Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms.

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.

Transgenic plants: an emerging approach to pest control.

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.

Hsp70 heat shock protein cognate is expressed and stored in developing tomato pollen.

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.

Autophosphorylation of the pea mitochondrial heat-shock protein homolog.

Highly purified mitochondria isolated from 14-day-old pea (Pisum sativum L., cv Little Marvel) seedlings contain a homolog of the 70,000 molecular weight heat-shock protein. The amount of this heat-shock cognate (Hsc70) was not reduced by limited proteolysis of intact mitochondria or by preparation of mitoplasts, indicating that the protein is located within the matrix compartment. Pea mitochondrial Hsc70 binds to immobilized ATP and reacts on western blots with anti-tomato Hsc70 antiserum. When a mitochondrial matrix fraction was incubated with [gamma-(32)P]ATP, there was phosphorylation of Hsc70. The extent of phosphorylation was increased by including calcium chloride in the reactions. Phospho amino acid analysis of purified mitochondrial Hsc70, phosphorylated in the calcium-stimulated reaction, revealed only phosphothreonine. Pea mitochondrial Hsc70, purified by a combination of ATP-agarose affinity chromatography and gel permeation chromatography, was labeled when incubated with ATP plus calcium, suggesting autophosphorylation rather than phosphorylation by an associated kinase. In analogy to mammalian cells and yeast, it is likely that mitochondrial Hsc70 acts as a molecular chaperone, and it is possible that phosphorylation plays a role in chaperone function.

The 70-Kilodalton Heat Shock Cognate Can Act as a Molecular Chaperone during the Membrane Translocation of a Plant Secretory Protein Precursor.

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.

Heat shock protein hsp70 cognate gene expression in vegetative and reproductive organs of Lycopersicon esculentum.

We have detected hsc70 gene expression (heat shock protein hsp70 cognate) during vegetative growth and reproductive development in tomato (Lycopersicon esculentum). Using RNA from a tomato hsc70 cDNA as a probe in in situ hybridizations, we have determined expression patterns of hsc70 in nonstressed tomato roots, stems, leaves, flowers, and developing fruits. We have localized high levels of hsc70 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 hsp70 family members is developmentally regulated.