AEM Accepts, published online ahead of print on 6 April 2007

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Appl. Environ. Microbiol. doi:10.1128/AEM.01056-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Binding and pore formation of Cyt1Aa and Cry11Aa toxins of Bacillus thuringiensis israelensis to brush border membrane vesicles of Tipula paludosa (Diptera: Nematocera)

Jesko Oestergaard, Ralf-Udo Ehlers^*, Amparo C Martínez-Ramírez, and Maria Dolores Real

Institute for Phytopathology, Dep. for Biotechnology and Biological Control, Christian-Albrechts-University, Kiel, Germany; Departamento de Genetica, Facultat de Ciències Biològiques, Universitat de Valencia, Burjassot (Valencia), Spain

^* To whom correspondence should be addressed. Email: ehlers{at}biotec.uni-kiel.de.

Bacillus thuringiensis svar. israelensis (Bti) produces four insecticidal crystal proteins (Cry4A, Cry4B, Cry11A and Cyt1A). Toxicity of recombinant Bti strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein whereas Cry4A, Cry4B and Cry11A were virtually non-toxic. Synergistic effects were recorded when either Cry4A and/or Cry4B were combined with Cyt1A, but not with Cry11A. The binding and pore formation are key steps in the mode of action of Bti insecticidal crystal proteins (ICPs). Binding and pore forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analysed. Solubilization of Cry11Aa resulted in two fragments with apparent molecular weights of 32 and 36 kDa. No binding of the 36 kDa fragment to T. paludosa BBMVs was detected, whereas the 32 kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments indicating a low specificity of the binding. In contrast to mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase-K, previously reported to activate these toxins respectively, had the opposite effect. Higher efficiency in pore formation was observed when Cyt1A was proteinase-K treated, while the activity of trysin treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. padulosa.