Autophagy Induced By Bt Toxin And Its Mechanism

The Bt toxin protein produced by Bacillus thuringiensis can kill a variety of agricultural pests.Not only does activated Bacillus thuringiensis crystal toxin act on midgut cells of target insects,but also on some cultured insect cell lines.Although it is well known that activated crystal toxin kills midgut cells through forming pores on brush border membrane by oligotoxin,it is unclear that other influences of low levels of toxin on cells.Determining a series of physiological and biochemical reactions and the mechanisms of Bt toxin resistance in Bt toxin toxic insecticidal cells will help improve our ability to control pests and our understanding of bacterial toxicology.Research has shown that autophagy plays a key role in the innate immune defense of intracellular pathogens(autophagy against intracellular pathogens is also referred to as xeno-autophagy).Autophagy has recently involved responses to various PFTs,but it is not clear how to cause this process and the significance of this phenomenon is also unclear.In this study,we investigated the effects of low levels of activated Cry1 Ac and Cry1Ca toxins on cell autophagy in cultured insect and mammal cells,and explored the role of autophagy in Bt toxin resistance in insect cells.All the tested insect cells(Hi5,Sl-HP and Sf9)were susceptible to activated Cry1Ca,but only Sl-HP cells were susceptible to activated CrylAc.Both activated CrylAc and Cry1Ca is not toxic to mammalian cell 293T.Cry1Ca enhanced autophagy of Hi5,Sl-HP and Sf9 cells.And CrylAc had only significant effects on autophagy of Sl-HP cells.And the receptors of CrylAc were over-expressed in Hi5 cells,Helicoverpa armigera HaCadherin and HaABCC2,and the activated Cry1Ac also had significant influence on autophagy of the cells.Both activated CrylAc and Cry1Ca had no effect on autophagy of 293T cells.This indicates that the induction of autophagy by toxins requires the toxin receptor to mediate and is positively correlated with the sensitivity of cells to toxins.In experiment,we treated Hi5 cells with four autophagy inhibitors(Baf/CQ/ConA/3-MA),western blot analysis showed that three autophagy inhibitors Baf/CQ/ConA could inhibit the conversion of Atg8 to Atg8-PE induced by Cry1Ca toxin,and caused the decreased susceptibility to Cry1Ca.In addition,we interfered gene expression with the autophagy-related genes Hi5 Atg5/Atg13 dsRNA,and found that the RNAi caused the decreased susceptibility to Cry1Ca in Hi5 cells.Therefore,autophagy plays an important role in mediating the toxicity of Cry1Ca to Hi5 cells.We treated three insect cells(Hi5/Sl-HP/Sf9)with Jnk/P38/PI3K inhibitors,which could inhibit the autophagic level of Cry1Ca toxin-enhanced cells,but the results showed that the three insect cells treated with Jnk inhibitors to Cry toxins have the most significant decrease in sensitivity to the toxin,which is the same effect as using Jnk inhibitors.RNA interference(RNAi)knockdown of SIJnk in Sl-HP cells resulted in the same effect as the treatment with Jnk inhibitor.These results demonstrate that the enhanced autophagic levels of insect cells induced by Cry1 Ac and Cry1Ca toxins can be blocked by classic autophagy inhibitors,and that depending on Atg5 and Atg13,Jnk signaling plays an important role in this process,and autophagy affects the sensitivity of cells to Bt toxins to a certain extent.The establishment of the insect cell autophagy induction and blocking system is of great significance for further investigation of the autophagic molecular mechanism of insect cells,and will promote research on insect developmental biology and pest control.