Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.     

The regulation landscape of MAPK signaling cascade for thwarting Bacillus thuringiensis infection in an insect host

Host-pathogen interactions are central components of ecological networks where the MAPK signaling pathways act as central hubs of these complex interactions. We have previously shown that an insect hormone modulated MAPK signaling cascade participates as a general switch to trans-regulate differential expression of diverse midgut genes in the diamondback moth, Plutella xylostella (L.) to cope with the insecticidal action of Cry1Ac toxin, produced by the entomopathogenic bacterium Bacillus thuringiensis (Bt). The relationship between topology and functions of this four-tiered phosphorylation signaling cascade, however, is an uncharted territory. Here, we carried out a genome-wide characterization of all the MAPK orthologs in P. xylostella to define their phylogenetic relationships and to confirm their evolutionary conserved modules. Results from quantitative phosphoproteomic analyses, combined with functional validations studies using specific inhibitors and dsRNAs lead us to establish a MAPK “road map”, where p38 and ERK MAPK signaling pathways, in large part, mount a resistance response against Bt toxins through regulating the differential expression of multiple Cry toxin receptors and their non-receptor paralogs in P. xylostella midgut. These data not only advance our understanding of host-pathogen interactions in agricultural pests, but also inform the future development of biopesticides that could suppress Cry resistance phenotypes.     

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism.     

Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the midgut of silkworm

Identification of the resistance mechanism of insects against Bacillus thuringiensis Cry1A toxin is becoming an increasingly challenging task. This fact highlights the need for establishing new methods to further explore the molecular interactions of Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their wider application as biopesticides and a gene source for gene-modified crops. In this contribution, a quantum dot-based near-infrared fluorescence imaging method has been applied for direct dynamic tracking of the specific binding of Cry1A toxins, CrylAa and CrylAc, to the midgut tissue of silkworm. The in vitro fluorescence imaging displayed the higher binding specificity of CrylAa–QD probes compared to CrylAc–QD to the brush border membrane vesicles of midgut from silkworm. The in vivo imaging demonstrated that more CrylAa–QDs binding to silkworm midgut could be effectively and distinctly monitored in living silkworms. Furthermore, frozen section analysis clearly indicated the broader receptor-binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor-binding sites, and this scatheless and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism to maintain the insecticidal activity of B. thuringiensis toxins.     

A Spodoptera exigua Cadherin Serves as a Putative Receptor for Bacillus thuringiensis Cry1Ca Toxin and Shows Differential Enhancement of Cry1Ca and Cry1Ac Toxicity

Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca.     

Mechanism of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Greenhouse Population of the Cabbage Looper, Trichoplusia ni

The cabbage looper, Trichoplusia ni, is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni. This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.     

Variability in the cadherin gene in an Ostrinia nubilalis strain selected for Cry1Ab resistance

Transgenic corn expressing Cry1Ab (a Bacillus thuringiensis toxin) is highly effective in the control of Ostrinia nubilalis. For its toxic action, Cry1Ab has to bind to specific insect midgut proteins. To date, in three Lepidoptera species resistance to a Cry1A toxin has been conferred by mutations in cadherin, a protein of the Lepidoptera midgut membrane. The implication of cadherin in the resistance of an Ostrinia nubilalis colony (Europe-R) selected with Bacillus thuringiensis Cry1Ab protoxin was investigated. Several major mutations in the cadherin (cdh) gene were found, which introduced premature termination codons and/or large deletions (ranging from 1383 to 1701 bp). The contribution of these major mutations to the resistance was analyzed in resistant individuals that survived exposure to a high concentration of Cry1Ab protoxin. The results indicated that the presence of major mutations was drastically reduced in individuals that survived exposure. Previous inheritance experiments with the Europe-R strain indicated the involvement of more than one genetic locus and reduced amounts of the cadherin receptor. The results of the present work support a polygenic inheritance of resistance in the Europe-R strain, in which mutations in the cdh gene would contribute to resistance by means of an additive effect.     

