Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

Background

Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F₂ screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac.

Methodology/Principal Findings

Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with ¹²⁵I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in ¹²⁵I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins.

Conclusion/Significance

This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.     

Field-evolved resistance to Bt maize by western corn rootworm

Background

Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are planted on millions of hectares annually, reducing the use of conventional insecticides and suppressing pests. However, the evolution of resistance could cut short these benefits. A primary pest targeted by Bt maize in the United States is the western corn rootworm Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae).

Methodology/Principal Findings

We report that fields identified by farmers as having severe rootworm feeding injury to Bt maize contained populations of western corn rootworm that displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays than did western corn rootworm from fields not associated with such feeding injury. In all cases, fields experiencing severe rootworm feeding contained Cry3Bb1 maize. Interviews with farmers indicated that Cry3Bb1 maize had been grown in those fields for at least three consecutive years. There was a significant positive correlation between the number of years Cry3Bb1 maize had been grown in a field and the survival of rootworm populations on Cry3Bb1 maize in bioassays. However, there was no significant correlation among populations for survival on Cry34/35Ab1 maize and Cry3Bb1 maize, suggesting a lack of cross resistance between these Bt toxins.

Conclusions/Significance

This is the first report of field-evolved resistance to a Bt toxin by the western corn rootworm and by any species of Coleoptera. Insufficient planting of refuges and non-recessive inheritance of resistance may have contributed to resistance. These results suggest that improvements in resistance management and a more integrated approach to the use of Bt crops may be necessary.     

A meta-analysis of effects of Bt crops on honey bees (Hymenoptera: Apidae)

Background

Honey bees (Apis mellifera L.) are the most important pollinators of many agricultural crops worldwide and are a key test species used in the tiered safety assessment of genetically engineered insect-resistant crops. There is concern that widespread planting of these transgenic crops could harm honey bee populations.

Methodology/Principal Findings

We conducted a meta-analysis of 25 studies that independently assessed potential effects of Bt Cry proteins on honey bee survival (or mortality). Our results show that Bt Cry proteins used in genetically modified crops commercialized for control of lepidopteran and coleopteran pests do not negatively affect the survival of either honey bee larvae or adults in laboratory settings.

Conclusions/Significance

Although the additional stresses that honey bees face in the field could, in principle, modify their susceptibility to Cry proteins or lead to indirect effects, our findings support safety assessments that have not detected any direct negative effects of Bt crops for this vital insect pollinator.     

Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb Toxicities to Coleopteran Larvae by a Toxin-Binding Fragment of an Insect Cadherin

