Transcriptomic analysis of Spodoptera frugiperda Sf9 cells resistant to Bacillus thuringiensis Cry1Ca toxin, reveals that extracellular Ca2+, Mg2+ and production of cAMP are involved in toxicity

Abstract

Bacillus thuringiensis (Bt) produces pore forming toxins that have been used for pest control in agriculture for many years. However, their molecular and cellular mode of action is still unclear. While a first model, referred to as the pore forming model, is the most widely accepted scenario, a second model proposed that toxins could trigger an Mg2+- dependent intracellular signalling pathway leading to cell death. Although Cry1Ca has been shown to form ionic pores in the plasma membrane leading to cell swelling and death, we investigated the existence of other cellular or molecular events involved in Cry1Ca toxicity. The Sf9 insect cell line, derived from Spodoptera frugiperda, is highly and specifically sensitive to Cry1Ca. Through a selection program we developed various levels of laboratory-evolved Cry1Ca resistant Sf9 cell lines. Using a specific S. frugiperda microarray we performed a comparative transcriptomic analysis between sensitive and resistant cells and revealed genes differentially expressed in resistant cells and related to cation-dependent signalling pathways. Ion chelators protected sensitive cells from Cry1Ca toxicity suggesting the necessity of both Ca2+ and/or Mg2+ for toxin action. Selected cells were highly resistant to Cry1Ca while toxin binding onto their plasma membrane was not affected. This suggested a resistance mechanism different from the classical “loss of toxin binding”. We observed a correlation between Cry1Ca cytotoxicity and the increase of intracellular cAMP levels. Indeed, Sf9 sensitive cells produced high levels of cAMP upon toxin stimulation, while Sf9 resistant cells were unable to increase their intracellular cAMP. Together, these results provide new information about the mechanism of Cry1Ca toxicity and clues to potential resistance factors yet to discover.