SUMMARY During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Bacillus thuringiensis (Bt) is a valuable source of insecticidal proteins for use in conventional sprayable formulations and in transgenic crops, and it is the most promising alternative to synthetic insecticides. However, evolution of resistance in insect populations is a serious threat to this technology. So far, only one insect species has evolved significant levels of resistance in the field, but laboratory selection experiments have shown the high potential of other species to evolve resistance against Bt. We have reviewed the current knowledge on the biochemical mechanisms and genetics of resistance to Bt products and insecticidal crystal proteins. The understanding of the biochemical and genetic basis of resistance to Bt can help design appropriate management tactics to delay or reduce the evolution of resistance in insect populations.

SUMMARY The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.

Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor (insecticidal crystal ABSTRACTThe biochemical mechanism for resistance to Bacillus thuringiensis crystal proteins was studied in a field population of diamondback moths (Plutella xylostella) with a reduced susceptibility to the bioinsecticidal spray. The toxicity and binding characteristics of three crystal proteins [CryIA(b), CryIB, and CryICI were compared between the field population and a laboratory strain. The field population proved resistant (>200-fold compared with the laboratory strain) to CryIA(b), one of the crystal proteins in the insecticidal formulation. Binding studies showed that the two strains differ in a membrane receptor that recognizes CryIA(b). This crystal protein did not bind to the brush-border membrane of the midgut epithelial cells of the field population, either because of strongly reduced binding affinity or because of the complete absence of the receptor molecule. Both strains proved fully susceptible to the CryIB and CryIC crystal proteins, which were not present in the B. thuringiensis formulation used in the field. Characteristics of CryIB and CryIC binding to brushborder membranes of midgut epithelial cells were virtually identical in the laboratory and the field population.

Binding studies were performed with two '251-labeled Bacillus thuringiensis -endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One S-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other 6-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis 6-endotoxins active against M. sexta compete for binding of 125I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.Bacillus thuringiensis produces crystalline parasporal inclusions containing insecticidal proteins called 8-endotoxins. Most S-endotoxins are protoxins, which are proteolytically activated in the insect midgut to smaller active toxins (1). 8-Endotoxins produced by different B. thuringiensis strains may exhibit different insecticidal spectra. Toxins active toward lepidopteran, dipteran, or coleopteran larvae have been described (2-4). Among &endotoxins specific for Lepidoptera, marked differences exist in the relative levels of toxicity toward different species of this order (5-7).Several factors such as the solubilization and proteolytic activation of the crystals in the insect midgut (5, 8) and the presence of specific cell membrane receptors for different 8-endotoxins (9,10) MATERIALS AND METHODSInsect Toxicity Assays. Insect toxicity assays on M. sexta and P. brassicae have been described (12). M. sexta was reared on an artificial diet (13); P. brassicae was reared on fresh cabbage leaves (Brassica oleracea var. gemnifera D.C.).B. thuringiensis 6-Endotoxins. Cloning of bt2, the gene encoding the 130-kDa 8-endotoxin (Bt2-protoxin) from B. thuringiensis subsp. berliner strain 1715, purification of recombinant Bt2-protoxin from Escherichia coli, and generation of the toxic 60-kDa tryptic fragment (Bt2-toxin) has been described by Hofte et al. (12). For further purification Bt2-toxin was precipitated in (NH4)2SO4 (70%) and was then dissolved in Tris buffer (20 mM Tris HCl/200 mM NaCl, pH 8.65) with 5% (vol/vol) glycerol. Streptomycin sulfate was added to a concentration of 0.2% to remove contaminating nucleic acids. The solution was allowed to stand for 60 min at 4°C and the precipitate was spun down in a Sorvall SS34 rot...