The Cry3Aa and Cry3Bb insecticidal proteins of Bacillus thuringiensis are used in biopesticides and transgenic crops to control larvae of leaf-feeding beetles and rootworms. Cadherins localized in the midgut epithelium are identified as receptors for Cry toxins in lepidopteran and dipteran larvae. Previously, we discovered that a peptide of a toxin-binding cadherin expressed in Escherichia coli functions as a synergist for Cry1A toxicity against lepidopteran larvae and Cry4 toxicity against dipteran larvae. Here we report that the fragment containing the three most C-terminal cadherin repeats (CR) from the cadherin of the western corn rootworm binds toxin and enhances Cry3 toxicity to larvae of naturally susceptible species. The cadherin fragment (CR8 to CR10 [CR8-10]) of western corn rootworm Diabrotica virgifera virgifera was expressed in E. coli as an inclusion body. By an enzyme-linked immunosorbent microplate assay, we demonstrated that the CR8-10 peptide binds α-chymotrypsin-treated Cry3Aa and Cry3Bb toxins at high affinity (11.8 nM and 1.4 nM, respectively). Coleopteran larvae ingesting CR8-10 inclusions had increased susceptibility to Cry3Aa or Cry3Bb toxin. The Cry3 toxin-enhancing effect of CR8-10 was demonstrated for Colorado potato beetle Leptinotarsa decemlineata, southern corn rootworm Diabrotica undecimpunctata howardi, and western corn rootworm. The extent of Cry3 toxin enhancement, which ranged from 3- to 13-fold, may have practical applications for insect control. Cry3-containing biopesticides that include a cadherin fragment could be more efficacious. And Bt corn (i.e., corn treated with B. thuringiensis to make it resistant to pests) coexpressing Cry3Bb and CR8-10 could increase the functional dose level of the insect toxic activity, reducing the overall resistance risk.The Cry3 class of Bacillus thuringiensis Cry proteins is known for toxicity to coleopteran larvae in the family Chrysomelidae. Cry3Aa and Cry3Bb proteins are highly toxic to Colorado potato beetle (CPB) Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), and both were used for the development of Bt crops (crops treated with B. thuringiensis to make them resistant to pests) and Bt biopesticides. Due to the limited efficacy of Cry3-based biopesticides/plants and the success of competing chemical pesticides, these biopesticides have had limited usage and sales (12). Cry3Bb is toxic to corn rootworms (8, 17), and a modified version is expressed in commercialized MON863 corn hybrids (26).Cry3 toxins have a mode of action that is similar to, yet distinct from, the action of lepidopteran-active Cry1 toxins. The Cry3A protoxin (73 kDa) lacks the large C-terminal region of the 130-kDa Cry1 protoxins, which is removed by proteases during activation to toxin. The Cry3A protoxin is activated to a 55-kDa toxin and then further cleaved within the toxin molecule (5, 18). Activated Cry3A toxin binds to brush border membrane vesicles with a K_d (dissociation constant) of ∼37 nM (19) and recognizes a 144-kDa binding protein in brush border membrane vesicles prepared from the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (2). Recently, Ochoa-Campuzano et al. (20) identified an ADAM metalloprotease as a receptor for Cry3Aa toxin in CPB larvae.Structural differences between Cry3Bb and Cry3Aa toxins must underlie the unique rootworm activities of Cry3Bb toxin. As noted by Galitsky et al. (11), differences in toxin solubility, oligomerization, and binding are reported for these Cry3 toxins. Recently, Cry3Aa was modified to have activity against western corn rootworm (WCRW) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) (27). Those authors introduced a chymotrypsin/cathepsin G cleavage site into domain 1 of Cry3Aa that allowed the processing of the 65-kDa form to a 55-kDa toxin that bound rootworm midgut.Cadherins function as receptors for Cry toxins in lepidopteran and dipteran larvae. A critical Cry1 toxin binding site is localized within the final cadherin repeat (CR), CR12, of cadherins from tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) and tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) (14, 28). Unexpectedly, a fragment of B. thuringiensis R₁ cadherin, the Cry1A receptor from M. sexta, not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (6). If the binding residues within CR12 were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist. Recently, we identified a cadherin from mosquito Anopheles gambiae (Diptera: Culicidae) that binds Cry4Ba toxin and probably functions as a receptor. We discovered a similar effect where a fragment of a cadherin from A. gambiae enhanced the toxicity of the mosquitocidal toxin Cry4Ba to mosquito larvae (15). Sayed et al. (22) identified a novel cadherin-like gene in WCRW and proposed this protein as a candidate Bt toxin receptor. The cadherin-like gene is highly expressed in the midgut tissue of larval stages. The encoded protein is conserved in structure relative to that of other insect midgut cadherins.In this study, we hypothesized that a fragment from a beetle cadherin that contains a putative Bt toxin binding region might enhance the insecticidal toxicities of Cry3Aa and Cry3Bb toxins. The region spanning CR8 to CR10 (CR8-10) of the WCRW cadherin (22) was cloned and expressed in E. coli. This cadherin fragment significantly enhanced the toxicities of Cry3Aa and Cry3Bb toxins to CPB and rootworms.     

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus

Background

Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling.

Results

Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster.

Conclusions

Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1763-2) contains supplementary material, which is available to authorized users.     

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

Background

Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.

Results

Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2^nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.

Conclusion

Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

Oligomerization of Cry11Aa from Bacillus thuringiensis Has an Important Role in Toxicity against Aedes aegypti

