous protease functions consists of integrating a cleavable, plant protease-sensitive linker between the proteins to express and targeting the resulting polyprotein precursor to the endoplasmic reticulum for processing by endogenous proteases along the cell secretory pathway  (Figure 1b). The antidigestive protease inhibitors cowpea trypsin inhibitor and rice cystatin I have been produced in Arabidopsis using this approach by expressing a single translational fusion product for the two proteins separated by a cleavable peptide from the plant metallothionein-like protein, PsMTa . In a similar way, a polyprotein precursor including the antimicrobial defensins DmAMP1 and RsAFP2 linked by a seed protein cleavable sequence from Impatiens balsamina was shown to drive the accumulation of free and active forms of the two defensins in the extracellular milieu of transgenic Arabidopsis leaves [123,124,125]. These findings, along with a recently described approach involving ubiquitin-derived linker sequences for processing by endogenous deubiquitinating proteases , underline the potential of cleavable polyproteins sensitive to host endogenous proteases for the coordinate expression and release of multiple recombinant proteins, including pesticidal proteins, in plants.
2.3. Polycistronic Constructs
Polycistronic DNA constructs for the expression of different proteins from a single mRNA transcript also show potential to generate transgenic plant lines producing multiple recombinant proteins . Unlike polyprotein-encoding constructs, polycistronic constructs drive the accumulation of free recombinant proteins in host tissues, with no need for an endogenous or recombinant accessory protease acting co- or post-translationally on the translated polypeptide product. Two main approaches have been described to express recombinant proteins in plants using polycistronic mRNAs, both of which make use of viral protein expression strategies (Figure 2). The first approach involves a viral structural RNA motif, the so-called internal ribosome entry site (IRES) . The second approach involves the 2A catalytic peptide of foot-and-mouth disease virus and related picornaviruses .
2.3.1. IRES-Mediated mRNA Translation
IRES motifs direct ribosomes to initiate translation in a cap-independent manner, at internal positions within polycistronic transcripts  (Figure 2a). A number of IRES motifs from plant [130,131] and animal  viruses have successfully been used to direct the expression of multiple recombinant proteins in plants and plant cells. Bicistronic constructs have been used, for instance, to engineer nematode pest resistance in tobacco by the co-expression of cowpea trypsin inhibitor and rice cystatin I , or to facilitate the implementation of abiotic stress tolerance in cell cultures and transgenic plants by linking the detection of a marker gene with the expression of a stress-related protein [134,135]. However, IRES-based approaches present practical limitations associated with the highly variable efficacy of IRES motifs in planta, their taxonomic origin and the plant tissue selected for protein expression [131,133,134,136,137]. Perhaps the most prominent limitation preventing the widespread use of IRES motifs in plant biotechnology is their relative inefficiency. Internally initiated translation is generally low compared to cap-dependent translation  and is, therefore, hardly compatible with plant protection strategies requiring sufficient and comparable amounts of different pesticidal proteins.
2.3.2. 2A Peptide-Mediated mRNA Translation
A straightforward way to ensure a balanced translation of polycistron-encoded proteins may be to use the 20-amino acids 2A peptide of foot-and-mouth disease virus [51,138]. This peptide mediates a “ribosomal skip” during viral transcript translation that results in a co-translational, non-proteolytic dissociation of the encoded proteins . This unique biochemical process prevents a peptide bond to form between the last two amino acids of the viral peptide and causes the nascent polypeptide to dissociate from the ribosomal translational complex while allowing mRNA translation to continue [139,140]. Polycistronic protein constructs involving the 2A peptide may be engineered in vitro by insertion of this peptide between the recombinant proteins to be expressed (Figure 2b). The correct processing of 2A peptides in plants was initially reported for transgenic tobacco lines expressing combinations of the model reporter proteins β-glucuronidase, chloramphenicol acetyltransferase and green fluorescent protein [141,142]. Several studies then reported the successful co-expression of useful proteins by this approach, for applications as diverse as the heterologous expression of vaccines and antibodies [143,144,145], the engineering of carotenoid biosynthetic pathways [137,146], storage protein enrichment to increase the nutritional value of plant foods , and the expression of stress or pesticidal proteins conferring abiotic stress tolerance  or pest resistance [125,149]. The presence of several non-cognate amino acids at the C-terminus of the upstream protein(s) following mRNA translation still represents a practical limitation to the 2A peptide technology , but strategies have been devised in recent years to remove this extension, relying on the addition of cleavage sites between the C-terminus and the viral peptide [150,151]. A number of factors should make the 2A peptide expression strategy particularly useful for the implementation of multiple protein resistance traits in plants [138,152]. The most obvious are the possibility of integrating several recombinant proteins and 2A peptide connections within the same construct [153,154], and the relatively efficient self-processing activity of 2A peptide-containing polyproteins in plant systems . Other factors are the greater efficiency of this expression system compared to the IRES motif strategy , and the possible targeting of recombinant proteins to different subcellular locations after their co-translational release from the ribosomal complex [153,154,155].
3. Concluding Remarks
Numerous studies have illustrated the potential of multimodal protein constructs for the implementation of multiple defense protein traits in plants. Expression strategies based on stable fusion proteins or cleavable polyprotein precursors have been described, as well as alternative strategies involving polycistronic DNA constructs for the co-translational release of functionally distinct proteins. Accumulating data in forthcoming years should further demonstrate the potential of these approaches for a durable control of plant pests and pathogens. One forthcoming issue will be to implement the resulting control tools in realistic conditions, not only in terms of efficiency and applicability, but also taking into account the possible impacts of multifunctional protein systems at the ecosystem level . Plant protection strategies involving pesticidal proteins such as the Cry toxins have the obvious advantage of being highly specific to target pests. Hybrid protein constructs integrating multiple, and sometimes less specific, recombinant traits could by contrast exert undue effects on non-target organisms. A further challenge in the field will be to find the right balance between the efficacy, durability and specificity requirements of each pest control problem.
Work supported by the Natural Science and Engineering Research Council of Canada and the Fonds Québécois de la Recherche sur la Nature et les Technologies.
Conflict of Interest
The authors declare no conflict of interest.
- James, C. Brief 42: Global Status of Commercialized Biotech/GM Crops: 2010; ISAAA: Ithaca, New York, NY, USA, 2010.
