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The usefulness of Bacillus thuringiensis insecticidal crystal protein (ICP) is studied. An alternative method is established by it for the screening of novel ICPs toxic to pest insects, and for ICP receptor characterization. The results of heterologous competition experiments performed with biotinylated and native ICPs confirms the relationship between different ICP receptor types in Plutella xylostella which is inferred from radioligand binding studies.

The presence of insecticidal crystal protein (ICP) receptors in the European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Pyralidae), the major lepidopteran pest of maize in southern Europe and North America, is studied. Two independent ICP receptors are present in the brush border of O.nubilalis gut epithelium. The interactions of B-thuringiensis ICPs with the larval gut of O.nubilalis is investigated by the experiments performed to determine binding of ICPs to both isolated brush border membrane vesicles and tissue sections.

Chemical crop protection is widely used to control plant diseases. However, the adverse effects of pesticide use on human health and environment, resistance development and the impact of regulatory requirements on the crop protection market urges the agrochemical industry to explore innovative and alternative approaches. In that context, we demonstrate here the potential of camelid single domain antibodies (VHHs) generated against fungal glucosylceramides (fGlcCer), important pathogenicity factors. To this end, llamas were immunized with purified fGlcCer and a mixture of mycelium and spores of the fungus Botrytis cinerea , one of the most important plant pathogenic fungi. The llama immune repertoire was subsequently cloned in a phage display vector to generate a library with a diversity of at least 10 8 different clones. This library was incubated with fGlcCer to identify phages that bind to fGlcCer, and VHHs that specifically bound fGlcCer but not mammalian or plant-derived GlcCer were selected. They were shown to inhibit the growth of B. cinerea in vitro , with VHH 41D01 having the highest antifungal activity. Moreover, VHH 41D01 could reduce disease symptoms induced by B. cinerea when sprayed on tomato leaves. Based on all these data, anti-fGlcCer VHHs show the potential to be used as an alternative approach to combat fungal plant diseases.

Background: Rainfall greatly affects the retention of foliar-applied agroformulations. Improving their resistance to wash-off is therefore of great importance in spray applications. When developing such formulations, small-scale laboratory assays are generally required. A set-up for retention studies using only small amounts of agroformulations (<0.5 L) was developed. The set-up consists of a spray device and a rainfall simulator. The effect of rain quantity (1, 3, 6 mm) on the spray retention of agroformulations was evaluated using this set-up., Results: The data showed that uniform and repeatable spraying was achieved with the small-scale spray device (coefficient of variation 23.4%) on potato pot plants (Solanum tuberosum L.). Rain quantity significantly affected the spray retention. Approximately 40% of the initial deposition was lost after 1 mm of rain at an intensity of 25 mm h -1 . Additional losses decreased with increasing volumes of rain (65 and 80% loss after 3 and 6 mm of rain respectively)., Conclusion: Future studies could implement the set-up to evaluate the effect of different rainfall characteristics and formulations on spray retention in order to improve the rainfastness of agroformulations. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Binding of several Bacillus thuringiensis delta-endotoxins was studied on histological midgut sections of larvae of coffee leaf miner Perileucoptera coffeella from Brazil and Perileucoptera sp from Madagascar. CryIA(a), CryIA(b), CryIA(c), CryIB, CryIE, and CryIIA were tested for binding, and only CryIA(c), CryIB, and CryIE yielded a positive response. The toxins bound to the whole midgut, and the result was identical on both insect populations. The same toxins, to the number of which CryIC was added, were tested on larvae of P. coffeella. CryIA(c) and CryIB were toxic with an LC50 of 1.47 &mgr;g/ml and 21.93 &mgr;g/ml, respectively. CryIE was not toxic to P. coffeella. CryIA(c) and CryIB were tested for synergistic activity and were shown to act by cumulative effect when delivered to the insect larvae as a mixture.

We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1Ab5, an insecticidal crystal protein [ICP] from Bacillus thuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning of an aminopeptidase N from the midgut brush-border membrane of Plutella xylostella, an insect species of which some populations acquired resistance against Cry1Ab5. Affinity chromatography on a Cry1Ab5 matrix was used to isolate a 120-kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1Ab5 and the coleopteran-specific Cry3A delta-endotoxin. Internal amino acid sequences from the 120-kDa protein were used for the design of degenerate oligonucleotides. From a nested PCR with M. sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes. This PCR fragment was used to screen cDNA libraries of larval midguts from M. sexta and P. xylostella. From the M. sexta midgut cDNA library a 2973-bp nucleotide sequence was cloned. The ORF of the sequence encodes a 942-residue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions. The NH2-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins. Low-stringency hybridization of the P. xylostella midgut cDNA library with M. sexta apn2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein. This aminopeptidase N (P. xylostella Apn1) displays 61% amino acid identity to M. sexta Apn2 and contains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addition. Both M. sexta Apn2 and P. xylostella Apn1 contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P. xylostella apn1 gene with PtdIns-specific phospholipase C demonstrated that P. xylostella Apn1 is attached to the insect cell membrane by a glycosyl-PtdIns anchor.