Cry11Aa and Cyt1Aa of Bacillus thuringiensis are active against mosquitoes and show synergism. Cyt1Aa functions as a membrane receptor inducing Cry11Aa oligomerization. Here we characterized Cry11Aa helix α-3 mutants impaired in oligomerization and toxicity against Aedes aegypti, indicating that oligomerization of Cry11Aa is important for toxin action. Cyt1Aa did not recover the insecticidal activity of Cry11Aa mutants.Bacillus thuringiensis subsp. israelensis has been used worldwide for the control of different mosquitoes that are vectors of several human diseases (10, 11). This bacterium produces different toxins that individually show activity against mosquitoes, i.e., Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa (2). The toxicity of Cry11Aa and Cry4 toxins against Aedes aegypti is greatly increased in the presence of sublethal concentrations of Cyt1Aa (14). Also, Cyt1Aa overcomes the resistance of the Culex quinquefasciatus population to Cry11Aa (12, 13). Cyt1Aa synergizes the toxic activity of Cry11Aa by functioning as a Cry11Aa receptor, facilitating the oligomerization of Cry11Aa and its pore formation activity (7, 8). Oligomerization is a complex event that involves interaction with a toxin receptor and further proteolysis of helix α-1 (3). In the case of the Cry1Ab toxin, helix α-3 of domain I contains coiled-coil structures that are important for oligomerization (4). Some point mutations in helix α-3 do not affect interaction with receptors but severely affected oligomerization, influencing pore formation and toxicity against Manduca sexta larvae (4).Since binding with Cyt1Aa facilitates Cry11Aa oligomerization, we hypothesize that Cry11Aa mutants unable to oligomerize would be affected in synergism with Cyt1Aa and in toxicity. In this report, we analyzed the effect of point mutations in helix α-3 of Cry11Aa on oligomerization, synergism with Cyt1Aa, and toxicity against A. aegypti larvae.Helix α-3 of Cry11Aa potentially forms coiled-coil structures, as determined by the program COILS, which calculates the probability that a sequence will adopt a coiled-coil conformation (6). The coiled-coil structures are characterized by heptads of residues (abcdefg), where positions a and d are occupied mostly by apolar residues and g and e by charged residues. Here we mutagenized some residues located at positions g and a of the predicted coiled-coil (Fig. ​(Fig.1).1). Substitutions R90E, E97A, Y98E, V104E, and S105E were produced by site-directed mutagenesis (Quick Change; Stratagene, La Jolla, CA) using the pCG6 plasmid (1) as a template and appropriate mutagenic oligonucleotides. Point mutations were verified by automated DNA sequencing at Instituto de Biotecnología-UNAM and transformed into the acrystalliferous B. thuringiensis 407 strain. B. thuringiensis strains were grown in solid nutrient broth sporulation medium supplemented with 10 μg/ml erythromycin (5). Crystal inclusions were purified as described previously (8) and solubilized in 100 mM NaOH for 1 h at 4°C. After solubilization, the Cry11Aa protoxins were dialyzed for 12 h against 50 mM Na₂CO₃, pH 10.5. The pH was equilibrated at pH 8.6 with equal volumes of 1 M Tris-HCl, pH 8, and protoxins were activated with trypsin (1:50, wt/wt) for 2 h at 25°C. All mutants, with the exception of the V104E mutant, which was not analyzed further, produced crystal inclusions similar to those for the wild-type toxin, composed of a 70-kDa protoxin (Fig. ​(Fig.2A).2A). After trypsin activation, all mutants produced two polypeptides of 32 and 36 kDa, similarly to the Cry11Aa toxin, suggesting that these mutations did not cause a major structural disturbance (Fig. ​(Fig.2B).2B). The Cry11Aa and mutant activated toxins were analyzed by circular dichroism spectroscopy (Fig. ​(Fig.2C).2C). The activated toxins were dialyzed against 10 mM Na₂HPO₄, 50 mM NaF, pH 9, and then purified by anion-exchange chromatography with HiTrap Q-Sepharose (Pharmacia LKB Biotechnology) in the same buffer, using a linear NaF gradient from 50 to 400 mM. The similarities among the curves indicate that the mutant toxins have a structure similar to that of the wild-type toxin.Open in a separate windowFIG. 1.Schematic representation of the coiled-coil structures of the α-3 helices of Cry1Ab and Cry11Aa toxins. The positions of residues a, b, c, d, e, f, and g of the heptads are presented. The mutated residues in both toxins that affected oligomerization and toxicity are shown in boldface type (reference 4 and this work).Open in a separate windowFIG. 