- Sanahuja, G.; Banakar, R.; Twyman, R.M.; Capell, T.; Christou, P. Bacillus tuhringiensis: A century of research, development and commercial applications. Plant Biotechnol. J. 2011, 9, 283–300, doi:10.1111/j.1467-7652.2011.00595.x.
- Tabashnik, B.E.; Gassmann, A.J.; Crowder, D.W.; Carrière, Y. Insect resistance to Bt crops: Evidence versus theory. Nat. Biotechnol. 2008, 26, 199–202, doi:10.1038/nbt1382.
- Tabashnik, B.E.; van Rensburg, J.B.J.; Carrière, Y. Field-evolved insect resistance to Bt crops: Definition, theory, and data. J. Econ. Entomol. 2009, 102, 2011–2025, doi:10.1603/029.102.0601.
- Huang, F.; Parker, R.; Leonard, B.R.; Yang, Y.; Liu, J. Frequency of resistance alleles to Bacillus thuringiensis-corn in Texas populations of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae). Crop Protect. 2009, 28, 174–180, doi:10.1016/j.cropro.2008.10.002.
- Downes, S.; Parker, T.L.; Mahon, R.J. Frequence of alleles conferring resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa punctigera (Lepidoptera: Noctuidae) from 2002 to 2006. J. Econ. Entomol. 2009, 102, 733–742, doi:10.1603/029.102.0234.
- Griffitts, J.S.; Aroian, R.V. Many roads to resistance: How invertebrates adapt to Bt toxins. Bioessays 2005, 27, 614–624, doi:10.1002/bies.20239.
- Meihls, L.N.; Higdon, M.L.; Ellersieck, M.; Hibbard, B.E. Selection for resistance to mCry3A-expressing transgenic corn in western corn rootworm. J. Econ. Entomol. 2011, 104, 1045–1054, doi:10.1603/EC10320.
- Oswald, K.J.; French, B.W.; Nielson, C.; Bagley, M. Selection for Cry3Bb1 resistance in a genetically diverse population of nondiapausing western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 2011, 104, 1038–1044, doi:10.1603/EC10312.
- Kruger, M.; van Rensburg, J.B.J.; van den Berg, J. Perspective on the development of stem borer resistance to Bt maize and refuge compliance at the Vaalharts irrigation scheme in South Africa. Crop Protect. 2009, 28, 684–689, doi:10.1016/j.cropro.2009.04.001.
- Bagla, P. Hardy cotton-munching pests are latest blow to GM crops. Science 2010, 327, doi:10.1126/science.327.5972.1439.
- Storer, N.P.; Babcock, J.M.; Schlenz, M.; Meade, T.; Thompson, G.D.; Bing, J.W.; Huckaba, R.M. Discovery and characterization of field resistance to Bt maize: Spodoptera frugiperda (Lepidoptera: Noctuidae) in Puerto Rico. J. Econ. Entomol. 2010, 103, 1031–1038, doi:10.1603/EC10040.
- Dhura, S.; Gujan, G.T. Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India. Pest Manag. Sci. 2011, 67, 898–903, doi:10.1002/ps.2127.
- Gassmann, A.J.; Petzold-Maxwell, J.L.; Keweshan, R.S.; Dunbar, M.W. Field-evolved resistance to Bt maize by western corn rootworm. PLoS One 2011, 6, doi:10.1371/journal.pone.0022629.
- Zhang, H.; Yin, W.; Zhao, J.; Jin, L.; Yang, Y.; Wu, S.; Tabashnik, B.E.; Wu, Y. Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China. PLoS One 2011, 6, doi:10.1371/journal.pone.0022874.
- Bates, S.L.; Zhao, J.-Z.; Roush, R.T.; Shelton, A.M. Insect resistance management in GM crops: Past, present and future. Nat. Biotechnol. 2005, 23, 57–62, doi:10.1038/nbt1056.
- Naranjo, S.E. Impacts of Bt transgenic cotton on integrated pest management. J. Agric. Food Chem. 2011, 59, 5842–5851, doi:10.1021/jf102939c.
- Gray, M.E. Relevance of traditional integrated pest management (IPM) strategies for commercial corn producers in a transgenic agroecosystem: A bygone era? J. Agric. Food Chem. 2011, 59, 5852–5858, doi:10.1021/jf102673s.
- Roush, R.T. Two-toxin strategies for management of insecticidal transgenic crops: Can pyramiding succeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. B 1998, 353, 1777–1786, doi:10.1098/rstb.1998.0330.
- Moar, W.J.; Anilkumar, K.J. The power of the pyramid. Science 2007, 318, 1561–1562, doi:10.1126/science.1151313.
- Gould, F. Bt resistance management—theory meets data. Nat. Biotechnol. 2003, 21, 1450–1451, doi:10.1038/nbt1203-1450.
- Zhao, J.-Z.; Cao, J.; Li, Y.; Collins, H.L.; Roush, R.T.; Earle, E.D.; Shelton, A.M. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nat. Biotechnol. 2003, 21, 1493–1497, doi:10.1038/nbt907.
- Zhao, J.-Z.; Cao, J.; Collins, H.L.; Bates, S.L.; Roush, R.T.; Earle, E.D.; Shelton, A.M. Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants. Proc. Natl. Acad. Sci. USA 2005, 102, 8426–8430, doi:10.1073/pnas.0409324102. 15939892
- Alyokhin, A. Scant evidence supports EPA’s pyramided Bt corn refuge size of 5%. Nat. Biotechnol. 2011, 29, 577–578, doi:10.1038/nbt.1911.
- Ives, A.R.; Glaum, P.R.; Ziebarth, N.L.; Andow, D.A. The evolution of resistance to two-toxin pyramid transgenic crops. Ecol. Appl. 2011, 21, 503–515, doi:10.1890/09-1869.1.
- Huang, F.; Ghimire, M.N.; Leonard, B.R.; Wang, J.; Daves, C.; Levy, R.; Cook, D.; Head, G.P.; Yang, Y.; Temple, J.; et al. F2 screening for resistance to pyramided Bacillus thuringiensis maize in Louisiana and Mississippi populations of Diatraea saccharalis (Lepidoptera: Crambidae). Pest Manag. Sci. 2011, 67, 1269–1276, doi:10.1002/ps.2182.
