Abstract
ABSTRACTBacillus 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.
Highlights
Development and characterization of Sf9 cells resistant to Cry1Ca toxin Sf9 cells are known to be susceptible to the Cry1C Bacillus thuringiensis (Bt) toxin (Kwa et al, 1998)
Since (i) divalent ions chelators appeared to have a protective effect upon Cry1Ca toxicity and (ii) numerous crosstalk have been described between cAMP pathway and calcium signal (Hofer, 2012), we incubated Sf9 cells with the toxin in media with or without chelators and we evaluated cAMP production (Fig. 6)
In this report, we describe Sf9 cells resistant to the Cry1Ca toxin obtained after a selection program
Summary
The first and well-established model, referred to as the pore-forming model, requires the sequential binding to two specific receptors localized at the plasma membrane of insect intestinal cells: a cadherin receptor protein (CADR) and a glycosylphosphatidylinositol (GPI) membrane-anchored aminopeptidase N (APN). This sequential binding allows pre-pore complex formation and membrane insertion where they act as functional cationicspecific pores causing osmolytic lysis of targeted cells (JiménezJuárez et al, 2007; Soberón et al, 2000; Zhuang et al, 2002). Zhang and colleagues showed that an Mg2+-dependent signalling pathway is essential to Cry1A-induced cell death This model starts with the binding of Cry1A to the primary receptor CADR triggering the recruitment and activation of a heterotrimeric G protein, activation of an adenylyl cyclase (AC), and elevation of intracellular cyclic AMP (cAMPi). The existence of these many potential receptors makes it more difficult to demonstrate a single mode of action of Cry toxins
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