2.SDS-PAGE analysis and circular dichroism spectra of Cry11Aa mutant toxins. (A) The Cry11Aa protoxins were solubilized at pH 10.5 and analyzed by SDS-PAGE (15% acrylamide). (B) SDS-PAGE analysis (15% acrylamide) of the activated toxins with trypsin. Both SDS-polyacrylamide gels were stained with Coomassie blue. Lanes 1, Cry11Aa; lanes 2, E97A mutant; lanes 3, Y98E mutant; lanes 4, R90E mutant; lanes 5, S105E mutant. (C) Analysis of the secondary-structure compositions of the mutants and Cry11Aa activated toxins. Circular dichroism spectra were recorded with a Jasco model J-715 spectropolarimeter equipped with a Peltier temperature control supplied by Jasco. Spectra were collected from 190 to 250 nm. Eight replicate spectra were collected for each sample to improve the signal-to-noise ratios. The final purified-protein concentration was 0.3 mg/ml, and spectra were collected in a 0.1-cm-pathlength cell. The secondary-structure prediction was performed using the CDSSTR algorithm (1a, 11a). Solid black line, Cry11Aa; dotted black line, E97A mutant; dashed black line, Y98E mutant; solid gray line, R90E mutant; dotted gray line, S105E mutant; MRE, mean residue ellipticity; [θ], ellipticity.The toxicity of spore/crystal suspensions of Cry11Aa or the individual mutants (75 to 10,000 ng/ml) was analyzed with bioassays against 10 fourth-instar A. aegypti larvae reared at 28°C, 87% humidity, and 12:12 light-dark conditions in 100 ml dechlorinated water, and mortality was scored after 24 h (four independent assays). The Cry11Aa toxin showed a mean lethal concentration of 355 ng/ml, with 95% confidence limits of 265 to 446 (Probit analysis using Polo-PC LeOra Software). In contrast, the R90E, E97A, Y98E, and S105E mutants were severely affected in toxicity against A. aegypti larvae, since no mortality was observed at the highest concentration used (10,000 ng/ml).We then analyzed the oligomerization of Cry11Aa toxins as previously described (8). Small unilamelar vesicles (SUV), composed of egg yolk phosphatidyl choline, cholesterol (Avanti Polar Lipids, Alabaster, AL), and stearylamine (Sigma, St. Louis, MO) at a 10:3:1 proportion, respectively, were used (8). Cyt1Aa was purified from the 4Q7/pWF45 strain (14) grown as described above. Cyt1Aa inclusions were purified by sucrose gradients, solubilized in 50 mM Na₂CO₃, 10 mM dithiothreitol, pH 10.5 (2 h at 30°C), and activated with 1:100 proteinase K (Sigma-Aldrich Co.), wt/wt, for 20 min at 30°C.For oligomerization assays, 2.5 μg soluble Cry11Aa or mutant protoxin was incubated for 2 h at 37°C in a 100-μl final volume of 50 mM Na₂CO₃, pH 10.5, with 200 μM SUV, 1:50 trypsin (wt/wt), and 0.5 μg Cyt1Aa activated toxin. After 2 h of incubation, 1 mM phenylmethylsulfonyl fluoride was added to stop the reaction, and the membrane fraction was separated by centrifugation (1 h at 100,000 × g). The pellet was suspended in the same buffer solution. Oligomeric structures of Cry toxins are highly stable after boiling as well as after urea denaturation (9). The suspension was boiled for 4 min, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8% acrylamide), and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The oligomeric and monomeric structures of Cry11Aa were detected using polyclonal anti-Cry11Aa antibody (1/15,000; 1 h) and a secondary antibody coupled with horseradish peroxidase (Sigma, St. Louis, MO) (1/5,000; 1 h) followed by luminol (ECL; Amersham Pharmacia Biotech) as described by the manufacturers. Figure ​Figure33 shows that only the Cry11Aa wild-type toxin was able to oligomerize, while the mutants were severely impaired in oligomerization.Open in a separate windowFIG. 3.Analysis of Cry11Aa oligomer formation. Soluble Cry11Aa protoxin was activated with trypsin for 2 h at 37°C in the presence of SUV and Cyt1Aa activated toxin. The membrane fraction was separated by ultracentrifugation, and the Cry11Aa protein was analyzed by Western blotting of the membrane pellet with polyclonal anti-Cry11A antibody. The sizes of the proteins were estimated from a molecular prestained plus standard, all blue (Bio-Rad). Lane 1, Cry11Aa; lane 2, R90E mutant; lane 3, Y98E mutant; lane 4, E97A mutant; lane 5, S105E mutant.Finally, the synergistic activity between Cyt1Aa and Cry11Aa was analyzed. A concentration of Cyt1Aa that produced 10% mortality was assayed in the presence of a protein concentration of wild-type Cry11A that produced 20% mortality. Larvae were examined 24 h after treatment, in three repetitions. This particular protein mixture produced a synergism factor of 8. Under these conditions, mortality was more than 80%, due to the synergistic activities of both toxins. Similar experiments were performed with the mutant toxins, using the same concentration of Cyt1Aa toxin and different concentrations (up to 6,000 ng/ml) of the mutant toxins. Cyt1A did not increase the toxicity of the Cry11Aa mutants, since only 10% mortality was observed, even at the highest concentration of the mutant toxins.Previously, helix α-3 of a lepidopteran-specific toxin (Cry1Ab) was subjected to mutagenesis. The R99E and Y107E mutants of the Cry1Ab toxin were severely impaired in oligomerization and toxicity, showing that oligomer formation is a necessary step to kill the larvae (4). The data presented here indicate that oligomer formation is also an essential step in the mechanism of toxicity of the mosquitocidal Cry11Aa toxin and that helix α-3 is involved in this process.     