- Chitkowski, R.L.; Turnispeed, S.G.; Sullivan, M.J.; Bridges, W.C., Jr. Field and laboratory evaluations of transgenic cottons expressing one or two Bacillus thuringiensis var. kurstaki Berliner proteins for management of noctuid (Lepidopteran) pests. J. Econ. Entomol. 2003, 96, 755–762, doi:10.1603/0022-0493-96.3.755.
- Jackson, R.E.; Bradley, J.R.; van Duyn, J.W. Performance of feral and Cry1Ac-selected Helicoverpa zea (Lepidoptera: Noctuidae) strains on transgenic cottons expressing one or two Bacillus thuringiensis ssp. kurstaki proteins under greenhouse conditions. J. Entomol. Sci. 2004, 39, 46–55.
- Tu, J.; Zhang, G.; Datta, K.; Xu, C.; He, Y.; Zhang, Q.; Khush, G.S.; Datta, S.K. Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis ∂-endotoxin. Nat. Biotechnol. 2000, 18, 1101–1104, doi:10.1038/80310.
- Bohorova, N.; Frutos, R.; Royer, M.; Estanol, P.; Pacheco, M.; Rascon, Q.; McLean, S.; Hoisington, D. Novel synthetic Bacillus thuringiensis cry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor. Appl. Genet. 2001, 103, 817–826, doi:10.1007/s001220100686.
- Naimov, S.; Dukiandjiev, S.; de Maagd, R.A. A hybrid Bacillus thuringiensis delta-endotoxin gives resistance against a coleopteran and a lepidopteran pest in transgenic potato. Plant Biotechnol. J. 2003, 1, 51–57. 17147680
- Lee, M.K.; Walters, F.S.; Hart, H.; Palekar, N.; Chen, J.-S. The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab ∂-endotoxin. Appl. Environ. Microbiol. 2003, 69, 4648–4657, doi:10.1128/AEM.69.8.4648-4657.2003.
- Jackson, R.E.; Marcus, M.A.; Gould, F.; Bradley, J.R.; van Duyn, J.W. Cross-resistance responses of CryIAc-selected Heliothis virescens (Lepidoptera: Noctuidae) to the Bacillus thuringiensis protein vip3A. J. Econ. Entomol. 2007, 100, 180–186, doi:10.1603/0022-0493(2007)100[180:CROCHV]2.0.CO;2.
- Kurtz, R.W.; McCaffery, A.; O’Reilly, D. Insect resistance management for Syngenta’s VipCot™ transgenic cotton. J. Inverteb. Pathol. 2007, 95, 227–230, doi:10.1016/j.jip.2007.03.014.
- Burkness, E.C.; Dively, G.; Patton, T.; Morey, A.C.; Hutchison, W.D. Novel Vip3A Bacillus thuringiensis (Bt) maize approaches high-dose efficacy against Helicoverpa zea (Lepidoptera: Noctuidae) under field conditions: Implications for resistance management. GM Crops 2010, 1, 337–343, doi:10.4161/gmcr.1.5.14765.
- Maqbool, S.B.; Riazzudin, S.; Loc, N.T.; Gatehouse, A.M.R.; Gatehouse, J.A.; Christou, P. Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests. Mol. Breed. 2001, 7, 85–93, doi:10.1023/A:1009644712157.
- Yang, G.D.; Zhu, Z.; Li, Y.O.; Zhu, Z.J. Transformation of Bt-CpTi fusion protein gene to cabbage (Brassica oleracea var. capitata) mediated by Agrobacterium tumefaciens and particle bombardment. Shi Yan Sheng Wu Xue Bao 2002, 35, 117–122. 15344329
- Datta, K.; Baisakh, N.; Thet, K.M.; Tu, J.; Datta, S.K. Pyramiding transgenes for multiple resistance in rice against bacterial blight, yellow stem borer and sheath blight. Theor. Appl. Genet. 2002, 106, 1–8. 12582865
- Su, X.; Chu, Y.; Li, H.; Hou, Y.; Zhang, B.; Huang, Q.; Hu, Z.; Huang, R.; Tian, Y. Expression of multiple resistance genes enhances tolerance to environmental stressors in transgenic poplar (Populus x euramericana ‘Guariento’). PLoS One 2011, 6, doi:10.1371/journal.pone.0024614.
- Zhang, B.; Chen, M.; Zhang, X.; Luan, H.; Diao, S.; Tian, Y.; Su, X. Laboratory and field evaluation of the transgenic Populus alba ×Populus glandulosa expressing double coleopteran-resistance genes. Tree Physiol. 2011, 31, 567–573, doi:10.1093/treephys/tpr032.
- Moellenbeck, D.J.; Peters, M.L.; Bing, J.W.; Rouse, J.R.; Higgins, L.S.; Sims, L.; Nevshemal, T.; Marshall, L.; Ellis, R.T.; Bystrak, P.G.; et al. Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms. Nat. Biotechnol. 2001, 19, 668–672, doi:10.1038/90282.
- Boulter, D.; Edwards, G.A.; Gatehouse, A.M.R.; Gatehouse, J.A.; Hilder, V.A. Additive protective effects of different plant-derived insect resistance genes in transgenic tobacco plants. Crop Protect. 1990, 9, 351–354, doi:10.1016/0261-2194(90)90005-R.
- Tang, K.; Tinjuangjun, P.; Xu, Y.; Sun, X.; Gatehouse, J.A.; Ronald, P.C.; Qi, H.; Lu, X.; Christou, P.; Kohli, A. Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests. Planta 1999, 208, 552–563, doi:10.1007/s004250050593.
- Campbell, M.A.; Fitzgerald, H.A.; Ronald, P.C. Engineering pathogen resistance in crop plants. Transg. Res. 2002, 11, 599–613, doi:10.1023/A:1021109509953.
- Abdeen, A.; Virgos, A.; Olivella, E.; Villanueva, J.; Aviles, X.; Gabarra, R.; Prat, S. Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors. Plant Mol. Biol. 2005, 57, 189–202, doi:10.1007/s11103-004-6959-9. 15821877
- Senthilkumar, R.; Cheng, C.-P.; Yeh, K.-W. Genetically pyramiding protease-inhibitor genes for dual braod-spectrum resistance against insect and phytopathogens in transgenic tobacco. Plant Biotechnol. J. 2010, 8, 65–75, doi:10.1111/j.1467-7652.2009.00466.x.
