Abstract
Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.
Highlights
Insecticidal crystal toxins (Cry) are pore-forming toxins (PFT) produced by Bacillus thuringiensis (Bt) bacteria as crystalline inclusions during the sporulation phase of growth [1]
Many other PFT are produced by different pathogenic bacteria that kill their targets by making pores in the cell membrane of their target cells, affecting cell permeability and disrupting cellular integrity [4]
Studies of the Caenorhabditis elegans response to Cry5 toxin, such as microarrays and a genome-wide RNA interference (RNAi) analysis, showed that the C. elegans response is quite complex since 0.5% of the genome of this animal participates in the protection from PFT attack, with MAPK and JNK having pivotal roles in activating transcriptional and functional responses [6,7]
Summary
Insecticidal crystal toxins (Cry) are pore-forming toxins (PFT) produced by Bacillus thuringiensis (Bt) bacteria as crystalline inclusions during the sporulation phase of growth [1]. The Cry toxins are highly specific against different insect orders such as Lepidoptera, Diptera, Coleoptera, or Hymenoptera, as well as to nematodes. The Cry proteins show a complex mechanism of action involving multiple and sequential binding interactions with specific protein receptors located in the microvilli of midgut epithelial cells. The interaction with these receptors depends on a change in the oligomeric state of the toxin, from monomeric to oligomeric, leading to insertion of the oligomeric form of Cry toxin into the membrane, forming lytic pores that causes cell swelling, lysis and insect death [2,3]. Studies of the Caenorhabditis elegans response to Cry toxin, such as microarrays and a genome-wide RNA interference (RNAi) analysis, showed that the C. elegans response is quite complex since 0.5% of the genome of this animal participates in the protection from PFT attack, with MAPK and JNK having pivotal roles in activating transcriptional and functional responses [6,7]
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