Transgenic Cry1Ab rice does not impact ecological fitness and predation of a generalist spider

Background

The commercial release of rice genetically engineered to express a Cry1Ab protein from Bacillus thuringiensis (Bt) for control of Lepidoptera in China is a subject of debate. One major point of the debate has focused on the ecological safety of Bt rice on nontarget organisms, especially predators and parasitoids that help control populations of insect pests.

Methodology/Principal Findings

A tritrophic bioassay was conducted to evaluate the potential impact of Cry1Ab-expressing rice on fitness parameters of a predaceous ground spider (Pardosa pseudoannulata (Bösenberg et Strand)) that had fed on Bt rice-fed brown planthopper (Nilaparvata lugens (Stål)) nymphs. Survival, development time and fecundity of this spider were not different when they were fed with Bt rice-fed or non-Bt rice-fed prey. Furthermore, ELISA and PCR gut assays, as well as a functional response trial, indicated that predation by P. pseudoannulata was not significantly different in Bt rice or non-Bt rice fields.

Conclusions/Significance

The transgenic Cry1Ab rice lines tested in this study had no adverse effects on the survival, developmental time and fecundity of P. pseudoannulata in the laboratory or on predation under field conditions. This suggests that this important predator would not be harmed if transgenic Cry1Ab rice were commercialized.     

Neonicotinoid insecticide imidacloprid causes outbreaks of spider mites on elm trees in urban landscapes

Background

Attempts to eradicate alien arthropods often require pesticide applications. An effort to remove an alien beetle from Central Park in New York City, USA, resulted in widespread treatments of trees with the neonicotinoid insecticide imidacloprid. Imidacloprid''s systemic activity and mode of entry via roots or trunk injections reduce risk of environmental contamination and limit exposure of non-target organisms to pesticide residues. However, unexpected outbreaks of a formerly innocuous herbivore, Tetranychus schoenei (Acari: Tetranychidae), followed imidacloprid applications to elms in Central Park. This undesirable outcome necessitated an assessment of imidacloprid''s impact on communities of arthropods, its effects on predators, and enhancement of the performance of T. schoenei.

Methodology/Principal Findings

By sampling arthropods in elm canopies over three years in two locations, we document changes in the structure of communities following applications of imidacloprid. Differences in community structure were mostly attributable to increases in the abundance of T. schoenei on elms treated with imidacloprid. In laboratory experiments, predators of T. schoenei were poisoned through ingestion of prey exposed to imidacloprid. Imidacloprid''s proclivity to elevate fecundity of T. schoenei also contributed to their elevated densities on treated elms.

Conclusions/Significance

This is the first study to report the effects of pesticide applications on the arthropod communities in urban landscapes and demonstrate that imidacloprid increases spider mite fecundity through a plant-mediated mechanism. Laboratory experiments provide evidence that imidacloprid debilitates insect predators of spider mites suggesting that relaxation of top-down regulation combined with enhanced reproduction promoted a non-target herbivore to pest status. With global commerce accelerating the incidence of arthropod invasions, prophylactic applications of pesticides play a major role in eradication attempts. Widespread use of neonicotinoid insecticides, however, can disrupt ecosystems tipping the ecological balance in favor of herbivores and creating pest outbreaks.     

Unique Mechanism of the Interaction between Honey Bee Toxin TPNQ and rKir1.1 Potassium Channel Explored by Computational Simulations: Insights into the Relative Insensitivity of Channel towards Animal Toxins

Background

The 21-residue compact tertiapin-Q (TPN_Q) toxin, a derivative of honey bee toxin tertiapin (TPN), is a potent blocker of inward-rectifier K⁺ channel subtype, rat Kir1.1 (rKir1.1) channel, and their interaction mechanism remains unclear.