- Cao, J.; Zhao, J.-Z.; Tang, J.D.; Shelton, A.M.; Earle, E.D. Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins. Theor. Appl. Genet. 2002, 105, 258–264, doi:10.1007/s00122-002-0942-0.
- Yang, Z.; Chen, H.; Tang, W.; Hua, H.; Lin, Y. Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes. Pest Manag. Sci. 2011, 67, 414–422, doi:10.1002/ps.2079.
- Ramesh, S.; Nagadhara, D.; Pasalu, I.C.; Padma Kumari, A.; Sarma, N.P.; Reddy, V.D.; Rao, K.V. Development of stem borer resistant transgenic parental lines involved in the production of hybrid rice. J. Biotechnol. 2004, 111, 131–141, doi:10.1016/j.jbiotec.2004.04.004. 15219400
- Vemanna, R.S.; Chandrashekar, B.K.; Hanumantha Rao, H.M.; Sathyanarayanagupta, S.K.; Sarangi, K.S.; Nataraja, K.N.; Udayakumar, M. A modified multisite Gateway cloning strategy for consolidation of genes in plants. Mol. Biotechnol. 2012, doi:10.1007/s12033-012-9499-6.
- Halpin, C. Gene stacking in transgenic plants–the challenge for 21st century plant biotechnology. Plant Biotechnol. J. 2005, 3, 141–155, doi:10.1111/j.1467-7652.2004.00113.x.
- Dafny-Yelin, M.; Tzfira, T. Delivery of multiple transgenes to plant cells. Plant Physiol. 2007, 145, 1118–1128, doi:10.1104/pp.107.106104.
- Naqvi, S.; Farre, G.; Sanahuja, G.; Capell, T.; Zhu, C.; Christou, P. When more is better: Multigene engineering in plants. Trends Plant Sci. 2009, 15, 48–56. 19853493
- Saxena, P.; Hsieh, Y.C.; Alvarado, V.Y.; Sainsbury, F.; Saunders, K.; Lomonossoff, G.P.; Scholthof, H.B. Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless. Plant Biotechnol. J. 2011, 9, 703–712, doi:10.1111/j.1467-7652.2010.00574.x.
- Miki, B.; Abdeen, A.; Manaba, Y.; MacDonald, P. Selectable marker genes and unintended changes to the plant transcriptome. Plant Biotechnol. J. 2009, 7, 211–218, doi:10.1111/j.1467-7652.2009.00400.x.
- Uhlen, M.; Forsberg, G.; Moks, T.; Hartmanis, M.; Nilsson, B. Fusion proteins in biotechnology. Curr. Opin. Biotechnol. 1992, 3, 363–369, doi:10.1016/0958-1669(92)90164-E. 1368437
- Bouchard, É.; Cloutier, C.; Michaud, D. Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant. Mol. Ecol. 2003, 12, 2439–2446, doi:10.1046/j.1365-294X.2003.01919.x.
- Waldron, C.; Wegrich, L.M.; Owens Merlo, P.A.; Walsh, T.A. Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor. Plant Mol. Biol. 1993, 23, 801–812, doi:10.1007/BF00021535.
- Benchabane, M.; Schlüter, U.; Vorster, J.; Goulet, M.-C.; Michaud, D. Plant cystatins. Biochimie 2010, 92, 1657–1666, doi:10.1016/j.biochi.2010.06.006.
- Kiggundu, A.; Goulet, M.-C.; Goulet, C.; Dubuc, J.-F.; Rivard, D.; Benchabane, M.; Pépin, G.; van der Vyver, C.; Kunert, K.; Michaud, D. Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites. Plant J. 2006, 48, 403–413, doi:10.1111/j.1365-313X.2006.02878.x.
- Goulet, M.-C.; Dallaire, C.; Vaillancourt, L.-P.; Khalf, M.; Badri, A.M.; Preradov, A.; Duceppe, M.-O.; Goulet, C.; Cloutier, C.; Michaud, D. Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites. Plant Physiol. 2008, 146, 1010–1019, doi:10.1104/pp.108.115741.
- Walsh, T.A.; Strickland, J.A. Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains. Plant Physiol. 1993, 103, 1227–1234, doi:10.1104/pp.103.4.1227. 8290629
- de Maagd, R.A.; Kwa, M.S.G.; van der Klei, H.; Yamamoto, T.; Schipper, B.; Vlak, J.M.; Stiekema, W.J.; Bosch, D. Domain III substitution in Bacillus thuringiensis delta-endotoxin Cry1A(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition. Appl. Environ. Microbiol. 1996, 62, 1537–1543. 8633853
- Walters, F.S.; deFontes, C.M.; Hart, H.; Warren, G.W.; Chen, J.S. Lepidopteran-active variable-region sequence imparts Coleopteran activity in eCry3.1Ab, an engineered Bacillus thuringiensis hybrid insecticidal protein. Appl. Environ. Microbiol. 2010, 76, 3082–3088, doi:10.1128/AEM.00155-10. 20305020
- de Maagd, R.A.; Weemen-Hendriks, M.; Stiekema, W.J.; Bosch, D. Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1–Cry1C hybrids. Appl. Environ. Microbiol. 2000, 66, 1559–1563, doi:10.1128/AEM.66.4.1559-1563.2000.
- Naimov, S.; Weemen-Hendriks, M.; Dukiandjiev, S.; de Maagd, R.A. Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle. Appl. Environ. Microbiol. 2001, 67, 5328–5330, doi:10.1128/AEM.67.11.5328-5330.2001.
- Karlova, R.; Weemen-Hendriks, M.; Naimov, S.; Ceron, J.; Dukiandjiev, S.; de Maagd, R.A. Bacillus thuringiensis ∂-endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1–Cry1Ac hybrids. J. Invertebr. Pathol. 2005, 88, 169–172, doi:10.1016/j.jip.2004.11.004.
- Singh, P.K.; Kumar, M.; Chaturvedi, C.P.; Yadav, D.; Tuli, R. Development of a hybrid ∂-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura. Transg. Res. 2004, 13, 397–410, doi:10.1007/s11248-004-4908-7.
