Bacillus thuringiensis and its close relatives have proved to be a valuable source of insecticidal genes. Nevertheless, after the screening of many thousands of isolates, new types of insecticidal Bt genes have been difficult to find. The commercial and environmental successes of Bt-crops have provided the impetus for a widespread search for other potent insecticidal genes from microbes, insects, plants, and even animals. These new insecticidal genes could be used instead of Cry-toxins or used to augment Cry-toxins to strengthen resistance management programs for the current generation of transgenic crops that rely on a single insecticidal activity. The main requirements for these new genes are that the insecticidal agents must be orally active and preferably encoded by relatively few genes as transformation technologies can still handle only a couple of genes at a time. Novel insecticidal genes have been found using mass screening or the rational approach of seeking specific inhibitors of an insect's digestive or neural physiology. Several potential new technologies have emerged (reviewed in Ref. 9), but none of these has yet had the commercial impact of the Cry genes.
Different Cry genes have been isolated, covering a spectrum of insecticidal activities, but many important pests still cannot be controlled by Cry proteins. Most mass screening efforts to identify novel Bacillus strains have concentrated on the identification of new crystal proteins and hence have been carried out with sporulating cultures. B. thuringiensis isolates with high activity against corn rootworms (Diabrotica sp.), for example, have been found only recently, but a novel screening approach has been used to discover a rootworm-active insecticidal complex produced during the vegetative growth phase of B. cereus (45). One non-crystal-forming isolate, AB78, contained a proteinaceous insecticidal activity produced during log phase growth that was absent at sporulation. This was traced to the presence of two proteins designated ViplA and Vip2A (vegetative insecticidal protein) that, together, appear to have activity against corn rootworms. The corresponding Vip genes were cloned and were found to be widespread in Bacillus species (46). Vip2 has been crystallized and has been shown to be an ADP-ribosylating toxin (47). During the vegetative phase screening, a strain of B. thuringiensis, AB88, was also identified. This strain had high activity against many lepidopteran pests but no activity against coleopterans, and from this strain a third type of vegetative insecticidal protein, Vip3A, has been isolated. Vip3 has no homology to the Cry genes or any other protein in the sequence databases and thus represents a totally new class of insecticidal genes. Vip3 expressed in transgenic tobacco achieved partial protection against Spodoptera litura (48), but no extensive analyses of transgenic plants expressing this or other Vip genes have yet been published. The existence of the Vip genes will encourage a new round of screening of the existing Bacillus strain collections.
The venom of many insect predators such as spiders and scorpions contains potent neurotoxins, many of which are insect specific. These have been used to enhance the speed of kill of insect viruses (e.g., Ref. 49) but seem unlikely candidates for expression in plants because of the food safety concerns, real or imagined, that they would arouse in consumers (50).
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