Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

Abstract

bBacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu 47 for the 70-kDa form or Ile 88 for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) from C. puncticollis larvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective.

Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

“…Indeed, field monitoring of pest susceptibility, isolation of new toxins, and the study of resistance mechanisms have been widely pursued by the scientific community (10,15). The growing evidence that the alteration of toxin binding is the most common resistance mechanism (6) emphasizes the importance of binding models to predict the effective toxin combinations for successful implementation of gene pyramiding strategies (12,(16)(17)(18)(19).…”

Unshared binding sites for Bacillus thuringiensis Cry3Aa and Cry3Ca proteins in the weevil Cylas puncticollis (Brentidae)

“…The mode of action of Cry proteins from B. thuringiensis has been extensively studied, especially for lepidopteran-active Cry proteins (Bravo et al., 2007, Vachon et al., 2012; Adang et al., 2014), whereas much less is known for coleopteran-active Cry proteins (Hernández-Martínez et al., 2014, Ochoa-Campuzano et al., 2012, Rausell et al., 2004, Rausell et al., 2007, Slaney et al., 1992). The proposed model starts after the ingestion of the crystals by susceptible insect larvae, followed by crystal solubilization and protease activation in the midgut environment.…”

“…Recently, the existence of common binding sites for three B. thuringiensis proteins, Cry3Ca, Cry3Bb, and Cry7Aa proteins to C. puncticollis brush border membrane vesicles (BBMV) has been proposed (Hernández-Martínez et al., 2014). Thus, from a resistant management standpoint, combinations of these three proteins do not seem to be suitable for development of Bt sweetpotato plants.…”

“…Previous studies (Slaney et al., 1992, Rausell et al., 2004) have shown that some Cry3 proteins are able to bind to BBMV from some coleopteran insect pest including C. puncticollis (Hernández-Martínez et al., 2014). Interestingly, some reports have shown that only the chymotrypsin-activated Cry3Aa, and not the trypsin-activated, was able to bind specifically to BBMV from L. decemlineata (Martínez-Ramírez and Real, 1996).…”

“…Thus, an alteration of the shared binding site in C. puncticollis might not confer resistance to both proteins, since both Cry3 proteins have an unshared binding site. The occurrence of shared binding sites on C. puncticollis BBMV for two Cry3 proteins (Cry3Bb and Cry3Ca) was previously described (Hernández-Martínez et al., 2014). Moreover, this common binding site is also shared with the Cry7Aa protein.…”