Binding of different Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Spodoptera frugiperda larvae was characterized by binding experiments with midgut tissue sections and isolated brush border membrane vesicles. Our results show that ICPs interact with the microvilli of epithelial cells of S. frugiperda in two different ways. The first is typical of highly toxic proteins (like CryIC and CryID); this interaction is saturable and specific. In contrast, some nontoxic proteins (like CryIAb) interact nonspecifically with the microvilli, since the binding of this toxin is not affected by the presence of high concentrations of homologous competitor. The CryIC toxin binds to two brush border proteins of 40 and 44 kDa and the CryIAb toxin binds to a single protein of 150 kDa. Immunological detection of ingested B. thuringiensis ICPs on gut sections of S. frugiperda larvae revealed that CryIC and CryID toxins bound along the epithelial brush border microvilli membrane. Binding of the nontoxic protein CryIAb was also observed in the epithelial brush border membrane of fed larvae, but it was extremely weak, implying that this type of interaction occurs also in vivo although it is not related to toxicity.

A novel Bacillus thuringiensis crystal protein with a silent activity against the Colorado potato beetle is described. The crystal proteins are produced as bipyramidal crystals. These crystals contain a protein of 129 kDa with a trypsin-resistant core fragment of 72 kDa. Neither a spore-crystal mixture nor in vitro-solubilized crystals are toxic to any of several Lepidoptera and Coleoptera species tested. In contrast, a trypsin-treated solution containing the 72-kDa tryptic core fragment of the protoxin is highly toxic to Colorado potato beetle larvae. The crystal protein-encoding gene was cloned and sequenced. The inferred amino acid sequence of the putative toxic fragment has 37, 32, and 33% homology to the CryIIIA, CryIIIB, and CryIIID toxins, respectively. Interestingly, the 501 C-terminal amino acids show 41 to 48% amino acid identity with corresponding C-terminal amino acid sequences of other crystal proteins. Because of the toxicity of the fragment to the Colorado potato beetle and because of the distinct similarities of the toxic fragment with the other CryIII proteins, this gene was given a new subclass name (cryIIIC) within the CryIII class of coleopteran-active crystal proteins. CryIIIC represents the first example of a crystal protein with a silent activity towards coleopteran insect larvae. Natural CryIIIC crystals are not toxic. Toxicity is revealed only after an in vitro solubilization and activation step.

The nucleotide sequence of a novel insecticidal crystal protein(Cry)-encoding gene from a Bacillus thuringiensis serotype kurstaki isolate is described. The gene is related to the known coleopteran-active cryIII genes and encodes a CryIIID that is much more active against Colorado potato beetle than other CryIII.

The first system we used to transfer proteins from polyacrylamide gels onto nitrocellulose was a capillary blotting system, based on the work of Renart et al. (1). Although we were very enthusiastic about the wide range of possible applications created by the protein blotting technique, we were unsatisfied with the slow and inefficient transfer. Electroblotting (2,3) seemed to be much more efficient, but at that time there were serious financial limitations in the department.

Since its introduction by Towbin et al. (1), electro-blotting has become one of the most valuable analytical procedures in protein research. Conventionally, the gel and the immobilizing matrix are sandwiched between porous pads and inserted between two electrodes in a buffer tank. Electrodes of platinum wire are mounted in such a way that, with a minimum amount of material, they still generate a uniform electrical field in the center of the buffer chamber, with the minimum amount of material. On the other hand, transfer efficiency is largely dependent of the voltage gradient between the two electrodes. High voltages generate high currents and high temperatures, whereas reducing the distance between wire electrodes seriously disturbs the electrical field (2). Plate electrodes yield essentially homogeneous electrical fields, even when they are moved close together. Vaessen et al. (3) were the first to experiment with plate electrodes. Kyhse-Andersen (4) introduced horizontal semidry blotting in which the transfer takes place in a stack of buffer-saturated filter papers, inserted between two graphite plates.