A novel vegetative insecticidal gene, vip3A(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), and corn earworm (Helicoverpa zea) has been isolated from BaciUlus thuringiensis strain AB88. VIP3-insecticidal gene homologues have been detected in "15% of Bacillus strains analyzed. The isolation and characterization of new insecticidal activities is the basis of many pest control programs. Bacillus thuringiensis, a gram-positive soil bacterium, is well known for its ability to produce crystalline inclusions during sporulation which contain insecticidal proteins called S-endotoxins. These inclusions are solubilized in insect midguts, releasing the 6-endotoxins that, upon proteolytic activation, exhibit a highly specific insecticidal activity (1). In the past decades, many B. thuringiensis strains with different insect host spectra have been identified and their $-endotoxins used in formulations for biopesticides (2). Recently, the cloning of $-endotoxin genes (3) and their expression in transgenic plants (4) has provided an alternative strategy for crop protection against insect damage.Although B. thuringiensis 8-endotoxins are effective insecticidal proteins, there are several agronomically important insects that are less sensitive to their action (5). The lepidopteran black cutworm (BCW, Agrotis ipsilon) is an example. BCW is a worldwide pest that attacks more than 50 crops, including cereal grains (6). This pest is difficult to control because by the time the infestations are apparent, the susceptible stages (i.e., larvae) are past and damage may already be serious and irreversible.Extensive screening programs are being carried out by various groups to search for B. thuringiensis strains with new insecticidal spectra. These evaluations have focused mainly on the identification of new 6-endotoxins that are expressed during sporulation (7). Our experimental approach focused on bacterial stages before sporulation, and has led to the identification of non-S-endotoxins with insecticidal activities. We describe here the cloning and characterization of vip3A (a) (pH 7.5) and dialyzed overnight at 4°C. The dialysate was titrated to pH 4.5 using 20 mM sodium citrate (pH 2.5). After 30 min incubation at room temperature, the sample was centrifuged at 3000 x g for 10 min. The resulting protein pellet was redissolved in 20 mM BisTris-Propane (pH 9.0) and fractionated on a Poros HQ/N anion exchange column (PerSeptive Biosystems, Framingham, MA) using a linear gradient from 0 to 500 mM NaCl in 20 mM Bis-Tris-Propane (pH 9.0) at a flow rate of 4 ml/min. The insecticidal protein eluted at 250 mM NaCl.Peptide Analysis and Oligonucleotide Synthesis. Fractions with insecticidal activity were fractionated in SDS/8-16% polyacrylamide gradient gels and transferred to poly(vinylidene difluoride) membranes (8). The most abundant protein band (molecular...

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Insect pests are a major cause of damage to the world's commercially important agricultural crops. Current strategies aimed at reducing crop losses rely primarily on chemical pesticides. Alternatively transgenic crops with intrinsic pest resistance offer a promising alternative and continue to be developed. The first generation of insect-resistant transgenic plants are based on insecticidal proteins from Bacillus thuringiensis (Bt). A second generation of insect-resistant plants under development include both Bt and non-Bt proteins with novel modes of action and different spectra of activity against insect pests.

A rapid analysis of Bacillus thuringiensis strains predictive of insecticidal activity was established by using polymerase chain reaction (PCR) technology. Primers specific to regions of high homology within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each insecticidal class. Predictions of insecticidal activity were made on the basis of the electrophoretic patterns of the PCR products. Included in the screen were PCR primers specific for cryl, cryIII, and cryIV genes, which are insecticidal for lepidopterans, coleopterans, and dipterans, respectively. Known B. thuringiensis strains as well as unidentified strains isolated from soil and insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a single PCR reaction containing 12 to 20 primers capable of distinguishing between the different insecticidal genes. Insecticidal activity predicted by the PCR screen was found to correspond with the insecticidal activity of insect bioassays. In addition to identifying strains with known insecticidal genes, the PCR screen can identify strains with altered electrophoretic patterns containing potentially novel genes.

The Vip3A protein is a member of a newly discovered class of vegetative insecticidal proteins with activity against a broad spectrum of lepidopteran insects. Histopathological observations indicate that Vip3A ingestion by susceptible insects such as the black cutworm (Agrotis ipsilon) and fall armyworm (Spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm 2 of diet and complete lysis of gut epithelium cells resulting in larval death at concentrations above 40 ng/cm 2 . The European corn borer (Ostrinia nubilalis), a nonsusceptible insect, does not develop any pathology upon ingesting Vip3A. While proteolytic processing of the Vip3A protein by midgut fluids obtained from susceptible and nonsusceptible insects is comparable, in vivo immunolocalization studies show that Vip3A binding is restricted to gut cells of susceptible insects. Therefore, the insect host range for Vip3A seems to be determined by its ability to bind gut cells. These results indicate that midgut epithelium cells of susceptible insects are the primary target for the Vip3A insecticidal protein and that their subsequent lysis is the primary mechanism of lethality. Disruption of gut cells appears to be the strategy adopted by the most effective insecticidal proteins.