Abstract
Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.
Highlights
The western corn rootworm (WCR; Diabrotica virgifera virgifera) causes annual economic losses of over 1 billion dollars in the USA [1,2]
The sphingomyelin:ceramide phosphoethanolamine (CPE) relative molar ratio was estimated as ~1.7 by peak area integration, which indicated a preference for ceramide linked to the phosphocholine moiety with respect to ceramide linked to the phosphoethanolamine moiety (CPE)
We have shown here that the Ostreolysin A6 (OlyA6)/pleurotolysin B (PlyB) insecticidal protein complex from Pleurotus mushrooms can form transmembrane pores in the following systems: (i) lipid vesicles made from commercial lipids that contain physiologically relevant concentrations of the insect-specific sphingolipid CPE; (ii) large unilamellar vesicles (LUVs) reconstituted from non-polar lipid extracts from WCR larvae; and (iii) Brush border membrane vesicles (BBMVs) from WCR larvae
Summary
The western corn rootworm (WCR; Diabrotica virgifera virgifera) causes annual economic losses of over 1 billion dollars in the USA [1,2]. Resistance to Cry toxins can occur at any stage of the toxic pathway, but it is most commonly linked to the mutation of the protein receptor gene or the development of regulatory mechanisms that disrupt the production of the toxin receptors [11,12]. This development of resistance against Bt maize by the WCR has recently promoted the search for alternative biopesticides and approaches to combat this pest. Several new bioinsecticides of bacterial origin have been proposed recently [13,14,15,16,17]
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