- Lopez-Pazos, S.A.; Rojas Arias, A.C.; Ospina, S.A.; Ceron, J. Activity of Bacillus thuringiensis hybrid protein against a lepidopteran and a coleopteran pest. FEMS Microbiol. Lett. 2010, 302, 93–98, doi:10.1111/j.1574-6968.2009.01821.x.
- Inanaga, H.; Kobayashi, D.; Kouzuma, Y.; Aoki-Yasunaga, C.; Kimura, M. Protein engineering of novel proteinase inhibitors and their effects on the growth of Spodoptera exigua larvae. Biosci. Biotechnol. Biochem. 2001, 65, 2259–2264, doi:10.1271/bbb.65.2259.
- Mehlo, L.; Gahakwa, D.; Nghia, P.T.; Loc, N.T.; Capell, T.; Gatehouse, J.A.; Gatehouse, A.M.R.; Christou, P. An alternative strategy for sustainable pest resistance in genetically enhanced crops. Proc. Natl. Acad. Sci. USA 2005, 102, 7812–7816, doi:10.1073/pnas.0502871102. 15908504
- Xia, L.; Long, X.; Ding, X.; Zhang, Y. Increase in insecticidal toxicity by fusion of the cry1Ac gene from Bacillus thuringiensis with the neurotoxin gene hwtx-I. Curr. Microbiol. 2009, 58, 52–57, doi:10.1007/s00284-008-9265-y. 18953606
- Xia, L.; Zheng, Z.; Ding, X.; Huang, F. The expression of a recombinant cry1Ac gene with subtilisin-like protease CDEP2 gene in acrystalliferous Bacillus thuringiensis by Red/ET homologous recombination. Curr. Microbiol. 2009, 59, 386–392, doi:10.1007/s00284-009-9449-0.
- Seo, J.H.; Yeo, J.S.; Cha, H.J. Baculoviral polyhedrin—Bacillus thuringiensis toxin fusion protein: A protein-based bio-insecticide expressed in Escherichia coli. Biotechnol. Bioeng. 2005, 92, 166–172, doi:10.1002/bit.20592.
- Cao, C.-W.; Liu, G.-F.; Wang, Z.-Y.; Yan, S.-C.; Ma, L.; Yang, C.-P. Response of the gypsy moth, Lymantria dispar to transgenic poplar, Populus simonii ×P. nigra, expressing fusion protein gene of the spider insecticidal peptide and Bt-toxin C-peptide. J. Insect Sci. 2010, 10, doi:10.1673/031.010.20001.
- Zheng, S.S.; An, C.C.; Li, Q.R.; Chen, Z.L. Study of transferring fusion protein gene of the spider insecticidal peptide and Bt-toxin C-peptide into cotton. Cotton Sci. 2002, 14, 348–351.
- Fan, H.J.; Hu, C.X.; Wang, Z.Y.; Liu, G.F. Resistance of transgenic Xiaohei poplars with fusion protein gene of the spider insecticidal peptide and Bt-toxin C-peptide to Clostera anachoreta (Fabricius) (Lepidoptera; Notodontidae). Acta Entomol. Sin. 2006, 49, 780–785.
- Wang, Z.Y.; Xue, Z.; Fang, H.J.; Zhan, Y.G. Resistance of transgenic Betula platyphylla to the defoliator Lymantria dispar. Sci. Silvae Sci. 2007, 43, 116–120.
- Fitches, E.; Audsley, N.; Gatehouse, J.A.; Edwards, J.P. Fusion proteins containing neuropeptides as novel insect control agents: Snowdrop lectin delivers fused allatostatin to insect haemolymph following oral ingestion. Insect Biochem. Mol. Biol. 2002, 32, 1653–1661, doi:10.1016/S0965-1748(02)00105-4.
- Fitches, E.; Edwards, M.G.; Mee, C.; Grishin, E.; Gatehouse, A.M.R.; Edwards, J.P.; Gatehouse, J.A. Fusion proteins containing insect-specific toxins as pest control agents: Snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion. J. Insect Physiol. 2004, 50, 61–71, doi:10.1016/j.jinsphys.2003.09.010.
- Fitches, E.C.; Bell, H.A.; Powell, M.E.; Back, E.; Sargiotti, C.; Weaver, R.J.; Gatehouse, J.A. Insecticidal activity of scorpion toxin (ButalT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders. Pest Manag. Sci. 2010, 66, 74–83, doi:10.1002/ps.1833.
- Trung, N.P.; Fitches, E.; Gatehouse, J.A. A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects. BMC Biotechnol. 2006, 6, doi:10.1186/1472-6750-6-18.
- Down, R.E.; Fitches, E.C.; Wiles, D.P.; Corti, P.; Bell, H.A.; Gatehouse, J.A.; Edwards, J.P. Insecticidal spider venom toxin fused to snowdrop lectin is toxic to the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae) and the rice brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). Pest Manag. Sci. 2006, 62, 77–85, doi:10.1002/ps.1119. 16206236
- Zhu-Salzman, K.; Ahn, J.-E.; Salzman, R.A.; Koiwa, H.; Shade, H.; Balfe, S. Fusion of a soybean cysteine protease inhibitor and a legume lectin enhances anti-insect activity synergistically. Agric. For. Entomol. 2003, 5, 317–323, doi:10.1046/j.1461-9563.2003.00194.x.
- Outchkourov, N.S.; de Kogel, W.J.; Wiegers, G.L.; Abrahamson, M.; Jongsma, M.A. Engineered multidomain cysteine protease inhibitors yield resistance against western flower thrips (Frankliniella occidentalis) in greenhouse trials. Plant Biotechnol. J. 2004, 2, 449–458, doi:10.1111/j.1467-7652.2004.00089.x.
- Brunelle, F.; Girard, C.; Cloutier, C.; Michaud, D. A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases. Arch. Insect Biochem. Physiol. 2005, 60, 20–31, doi:10.1002/arch.20078.
- Benchabane, M.; Goulet, M.-C.; Dallaire, C.; Côté, P.-L.; Michaud, D. Hybrid protease inhibitors for pest and pathogen control–a functional cost for the fusion partners? Plant Physiol. Biochem. 2008, 46, 701–708, doi:10.1016/j.plaphy.2008.04.005.
- Michaud, D. Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol. 1997, 15, 4–6, doi:10.1016/S0167-7799(96)10072-X.
