Development of a hybrid delta-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura

Abstract

A hybrid δ-endotoxin protein was designed against a polyphagous lepidopteran insect pest Spodoptera litura, which is tolerant to most of the known δ-endotoxins. The hybrid δ-endotoxin was created by replacing amino acid residues 530-587 in a poorly active natural Cry1Ea protein, with a highly homologous 70 amino acid region of Cry1Ca in domain III. The truncated δ-endotoxins Cry1Ea, Cry1Ca and the hybrid protein Cry1EC accumulated in Escherichia coli to form inclusion bodies. The solubilised Cry1EC made from E. coli was 4-fold more toxic to the larvae of S. litura than Cry1Ca, the best known δ-endotoxin against Spodoptera sp. None of the two truncated toxins, solubilised from E. coli caused larval mortality. However, trypsinised Cry1Ca protoxin obtained from E. coli and solubilised from inclusion bodies caused mortality of S. litura with LC₅₀ 513 ng/ml semi synthetic diet. A synthetic gene coding for the hybrid δ-endotoxin Cry1EC was designed for high level expression in plants, taking into consideration several features found in the highly expressed plant genes. Transgenic, single copy plants of tobacco as well as cotton were developed. The selected lines expressed Cry1EC at 0.1-0.7% of soluble leaf protein. Such plants were completely resistant to S. litura and caused 100% mortality in all stages of larval development. Hence, unlike in E. coli, the hybrid δ-endotoxin folded into a functionally active conformation in both tobacco and cotton leaves. The truncated Cry1EC expressed in tobacco leaves was about 8-fold more toxic (LC₅₀ 58 ng/ml diet) compared to expression in E. coli.