The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM-1 min-1 to 8320 mM-1 min-1. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

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.

We have detected hsc7O gene expression (heat shock protein hsp7O cognate) during vegetative growth and reproductive development in tomato (Lycopersicon esculentum). Using RNA from a tomato hsc7O cDNA as a probe in in situ hybridizations, we have determined expression patterns of hsc7O in nonstressed tomato roots, stems, leaves, flowers, and developing fruits. We have localized high levels of hsc7O transcript to the vascular system of the ovary, dividing cells of the lateral root tips, and the inner integument of developing seeds. We also see expression in the transmitting tissue, in immature anthers, and in embryos. We cannot detect expression in mature pollen, xylem, or ovules. These data indicate that the expression of at least some tomato hsp7O family members is developmentally regulated.The heat shock response was first described in Drosphila in 1962 (1). Further analysis has shown that all organisms produce heat shock proteins (hsps) in response to heat, heavy metals, and a variety of other stresses (2); hsps range in size from 16 to 110 kDa, with the exact protein profile varying between organisms (3).The sequence of the 70-kDa hsp (hsp70) is evolutionarily highly conserved, and in some organisms this is the most abundantly expressed of the hsps (2). In all organisms investigated except chicken (4), hsp70s are encoded by multigene families. The multigene family members are differentially regulated (5), and members that are expressed in the absence of heat stress are referred to as cognate hsp70s (hsc70s), or "cognates" (6). Some hsc70s have been associated with specific functions such as clathrin uncoating (7, 8), glycosylation-related reactions (9), and transfemrn activities (10); other hsc70s seem to be developmentally regulated (11). hsc70s that are expressed during development have been detected during yeast sporulation (12), Drosophila oogenesis (13), in preimplantation mouse embryos (14), and during mouse, rat, and human spermatogenesis (15).Recent evidence indicates that some of the yeast hsc70s are involved in the transport of proteins across membranes (16,17). There is speculation (16) that the hsc70 unfolding activities used during transport may also be used in some of the other cognate activities. The localization studies presented in this paper indicate that there is nonstress, tissuespecific regulation of hsp70 family members in tomato. This work is the foundation for further analyses aimed at determining hsc70 functions during development and stress. Additionally, the promoters of such family members may be useful in chimeric gene constructions aimed at localizing foreign gene products in some of these tissues. call hsc-1, and the well-characterized petunia hsp70 (20) verified that hsc-1 is an hsp70 family member. MATERIALS AND METHODSTissue Preparation. The VF36 tomato plants used in all of these experiments were maintained in growth chambers at 18'C on a 9-hr day/15-hr night light/dark cycle. Buds were collected and measured from the zone of abcission to bud tip, and the correlati...

When a model secretory precursor was synthesized in vitro and analyzed by rate-zonal sedimentation, it appeared to be associated with other proteins present in a wheat germ extract. At least one of the associated proteins is a member of the 70-kD family of stress proteins. It was possible to immunoprecipitate the secretory precursor with anti-heat shock cognate 70 (Hsc70) antibodies in the absence but not in the presence of ATP, suggesting that the association was specific. ATP-sensitive association is one diagnostic characteristic of molecular chaperone-type proteins. Increasing incubation temperature decreased the amount of precursor associated with Hsc70. A method was developed for the removal of Hsc70 from a wheat germ in vitro translation mixture by immunoprecipitation. Cotranslational translocation and processing of the secretory precursor by maize endosperm microsomes were inefficient in the Hsc70-depleted system but were greatly stimulated by addition of purified preparations of various heat shock 70 proteins (Hsp70s). Cytosolic Hsc70 from maize endosperm was capable of autophosphorylation in vitro. Phosphorylated Hsc70 was much less efficient in promoting membrane translocation of the secretory precursor. These results suggest that chaperone function in vivo could be regulated by phosphorylation.

Pollen of angiosperms lacks the ability to respond to heat stress by synthesizing heat shock proteins (hsps). In tomato developing microspores were found to have 70 kDa heat shock proteins (hsp70s) present throughout development, even in the absence of heat stress. Heat shock protein family members expressed in the absence of heat stress are called cognate (hsc70) genes. Antisense RNA and antibody probes were used for in situ hybridizations which detected hsc70 expression in developing pollen of immature buds. Hsc70 mRNA transcripts and proteins were detected in nonstressed sporogenous tissues, microspores and in pre-tapetal layers during early pollen development. While immunoblot analysis detected hsc70 proteins stored in mature pollen, heat stress could not induce the synthesis of new hsp70 protein as measured by 35S-methionine labeling followed by immunoprecipitation.

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.

The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from a Chromobacterium piscinae strain and is specifically toxic to Diabrotica virgifera virgifera larvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here β-tripod. Using mutagenesis, we identified functionally important regions in the β-tripod domain, which may be involved in target recognition.