- Tajne, S.; Sanam, R.; Gundla, R.; Gandhi, N.S.; Mancera, R.L.; Boddupally, D.; Vudem, D.R.; Khareedu, V.R. Molecular modeling of Bt Cry1Ac (DI-DII)-ASAL (Allium sativum lectin)-fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta. J. Mol. Graph. Model. 2012, 33, 61–76, doi:10.1016/j.jmgm.2011.11.001.
- Upadhyay, S.K.; Saurabh, S.; Rai, P.; Singh, R.; Chandrashekar, K.; Verma, P.C.; Singh, P.K.; Tuli, R. SUMO fusion facilitates expression and purification of garlic leaf lectin but modifies some of its properties. J. Biotechnol. 2010, 146, 1–8, doi:10.1016/j.jbiotec.2010.01.013.
- Arai, R.; Ueda, H.; Kitayama, A.; Kamiya, N.; Nagamune, T. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Prot. Eng. 2001, 14, 529–532, doi:10.1093/protein/14.8.529.
- Wriggers, W.; Chakravarty, S.; Jennings, P.A. Control of protein functional dynamics by peptide linkers. Biopolymers 2005, 80, 736–746, doi:10.1002/bip.20291.
- Crasto, C.J.; Feng, J. LINKER: A program to generate linker sequences for fusion proteins. Prot. Eng. 2000, 13, 309–312, doi:10.1093/protein/13.5.309.
- Xue, F.; Gu, Z.; Feng, J. LINKER: A web server to generate peptide sequences with extended conformation. Nucl. Acids Res. 2004, 32, W562–W565, doi:10.1093/nar/gkh422.
- Kavoosi, M.; Creagh, A.L.; Kilburn, D.G.; Haynes, C.A. Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli. Biotechnol. Bioeng. 2007, 98, 599–610, doi:10.1002/bit.21396.
- Rawlings, N.D.; Morton, F.R.; Kok, C.Y.; Kong, J.; Barrett, A.J. MEROPS: The peptidase database. Nucleic.. Acids Res. 2006, 36, D320–D325.
- George, R.A.; Heringa, J. An analysis of protein domain linkers: Their classification and role in protein folding. Prot. Eng. 2003, 15, 871–879.
- Arai, R.; Wriggers, W.; Nishikawa, Y.; Nagamune, T.; Fujisawa, T. Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering. Proteins 2004, 57, 829–838, doi:10.1002/prot.20244.
- Peschen, D.; Li, H.-P.; Fischer, R.; Kreuzaler, F.; Liao, Y.-C. Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen. Nat. Biotechnol. 2004, 22, 732–738, doi:10.1038/nbt970.
- Dandekar, A.M.; Gouran, H.; Ibanez, A.M.; Uratsu, S.L.; Agüero, C.B.; McFarland, S.; Borhani, Y.; Feldstein, P.A.; Bruening, G.; Nascimento, R.; et al. An engineered innate immune defense protects grapevines from Pierce disease. Proc. Natl. Acad. Sci. USA 2012, 109, 3721–3725, doi:10.1073/pnas.1116027109. 22355130
- Urwin, P.E.; McPherson, M.J.; Atkinson, H.J. Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs. Planta 1998, 204, 472–479, doi:10.1007/s004250050281.
- Lu, P.; Feng, M.G. Bifunctional enhancement of a beta-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Appl. Microbiol. Biotechnol. 2008, 79, 579–587, doi:10.1007/s00253-008-1468-4.
- Zhao, H.L.; Yao, X.Q.; Xue, C.; Wang, Y.; Xiong, X.H.; Liu, Z.M. Increasing the homogeneity, stability and activity of human serum albumin and interferon-alpha 2b fusion protein by linker engineering. Protein Expr. Purif. 2008, 61, 73–77, doi:10.1016/j.pep.2008.04.013.
- Sauer, J.; Christensen, T.; Frandsen, T.P.; Mirgorodskaya, E.; McGuire, K.A.; Driguez, H.; Roepstorff, P.; Sigurskjold, B.W.; Svensson, B. Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger. Biochemistry 2001, 40, 9336–9346, doi:10.1021/bi010515i. 11478902
- Wu, Y.J.; Fan, C.Y.; Li, Y.K. Protein purification involving a unique auto-cleavage feature of a repeated EAAAK peptide. J. Biosci. Bioeng. 2009, 108, S63–S63.
- Maia, I.G.; Seron, K.; Haenni, A.L.; Bernardi, F. Gene expression from viral RNA genomes. Plant Mol. Biol. 1996, 32, 367–391, doi:10.1007/BF00039391.
- Dougherty, W.G.; Semler, B.L. Expression of virus-encoded proteinases: Functional and structural similarities with cellular enzymes. Microbiol. Rev. 1993, 57, 781–822. 8302216
- Adams, M.J.; Antoniw, J.F.; Beaudoin, F. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol. Plant Pathol. 2005, 6, 471–487, doi:10.1111/j.1364-3703.2005.00296.x.
- Marcos, J.F.; Beachy, R.N. In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide. Plant Mol. Biol. 1994, 24, 495–503, doi:10.1007/BF00024117.
- Saunders, K.; Sainsbury, F.; Lomonossoff, G.P. Efficient generation of cowpea mosaic virus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants. Virology 2009, 393, 329–337, doi:10.1016/j.virol.2009.08.023.
- Carrington, J.; Dougherty, W. Processing of the tobacco etch virus 49K protease requires autoproteolysis. Virology 1987, 160, 355–362, doi:10.1016/0042-6822(87)90006-7.
- Goldbach, R.W.; Wellink, J. Comovirus: Molecular Biology and Replication. In The Plant Viruses; Harrison, B.D., Murrant, A.F., Eds.; Plenum Press: New York, NY, USA, 1996; Volume 5, pp. 35–76.
- Carrington, J.; Dougherty, W. A viral cleavage site cassette: Identification of amino acid sequences required for tobacco etch virus polyprotein processing. Proc. Natl. Acad. Sci. USA 1988, 85, 3391–3395, doi:10.1073/pnas.85.10.3391.
