Molecular and insecticidal characterization of a Cry1I protein toxic to insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae

Abstract

The most notable characteristic of Bacillus thuringiensis is its ability to produce insecticidal proteins. More than 300 different proteins have been described with specific activity against insect species. We report the molecular and insecticidal characterization of a novel cry gene encoding a protein of the Cry1I group with toxic activity towards insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. PCR analysis detected a DNA sequence with an open reading frame of 2.2 kb which encodes a protein with a molecular mass of 80.9 kDa. Trypsin digestion of this protein resulted in a fragment of ca. 60 kDa, typical of activated Cry1 proteins. The deduced sequence of the protein has homologies of 96.1% with Cry1Ia1, 92.8% with Cry1Ib1, and 89.6% with Cry1Ic1. According to the Cry protein classification criteria, this protein was named Cry1Ia7. The expression of the gene in Escherichia coli resulted in a protein that was water soluble and toxic to several insect species. The 50% lethal concentrations for larvae of Earias insulana, Lobesia botrana, Plutella xylostella, and Leptinotarsa decemlineata were 21.1, 8.6, 12.3, and 10.0 microg/ml, respectively. Binding assays with biotinylated toxins to E. insulana and L. botrana midgut membrane vesicles revealed that Cry1Ia7 does not share binding sites with Cry1Ab or Cry1Ac proteins, which are commonly present in B. thuringiensis-treated crops and commercial B. thuringiensis-based bioinsecticides. We discuss the potential of Cry1Ia7 as an active ingredient which can be used in combination with Cry1Ab or Cry1Ac in pest control and the management of resistance to B. thuringiensis toxins.

FIG. 1.

SDS-PAGE of parasporal crystals obtained from the commercial biopesticides Dipel (lane 2) and Xentari (lane 3) and from the strain HU4-2 (lane 4) and purified by ultracentrifugation in a sucrose gradient as described in Materials and Methods. Molecular masses of the protein markers (lane 1) are given on the left.

FIG. 2.

SDS-PAGE showing the expression and purification of Cry1Ia7. (A) Lanes: 1, molecular mass markers; 2, E. coli BL21(DE3) native strain; 3, E. coli transformed with plasmid pPC-ire1 after IPTG-induced expression of the cloned cry1Ia7 gene; 4, pellet of the culture that expressed Cry1Ia7 after sonication; 5, supernatant of this culture after sonication; 6, Cry1Ia7 protein after nickel affinity column purification and Sephadex column buffer exchange. (B) Cry1Ia7 protein after purification (lane 2) and after trypsin digestion (lane 3). Molecular sizes of the markers (lanes 1) are given in kDa.

FIG. 3.

Comparison of the deduced amino acid sequences of Cry1I proteins. Conserved amino acid blocks for Cry1I proteins, predicted secretion signal peptides (PSSP), and starts of mature proteins (SMP) are shown. Letters highlighted in black are conserved amino acids, and those in gray are semiconserved.

FIG. 4.

Binding and competition experiments with biotinylated Cry1Ab and Cry1Ia7 with L. botrana BBMV. Lanes: 1, biotinylated Cry1Ab (control without BBMV); 2, biotinylated Cry1Ia7 (control without BBMV); 3 to 5, binding of biotinylated Cry1Ab to BBMV (lane 3) and in the presence of an excess of unlabeled Cry1Ab (lane 4) or Cry1Ia7 (lane 5); 6 to 8, binding of biotinylated Cry1Ia7 to BBMV (lane 6) and in the presence of an excess of unlabeled Cry1Ia7 (lane 7) or Cry1Ab (lane 8).

FIG. 5.

Binding and competition experiments with biotinylated Cry1Ab, Cry1Ac, and Cry1Ia7 with E. insulana BBMV. (A) Lanes: 1, biotinylated Cry1Ab (control without BBMV); 2 to 5, binding of biotinylated Cry1Ab to BBMV either without further addition (lane 2) or in the presence of an excess of unlabeled Cry1Ab (lane 3), Cry1Ia7 (lane 4), or Cry1Ac (lane 5); 6, biotinylated Cry1Ac (control without BBMV); 7 to 9, binding of biotinylated Cry1Ac to BBMV either without further addition (lane 7) or and in the presence of an excess of unlabeled Cry1Ac (lane 8) or Cry1Ia7 (lane 9). (B) Lanes: 1, biotinylated Cry1Ia7 (control without BBMV); 2 to 5, binding of biotinylated Cry1Ia7 to BBMV either without further addition (lane 2) or and in the presence of an excess of unlabeled Cry1Ia7 (lane 3), Cry1Ab (lane 4), or Cry1Ac (lane 5).