Principal Findings

Based on the flexible feature of potassium channel turrets, a good starting rKir1.1 channel structure was modeled for the accessibility of rKir1.1 channel turrets to TPN_Q toxin. In combination with experimental alanine scanning mutagenesis data, computational approaches were further used to obtain a reasonable TPN_Q toxin-rKir1.1 channel complex structure, which was completely different from the known binding modes between animal toxins and potassium channels. TPN_Q toxin mainly adopted its helical domain as the channel-interacting surface together with His12 as the pore-blocking residue. The important Gln13 residue mainly contacted channel residues near the selectivity filter, and Lys20 residue was surrounded by a polar “groove” formed by Arg118, Thr119, Glu123, and Asn124 in the channel turret. On the other hand, four turrets of rKir1.1 channel gathered to form a narrow pore entryway for TPN_Q toxin recognition. The Phe146 and Phe148 residues in the channel pore region formed strong hydrophobic protrusions, and produced dominant nonpolar interactions with toxin residues. These specific structure features of rKir1.1 channel vestibule well matched the binding of potent TPN_Q toxin, and likely restricted the binding of the classical animal toxins.

Conclusions/Significance

The TPN_Q toxin-rKir1.1 channel complex structure not only revealed their unique interaction mechanism, but also would highlight the diverse animal toxin-potassium channel interactions, and elucidate the relative insensitivity of rKir1.1 channel towards animal toxins.     

Bacillus thuringiensis Cry34Ab1/Cry35Ab1 Interactions with Western Corn Rootworm Midgut Membrane Binding Sites

Background

Bacillus thuringiensis (Bt) Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry) proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low.

Methodology/Principal Findings

Binding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV). Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that ¹²⁵I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances ¹²⁵I-Cry35Ab1 specific binding, and that ¹²⁵I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1) No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with ¹²⁵I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2) No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with ¹²⁵I-Cry3Aa, or ¹²⁵I-Cry8Ba.

Conclusions/Significance

Combining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba for deployment as insect resistance management pyramids for in-plant control of western corn rootworm.     

Tailored ?-Cyclodextrin Blocks the Translocation Pores of Binary Exotoxins from C. Botulinum and C. Perfringens and Protects Cells from Intoxication

Background

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components.

Methodology/Principal Findings

Here we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component.

Conclusions/Significance

The described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria.     

Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

Background

Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts.

Principal Findings

We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility.

Significance

Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.     

Cryptochrome 1 Overexpression Correlates with Tumor Progression and Poor Prognosis in Patients with Colorectal Cancer

Background

Clock genes drive about 5–15% of genome-wide mRNA expression, and disruption of the circadian clock may deregulate the cell''s normal biological functions. Cryptochrome 1 is a key regulator of the circadian feedback loop and plays an important role in organisms. The present study was conducted to investigate the expression of Cry1 and its prognostic significance in colorectal cancer (CRC). In addition, the function of Cry1 in human CRC was investigated in cell culture models.

Methods

Real-time quantitative PCR, Western blot analysis and immunohistochemistry were used to explore Cry1 expression in CRC cell lines and primary CRC clinical specimens. MTT and colony formation assays were used to determine effects on cellular proliferation ability. The animal model was used to explore the Cry1 impact on the tumor cellular proliferation ability in vivo. Transwell assays were performed to detect the migration ability of the cell lines. Statistical analyzes were applied to evaluate the diagnostic value and the associations of Cry1 expression with clinical parameters.

Results

Cry1 expression was up regulated in the majority of the CRC cell lines and 168 primary CRC clinical specimens at the protein level. Clinical pathological analysis showed that Cry1 expression was significantly correlated with lymph node metastasis (p = 0.004) and the TNM stage (p = 0.003). High Cry1 expression was associated with poor overall survival in CRC patients (p = 0.010). Experimentally, we found that up-regulation of Cry1 promoted the proliferation and migration of HCT116 cells, while down-regulation of Cry1 inhibited the colony formation and migration of SW480 cells.

Conclusions

These results suggest that Cry1 likely plays important roles in CRC development and progression andCry1 may be a prognostic biomarker and a promising therapeutic target for CRC.