- Phan, J.; Zdanov, A.; Evdokimov, A.G.; Tropea, J.E.; Peters, H.K., III; Kapust, R.B.; Li, M.; Wlodawer, A.; Waugh, D.S. Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 2002, 277, 50564–50572, doi:10.1074/jbc.M207224200. 12377789
- von Bodman, S.B.; Domier, L.L.; Farrand, S.K. Expression of multiple eukaryotic genes from a single promoter in Nicotiana. Bio/Technology 1995, 13, 587–591, doi:10.1038/nbt0695-587. 9634798
- Bedoya, L.; Martinez, F.; Rubio, L.; Daros, J.-A. Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector. J. Biotechnol. 2010, 150, 268–275. 20728479
- Dasgupta, S.; Collins, G.B.; Hunt, A.G. Co-ordinated expression of multiple enzymes in different subcellular compartments in plants. Plant J. 1998, 16, 107–116, doi:10.1046/j.1365-313x.1998.00255.x.
- Marcos, J.F.; Beachy, R.N. Transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide. J. Gen. Virol. 1997, 78, 1771–1778. 9225054
- Ceriani, M.F.; Marcos, J.F.; Hopp, H.E.; Beachy, R.N. Simultaneous accumulation of multiple viral coat proteins from a TEV-NIa based expression vector. Plant Mol. Biol. 1998, 36, 239–248, doi:10.1023/A:1005952001774.
- Powell, W.A.; Catranis, C.M.; Maynard, C.A. Design of self-processing antimicrobial peptides for plant protection. Lett. Appl. Microbiol. 2000, 31, 163–168. 10972721
- Liang, H.; Gao, H.; Maynard, C.A.; Powell, W.A. Expression of a self-processing, pathogen resistance-enhancing gene construct in Arabidopsis. Biotechnol. Lett. 2005, 27, 435–442, doi:10.1007/s10529-005-1884-9.
- Zhang, B.; Rapolu, M.; Huang, L.; Su, W.W. Coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p-like protease activity. Plant Biotechnol. J. 2011, 9, 970–981, doi:10.1111/j.1467-7652.2011.00607.x.
- François, I.E.J.A.; Dwyer, G.I.; De Bolle, M.F.C.; Goderis, I.J.W.M.; van Hemelrijck, W.; Proost, P.; Wouters, P.; Broekaert, W.F.; Cammue, B.P.A. Processing in transgenic Arabidopsis thaliana plants of polyproteins with linker peptide variants derived from the Impatiens balsamina antimicrobial polyprotein precursor. Plant Physiol. Biochem. 2002, 40, 871–879, doi:10.1016/S0981-9428(02)01440-7.
- François, I.E.J.A.; de Bolle, M.F.C.; Dwyer, G.; Goderis, I.J.W.M.; Woutors, P.F.J.; Verhaert, P.D.; Proost, P.; Schaaper, W.M.M.; Cammue, B.P.A.; Broekaert, W.F. Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins. Plant Physiol. 2002, 128, 1346–1358, doi:10.1104/pp.010794.
- François, I.E.J.A.; van Hemelrijck, W.; Aerts, A.M.; Wouters, P.F.J.; Proost, P.; Broekaert, W.F.; Cammue, B.P.A. Processing in Arabidopsis thaliana of a heterologous polyprotein resulting in differential targeting of the individual plant defensins. Plant Sci. 2004, 166, 113–121, doi:10.1016/j.plantsci.2003.09.001.
- Walker, J.M.; Vierstra, R.D. A ubiquitin-based vector for the co-ordinate synthesis of multiple proteins in plants. Plant Biotechnol. J. 2007, 5, 413–421, doi:10.1111/j.1467-7652.2007.00250.x.
- Hunt, A.G.; Maiti, I.B. Strategies for expressing multiple foreign genes in plants as polycistronic constructs. In Vitro Cell. Dev. Biol. Plant 2001, 37, 313–320, doi:10.1007/s11627-001-0056-4.
- Luke, G.A. Translating 2A Research into Practice. In Innovations in Biotechnology; Agbo, E.C., Ed.; InTech Open: Rijeka, Croatia, 2012; pp. 165–186.
- Martinez-Salas, E. Internal ribosome entry site biology and its use in expression vectors. Curr. Opin. Biotechnol. 1999, 10, 458–464, doi:10.1016/S0958-1669(99)00010-5.
- Toth, R.L.; Chapman, S.; Carr, F.; Santa Cruz, S. A novel strategy for the expression of foreign genes from plant virus vectors. FEBS Lett. 2001, 489, 215–219, doi:10.1016/S0014-5793(01)02091-9.
- Dorokhov, Y.L.; Skulachev, M.V.; Ivanov, P.A.; Zvereva, S.D.; Tjulkina, L.G.; Merits, A.; Gleba, Y.Y.; Hohn, T.; Atabekov, J.G. Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc. Natl. Acad. Sci. USA 2002, 99, 5301–5306, doi:10.1073/pnas.082107599. 11959981
- Groppelli, E.; Belsham, G.J.; Roberts, L.O. Identification of minimal sequences of the Rhopalosiphum padi virus 5' untranslated region required for internal initiation of protein synthesis in mammalian, plant and insect translation systems. J. Gen. Virol. 2007, 88, 1583–1588, doi:10.1099/vir.0.82682-0.
- Urwin, P.E.; Zubko, E.I.; Atkinson, H.J. The biotechnological application and limitation of IRES to deliver multiple defence genes to plant pathogens. Physiol. Mol. Plant Pathol. 2002, 61, 103–108.
- Ali, Z.; Schumacher, H.M.; Heine-Dobbernack, E.; El-Banna, A.; Hafeez, F.Y.; Jacobsen, H.J.; Kiesecker, H. Dicistronic binary vector system—A versatile tool for gene expression studies in cell cultures and plants. J. Biotechnol. 2010, 145, 9–16, doi:10.1016/j.jbiotec.2009.10.002. 19835918
- El-Banna, A.; Hajirezaei, M.R.; Wissing, J.; Ali, Z.; Vaas, L.; Heine-Dobbernack, E.; Jacobsen, H.J.; Schumacher, H.M.; Kiesecker, H. Over-expression of PR-10a leads to increased salt and osmotic tolerance in potato cell cultures. J. Biotechnol. 2010, 150, 277–287. 20854851
- Urwin, P.E.; Yi, L.; Martin, H.; Atkinson, H.J.; Gilmartin, P.M. Functional characterization of the EMCV IRES in plants. Plant J. 2000, 24, 583–589, doi:10.1046/j.1365-313x.2000.00904.x.
