Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.

uses a variant Bt Cry3Bb1 insecticidal protein (Donovan et al., 1992). Cry3Bb1 is known to be biologically active The corn rootworm (CRW; Diabrotica spp.) is one of the most against several species within the Coleopteran family serious pests of corn in the USA. Chemical insecticides and crop rotation have been the only two options available to growers for Chrysomelidae, including the western corn rootworm, managing CRW. Unfortunately, both of these tactics can be ineffective Diabrotica virgifera virgifera LeConte (Rupar et al., as a result of either resistance or behavioral modifications. In this 1991). The biological activity of this protein against D. paper, we describe transgenic maize (Zea mays L.) hybrids that control virgifera virgifera suggested its potential use in creating CRW. These hybrids were created with a Cry3Bb1 Bacillus thurintransgenic plants expressing Cry3Bb1 that would confer giensis (Bt) variant that is approximately eight times more lethal to protection to corn root tissue from larval feeding damcorn rootworm larvae than the wild-type protein. A DNA vector age. To further augment protection of the root system containing the modified cry3Bb1 gene was placed under control of a from larval feeding damage, modifications were introroot-enhanced promoter (4-AS1) and was introduced into embryonic duced in the cry3Bb1 gene that gave rise to an amino acid maize cells by microprojectile bombardment. Described here is the variant Cry3Bb1 protein with an eight-fold increase in molecular genetic characterization, protein expression levels, and field performance of the recently commercialized MON863 hybrids.

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The European corn borer [ECB; Ostrinia nubilalis (Hübner)] is an economically significant pest of corn (Zea mays L.). The ability to routinely transform corn has broadened the control options available to include the introduction of resistance genes from sexually incompatible species. In this study, microprojectile bombardment was used to introduce synthetic versions of cryIA insecticidal protein genes from Bacillus thuringiensis subsp, kurstaki (Btk) into embryogenitcis sue of the Hi‐II] (A188/B73 derivative) genotype of corn. Of 715 independent transgenic calli produced, 314 (44%) had insecticidal activity against tobacco hornworm (Manduca sexta L.) larvae. Plants were regenerated, self‐pollinated when possible, and crossed to B73. First‐generation progeny of 173 independent Btk‐protein expressing calli were evaluated under field conditions with artificial ECB infestations in 1992 or 1993. Approximately half (89/173) segregated in single‐gene manner for resistance to first‐generation ECB leaf‐feeding damage. All of the 89 lines evaluated in 1992 or 1993 for resistance to second‐generation ECB exhibited less stalk tunneling damage than the non‐transgenic controls. In 1993, 44% (34177) of the lines tested had ≤2.5 cm of tunneling, compared to severe damage (mean = 45.7 cm) in the B73 × Hi‐II controls. Experiments are in progress to evaluate the effect of the introduced genes on yield and other agronomic properties.

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This chapter examines the current management strategies and their limitations in USA maize production and the potential benefits of managing these pests with a developed corn rootworm (Diabrotica spp.)-protected maize. The topics covered include development of a genetic solution for corn rootworm management, characterization of YieldGard rootworm maize, food and feed safety of YieldGard rootworm maize, environmental safety of YieldGard rootworm maize, performance of YieldGard rootworm maize, and benefits of YieldGard rootworm maize.

The enzyme cholesterol oxidase (E.C. 1.1.3.6), purified from Streptomyces culture filtrate was previously found to have oral insecticidal activity on neonate larvae of the boll weevil (Anthonomus grandis grandis Boheman) from a laboratory population. In the present study, second instar larvae were also controlled by the enzyme at diet concentrations similar to those which control neonates (12 day LC50 = 2.4 μg.ml−1 in diet). Larvae from field‐collected adults were similarly susceptible to cholesterol oxidase in the diet. When ingested by adult females during the mating/pre‐oviposition period, cholesterol oxidase greatly reduced subsequent oviposition (83% reduction in eggs laid as compared to controls) and larval survival (97% reduction from controls). Dissection of treated adult females revealed poorly developed ovaries and few developing oöcytes. These studies were conducted to further evaluate the utility of cholesterol oxidase in a program to establish boll weevil‐resistant transgenic cotton.

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