Expression and genetic manipulation of Bacillus thuringiensis toxins for improved toxicity and development of a protocol for in vivo selection of toxin variants with improved activity

Bacillus thuringiensis (Bt) and its insecticidal toxins have been used in pest control for decades but there is a great concern about its future as a successful pest control agent due to the development of insect resistance and the narrow spectrum of activity of the toxins. To ensure the continuous relevance of Bt toxins in pest control, projects aimed at isolating novel Bt strains expressing toxins with improved activity are vigorously pursued and the genetic manipulation of existing toxins to improve their activity and overcome resistance is also undertaken. The aim of this project was to genetically manipulate the genes encoding Cry1Ah and Cry1Ie for improved activity aimed at countering resistance evolved by populations of Plutella xylostella. The project was also aimed at expressing cry30Ea and cry40Da genes cloned from highly mosquitocidal Bt strains S2160-1 and S2196 respectively and at developing a protocol for the in vivo selection of toxin variants with improved activity. Cry1Ah was successfully expressed in E. coli JM109 under the control of a cry1Ac promoter and ribosome binding site and in Bt IPS/78/11 under the control of the cyt1Aa promoter while Cry1Ie was also expressed in E. coli JM109. The expressed Cry1Ah and Cry1Ie toxins were found to be toxic to both susceptible (G88) and Cry1A resisitant (KARAK) populations of Plutella xylostella though there was significant cross resistance to Cry1Ah in KARAK. A genetically manipulated hybrid toxin CryAIA aimed at creating a novel toxin that captures the relatively broad spectrum of Cry1Ah but overcoming KARAK resistance was expressed but found to be non-toxic. Attempts to express cry30Ea and cry40Da were also not successful despite utilising different hosts and expression vector systems that have successfully been used in expressing other cry genes. Meanwhile, the strategy designed to enrich for more toxic Bt strains in vivo in from a mixed treatment in fact found that the non-toxic R128M strain dominated the toxic 431 strain.