Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 13, Num. 2, 1996

P210k: The putative binding protein for Bacillus thuringiensis CryIA(c)delta-endotoxin in the midgut brush-border membrane of the lepidopteran Diatraea saccharalis

Roberto I Vazquez, Dmitri L Prieto, Ariel Martinez and Gustavo A de la Riva

Department of Environmental Biotechnology, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba.

Code Number: BA96045
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Introduction

Bacillus thuringiensis is a gram-positive soil bacterium, able to produce insecticidal parasporal bodies during sporulation. These bodies -commonly of crystalline shape are composed of subunits called -endotoxins, or Cry proteins, encoded by cry genes, which form a large gene family. The Cry proteins are classified in classes and subclasses according to aminoacid sequence homology and target specificity (1).

The mode of action of B. thuringiensis insecticidal proteins involves several stages (2). Upon ingestion by the insect larvae, the Cry proteins are proteolytically processed in the midgut, and bind to high-affinity specific membrane receptors located on the brush-border epithelium. Then, cation-specific channels are formed causing an electrolytic imbalance that ultimately kills the larva. Several authors report the identification and purification of Cry protein receptors from various insect species, as well as cloning and sequencing of their genes.

Materials and Methods

Preliminary studies of the toxicity of CryI proteins against D. sacharalis were done employing recombinant CryIA(b) and CryIA(c) proteins expressed in Escherichia coli and 3-instar larvae at 10 per toxin concentration. Extracts from untransformed E. coli were used as negative control. The bioassay was standardised using B. thuringiensis var kurstaki HD-1delta -endotoxin.

To identify the toxin binding proteins in the midgut epithelial cell membranes of D. saccharalis, BBMV (3) proteins have been electrophoretically separated by SDS-PAGE and transferred to nitrocellulose membranes in a submarine transfer apparatus. The membrane was submitted to a ligand-blot analysis using activated natural CryIA(c) delta-endotoxin and immunopurified subclass-specific anti-CryIA polyclonal antibodies. Immunochemical recognition was detected by anti-rabbit peroxidase using ECL chemoluminiscent system. Preliminary competition assays were done.

Results and Discussion

Bioassays performed with recombinant CryIA(b) and CryIA(c) proteins and third instar D. saccharalis larvae, showed that the values of LD50 were 1,2 ug/ml diet for CryIA(b) and 7,8 ug/ml diet for CryIA(c). These results are correlated with the fact of strong self-pesticide activity, observed in transgenic sugarcane plants expressing recombinant CryIA(b) toxin.

The ligand-blot experiment allowed a well-defined band for a putative CryIA(c) binding protein with MW value of approximately 210 kDa. Preliminary competition experiments confirmed the possibility that this toxin binds to a putative receptor located on the brush-border membrane of the midgut epitelial cells. The protein with binding activity was called P210k.

Figure 1. Detection of P210 on the midgut brushborder membrane of D. saccharalis by Ligand Blotting. Lane 1: 10 ug total BBMV proteins not incubated with CryIA (c). Lane 2: 20 ug total BBMV proteins not incubated with CryIA (c). Lane 3: 10 ug total BBMV proteins incubated with CryIA (c). Lane 4: 20 ug total BBMV proteins incubated with CryIA (c). Lane 5: 10 ug trypsin-treated natural CryIA (c) toxin from B. thuringiensis HD- 73.

1. Hofte H and Whiteley H Microbiol. Rev 1989;53;242-255.

2. Knowles BH and Dow JAT Bio Essays 1993;15:469-476.

3. Wolfersberger MM et al. Comp. Biochem. Physiol 1987;86A:301-308.

Copyright 1996 Elfos Scientiae