Potency of the mosquitocidal Cry46Ab toxin produced using a 4AaCter-tag,which facilitates formation of protein inclusion bodies in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A Cry46Ab toxin derived from Bacillus thuringiensis strain TK-E6 shows mosquitocidal activity against Culex pipiens pallens Coquillett (Diptera: Culicidae) larvae as well as preferential cytotoxicity against human cancer cells. In B. thuringiensis cells, Cry46Ab is produced and accumulates as a protein crystal that is processed into the active 29-kDa toxin upon solubilization in the alkaline environment of the insect midgut. The Cry46Ab protoxin is 30 kDa, and is therefore thought to require an accessory protein such as P20 and/or ORF2 for efficient crystal formation. In the present study, the potency of the 4AaCter-tag was investigated for the production of alkali-soluble inclusion bodies of recombinant Cry46Ab in Escherichia coli. The 4AaCter-tag is a polypeptide derived from the C-terminal region of the B. thuringiensis Cry4Aa toxin and facilitates the formation of alkali-soluble protein inclusion bodies in E. coli. Fusion with the 4AaCter-tag enhanced both Cry46Ab production and the formation of Cry46Ab inclusion bodies. In addition, upon optimization of protein expression procedures, the Cry46Ab–4AaCter inclusion bodies showed mosquitocidal activity and stability in aqueous environments comparable to Cry46Ab without the 4AaCter-tag. Our study suggests that use of the 4AaCter-tag is a straightforward approach for preparing formulations of smaller-sized Cry toxins such as Cry46Ab in E. coli.     

Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.     

Toxicity, Binding, and Permeability Analyses of Four Bacillus thuringiensis Cry1 δ-Endotoxins Using Brush Border Membrane Vesicles of Spodoptera exigua and Spodoptera frugiperda

The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with ¹²⁵I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. ¹²⁵I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles from S. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.     

Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae

Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with ¹²⁵I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution.     

Lack of Detrimental Effects of Bacillus thuringiensis Cry Toxins on the Insect Predator Chrysoperla carnea: a Toxicological, Histopathological, and Biochemical Analysis

The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armigera larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated H. armigera larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from Spodoptera exigua but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.     

Cross-Resistance of Pink Bollworm (Pectinophora gossypiella) to Bacillus thuringiensis Toxins

Two strains of pink bollworm (Pectinophora gossypiella) selected in the laboratory for resistance to Bacillus thuringiensis toxin Cry1Ac had substantial cross-resistance to Cry1Aa and Cry1Ab but not to Cry1Bb, Cry1Ca, Cry1Da, Cry1Ea, Cry1Ja, Cry2Aa, Cry9Ca, H04, or H205. The narrow spectrum of resistance and the cross-resistance to activated toxin Cry1Ab suggest that reduced binding of toxin to midgut target sites could be an important mechanism of resistance.     

Southern analysis of BT-R 1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210?kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp. berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210?kDa protein, termed BT-R₁ (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R ₁ , the gene encoding the 210?kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R₁ as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R₁. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R₁, whereas the other is a closely related homologue.     

Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment

A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity.     

Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae

The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC₅₀ of Lip and HD1 were 33.27 and 128.61 μg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.     

Variant cry1Ab entomocidal Bacillus thuringiensis toxin gene facilitates the recovery of an increased number of lepidopteran insect resistant independent rice transformants against yellow stem borer (Scirpophaga incertulus) inflicted damage

The primary technical constraint plant scientists face in generating insect resistant transgenic crops with insecticidal Bacillus thuringiensis (Bt) crystal protein (Cry) genes remains failing to generate sufficiently large numbers of effective resistant transgenic plant lines. One possible means to overcome this challenge is through deployment of a Cry toxin gene that contains high levels of insecticidal specific activity for target insect pests. In the present study, we tested this hypothesis using a natural variant of the Cry1Ab toxin under laboratory conditions that possessed increased insecticidal potency against the yellow stem borer (YSB, Scirpophaga incertulus), one of the most damaging rice insect pests. Following adoption of a stringent selection strategy for YSB resistant transgenic rice lines under field conditions, results showed recovery of a significantly higher number of YSB resistant independent transgenic plant lines with the variant cry1Ab gene relative to transgenic plant lines harbouring cry1Ab berliner gene. Structural homology modelling of the variant toxin peptide with the Cry1Aa toxin molecule, circular dichroism spectral analysis, and hydropathy plot analysis indicated that serine substitution by phenylalanine at amino acid position 223 of the Cry1Ab toxin molecule resulted in a changed role for α-helix 7 in domain I of Cry1Ab for enhanced toxicity.