- Ha, S.-H.; Liang, Y.S.; Jung, H.; Ahn, M.-J.; Suh, S.-C.; Kweon, S.-J.; Kim, D.-H.; Kim, Y.-M.; Kim, J.-K. Application of two bicistronic systems involving 2A and IRES sequences to the biosynthesis of carotenoids in rice endosperm. Plant Biotechnol. J. 2010, 8, 928–938, doi:10.1111/j.1467-7652.2010.00543.x.
- de Felipe, P.; Luke, G.A.; Hughes, L.E.; Gani, D.; Halpin, C.; Ryan, M.D. E unum pluribus: Multiple proteins from a self-processing polyprotein. Trends Biotechnol. 2006, 24, 68–75, doi:10.1016/j.tibtech.2005.12.006.
- Donnelly, M.L.L.; Luke, G.; Mehrotra, A.; Li, X.; Hughes, L.E.; Gani, D.; Ryan, M.D. Analysis of the aphthovirus 2A/2B polyprotein “cleavage” mechanism indicates not a proteolytic reaction, but a novel translational effect: A putative ribosomal “skip”. J. Gen. Virol. 2001, 82, 1013–1025. 11297676
- Sharma, P.; Yan, F.; Doronina, V.A.; Escuin-Ordinas, H.; Ryan, M.D.; Brown, J.D. 2A peptides provide distinct solutions to driving stop-carry on translational recoding. Nucl. Acids Res. 2012, 40, 3143–3151, doi:10.1093/nar/gkr1176.
- Halpin, C.; Cooke, S.E.; Barakate, A.; El Amrani, A.; Ryan, M.D. Self-processing 2A-polyproteins—A system for co-ordinate expression of multiple proteins in transgenic plants. Plant J. 1999, 17, 453–459, doi:10.1046/j.1365-313X.1999.00394.x.
- Ma, C.; Mitra, A. Expressing multiple genes in a single open reading frame with the 2A region of foot-and-mouth disease virus as a linker. Mol. Breed. 2002, 9, 191–199, doi:10.1023/A:1019760127368.
- Smolenska, L.; Roberts, I.M.; Learmonth, D.; Porter, A.J.; Harris, W.J.; Michael, T.; Wilson, A.; Santa Cruz, S. Production of a functional single chain antibody attached to the surface of a plant virus. FEBS Lett. 1998, 441, 379–382, doi:10.1016/S0014-5793(98)01586-5.
- Sainsbury, F.; Lavoie, P.-O.; D’Aoust, M.-A.; Vézina, L.-P.; Lomonossoff, G.P. Expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus RNA-2. Plant Biotechnol. J. 2008, 6, 82–92. 17986176
- Marconi, G.; Albertini, E.; Barone, P.; DeMarchis, F.; Lico, C.; Marusic, C.; Rutili, D.; Veronesi, F.; Porceddu, A. In planta production of two peptides of the Classical Swine Fever Virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X. BMC Biotechnol. 2006, 6, doi:10.1186/1472-6750-6-29.
- Ralley, L.; Enfissi, E.M.A.; Misawa, N.; Schuch, W.; Bramley, P.M.; Fraser, P.D. Metabolic engineering of ketocarotenoid formation in higher plants. Plant J. 2004, 39, 477–486, doi:10.1111/j.1365-313X.2004.02151.x.
- Randall, J.; Sutton, D.; Ghoshroy, S.; Bagga, S.; Kemp, J.D. Co-ordinate expression of β- and ∂-zeins in transgenic tobacco. Plant Sci. 2004, 167, 367–372, doi:10.1016/j.plantsci.2004.04.005.
- Kwon, S.-J.; Hwang, E.-W.; Kwon, H.-B. Genetic engineering of drought resistant potato plants by co-introduction of genes encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Zygosaccharomyces rouxii. Kor. J. Genet. 2004, 26, 199–206.
- Geu-Flores, F.; Olsen, C.E.; Halkier, B.A. Towards engineering glucosinolates into non-cruciferous plants. Planta 2009, 229, 261–270, doi:10.1007/s00425-008-0825-y.
- Fang, J.; Qian, J.J.; Yi, S.; Harding, T.C.; Tu, G.H.; VanRoey, M.; Jooss, K. Stable antibody expression at therapeutic levels using the 2A peptide. Nat. Biotechnol. 2005, 23, 584–590, doi:10.1038/nbt1087.
- Fang, J.; Yi, S.; Simmons, A.; Tu, G.; Nguyen, M.; Harding, T.C.; VanRoey, M.; Jooss, K. An antibody delivery system for regulated expression of therapeutic levels of monoclonal antibodies in vivo. Mol. Ther. 2007, 15, 1153–1159. 17375065
- Luke, G.; Escuin, H.; de Filipe, P.; Ryan, M. 2A to the fore-research, technology and applications. Biotechnol. Genet. Eng. Rev. 2010, 26, 223–260. 21415883
- El Amrani, A.; Barakate, A.; Askari, B.M.; Li, X.; Roberts, A.G.; Ryan, M.D.; Halpin, C. Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene. Plant Physiol. 2004, 135, 16–24, doi:10.1104/pp.103.032649.
- Szymczak, A.L.; Workman, C.J.; Wang, Y.; Vignali, K.M.; Dilioglou, S.; Vanin, E.F.; Vignali, D.A.A. Correction of multi-gene deficiency in vivo using a single “self-cleaving” 2A peptide-based retroviral vector. Nat. Biotechnol. 2004, 22, 589–594, doi:10.1038/nbt957.
- de Felipe, P.; Luke, G.A.; Brown, J.D.; Ryan, M.D. Inhibition of 2A-mediated “cleavage” of certain artificial polyproteins bearing N-terminal signal sequences. Biotechnol. J. 2010, 5, 213–223, doi:10.1002/biot.200900134. 19946875
- Schlüter, U.; Benchabane, M.; Munger, A.; Kiggundu, A.; Vorster, J.; Goulet, M.-C.; Cloutier, C.; Michaud, D. Recombinant protease inhibitors for herbivore pest control: A multitrophic perspective. J. Exp. Bot. 2010, 61, 4169–4183, doi:10.1093/jxb/erq166.
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