Abstract
Plant-parasitic nematodes cause huge agricultural economic losses. Two major families of Bacillus thuringiensis crystal proteins, Cry5 and Cry6, show nematicidal activity. Previous work showed that binding to midgut receptors is a limiting step in Cry toxin mode of action. In the case of Cry5Ba, certain Caenorhabditis elegans glycolipids were identified as receptors of this toxin. However, the receptors for Cry6 toxin remain unknown. In this study, the C. elegans CUB-like-domain containing protein RBT-1, released by phosphatidylinositol-specific phospholipase C (PI-PLC), was identified as a Cry6Aa binding protein by affinity chromatography. RBT-1 contained a predicted glycosylphosphatidylinositol (GPI) anchor site and was shown to locate in lipid rafts in the surface of the midgut cells. Western ligand blot assays and ELISA binding analysis confirmed the binding interaction between Cry6Aa and RBT-1 showing high affinity and specificity. In addition, the mutation of rbt-1 gene decreased the susceptibility of C. elegans to Cry6Aa but not that of Cry5Ba. Furthermore, RBT-1 mediated the uptake of Cry6Aa into C. elegans gut cells, and was shown to be involved in triggering pore-formation activity, indicating that RBT-1 is required for the interaction of Cry6Aa with the nematode midgut cells. These results support that RBT-1 is a functional receptor for Cry6Aa.
Highlights
Bacillus thuringiensis (Bt) is a ubiquitous spore-forming bacterium, which produce a large family of environmentally friendly crystal proteins (Cry), with toxicity against target insects (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera, and Mallophaga) or nematodes [1]
Bacillus thuringiensis (Bt) crystal proteins belong to pore-forming toxins (PFTs), which display virulence against target hosts by forming holes in the cell membrane
We here demonstrate that the CUBlike-domain containing protein RBT-1 acts as a functional receptor of Cry6A, which mediates the intestinal cell interaction and nematicidal activity of this toxin
Summary
Bacillus thuringiensis (Bt) is a ubiquitous spore-forming bacterium, which produce a large family of environmentally friendly crystal proteins (Cry), with toxicity against target insects (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera, and Mallophaga) or nematodes [1]. Due to its highly and insecticidal activity, B. thuringiensis has been developed as the leading biopesticide as an alternative to synthetic chemical pesticides [2]. Some Cry protein genes have been used to generate transgenic crops for pest control [3]. One of the resistance-related mechanisms is linked to mutation of the Cry protein-binding cell surface receptors [5]. It is critical for insect and nematode biocontrol to identify the receptor genes of Cry proteins in different pests
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