A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae.

The full characterization of a novel insecticidal crystal protein, named Cry9Ca1 according to the revised nomenclatureforCryproteins,fromBacillusthuringiensisserovartolworthiisreported.Thecrystalproteinhas 1,157 amino acids and a molecular mass of 129.8 kDa. It has the typical features of the Lepidoptera-active crystal proteins such asfive conserved sequence blocks. Also, it is truncated upon trypsin digestion to a toxic fragment of 68.7 kDa by removal of 43 amino acids at the N terminus and the complete C-terminal half after conserved sequence block 5. The 68.7-kDa fragment is further degraded to a nontoxic 55-kDa fragment. The crystal protein has a fairly broad spectrum of activity against lepidopteran insects, including members of the families Pyralidae, Plutellidae, Sphingidae, and Noctuidae. A 50% lethal concentration of less than 100 ng/cm 2 of diet agar was found for diamondback moth, European corn borer, cotton bollworm, and beet armyworm. It is thefirst insecticidal crystal protein with activity against cutworms. No activity was observed against some beetles, such as Colorado potato beetle. The protein recognizes a receptor different from that recognized by Cry1Ab5 inOstrinia nubilalisandPlutella xylostella.InSpodoptera exiguaandP. xylostella, it binds to a receptor which is also recognized by Cry1Cax but with a lower affinity. In these insects, Cry1Cax probably binds with ahigheraffinitytoanadditionalreceptorwhichisnotrecognizedbyCry9Ca1.Eliminationofatrypsincleavage site which is responsible for the degradation to a nontoxic fragment did result in protease resistance but not in increased toxicity againstO. nubilalis. The major characteristic of the gram-positive, sporeforming bacteriumBacillusthuringiensisistheproductionofinsecticidal crystal proteins (ICPs) during sporulation. To date, nearly 100 distinct crystal protein gene sequences have been published either in the general scientific literature (5, 8) or in patent applications.Thesecrystalproteinshaveaspecifictoxicactivity against certain lepidopteran, dipteran, or coleopteran larvae. Several of the anti-Lepidoptera ICPs have a very high toxic activity against the early-instar larvae of agronomically important pest insects such as Heliothis and Helicoverpa spp. (budworms and bollworms),Spodopteraspp. (armyworms),Ostrinia nubilalis (European corn borer), and Plutella xylostella (diamondback moth) (12). However, some species of the family Noctuidae such asSpodoptera frugiperdaandAgrotisspp. (cutworms) are insensitive to ICPs. Therefore, we have screened a subset of our collection of 12,000 B. thuringiensis isolates for activity against these members of the Noctuidae family. This screening program resulted in the discovery of a novel crystal protein with broad-spectrum activity against members of the Lepidoptera, including several species of the Noctuidae family and some ICP-resistant insects. Here, we report the full characterization of this novel insecticidal crystal protein, including its gene and protein sequences, its homology with other crystal proteins, its activity spectrum, and the receptor-binding data. In addition, we describe the effect of elimination of a trypsin cleavage site on the activity of the crystal protein. (Thecrystalprotein,previouslynamedCryIH,wasdescribed in a patent application [9]. Preliminaryfindings were commu