In vitro template-change PCR to create single crossover libraries: a case study with B. thuringiensis Cry2A toxins

During evolution the creation of single crossover chimeras between duplicated paralogous genes is a known process for increasing diversity. Comparing the properties of homologously recombined chimeras with one or two crossovers is also an efficient strategy for analyzing relationships between sequence variation and function. However, no well-developed in vitro method has been established to create single-crossover libraries. Here we present an in vitro template-change polymerase change reaction that has been developed to enable the production of such libraries. We applied the method to two closely related toxin genes from B. thuringiensis and created chimeras with differing properties that can help us understand how these toxins are able to differentiate between insect species

Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case

Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples

In Vitro Uptake of 140 kDa Bacillus thuringiensis Nematicidal Crystal Proteins by the Second Stage Juvenile of Meloidogyne hapla

Plant-parasitic nematodes (PPNs) are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN) are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2) were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba). Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN

A Meta-Analysis of Effects of Bt Crops on Honey Bees (Hymenoptera: Apidae)

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.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.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

Comparative Proteomic Analysis of Aedes aegypti Larval Midgut after Intoxication with Cry11Aa Toxin from Bacillus thuringiensis

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes

Bacillus sphaericus Binary Toxin Elicits Host Cell Autophagy as a Response to Intoxication

Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways