Processing of Cry3Bb1 and Cry34Ab1/Cry35Ab1 in the Diabrotica midgut

Transcription

1 COST Action 862 Bacterial Toxins for Insect Control Working Group 3 Integrated Pest Management Workshop Bt-toxins against Diabrotica: Potential and prospective use in Europe University of Kiel, Salzau Castle, Germany 27 th March 2009 Processing of Cry3Bb1 and Cry34Ab1/Cry35Ab1 in the Diabrotica midgut Renate Kaiser-Alexnat Julius Kühn-Institute Institute for Biological Control Darmstadt, Germany

2 Potential resistance mechanisms Depending on the mode of action, the resistance of insects to B.t. toxins can occur at any step of the toxic pathway. This includes as its main steps the enzymatic digestion of B.t. toxins with proteases present in the midgut fluid and the binding of B.t. toxins to specific receptors of the midgut epithelium. Protease-mediated resistance qualitative and quantitative changes in protease activities in the midgut fluid Receptor-mediated resistance alternate toxin binding to specific receptors in the midgut epithelium

3 Studies on the processing of B.t. toxins in the midgut of Western corn rootworm third instar larvae Protease activities in the midgut fluid Proteolytic processing of B.t. toxins with midgut fluid Binding of B.t. toxins to receptors of the midgut epithelium (binding analysis)

4 Preparation of Diabrotica midguts foregut midgut hindgut Digestive system of Chrysomelidae The average of the ph values of the midgut fluid was 5.75.

5 Studies on the processing of B.t. toxins in the midgut of Western corn rootworm third instar larvae Protease activities in the midgut fluid Proteolytic processing of B.t. toxins with midgut fluid Binding of B.t. toxins to receptors of the midgut epithelium (binding analysis)

6 Photometrical tests pna substrate processing [nmol/ml/min] For the identification and quantification of protease activities in the midgut fluid, photometric tests with specific chromogenic substrates, mainly peptidyl-p-nitroanilide (pna) substrates, and specific inhibitors were conducted. Inhibitor concentration [µm]

7 Processing of pna substrates

8 Comparison of pna substrate processing activities Serine endopeptidases Cysteine endopeptidases Metallo endopeptidases Exopeptidases

9 Aspartic endopeptidase pepsin Pepsin-activity in midgut fluid of susceptible WCR 3 rd instar larvae

10 Studies on the processing of B.t. toxins in the midgut of Western corn rootworm third instar larvae Protease activities in the midgut fluid Proteolytic processing of B.t. toxins with midgut fluid Binding of B.t. toxins to receptors of the midgut epithelium (binding analysis)

11 Proteolytic processing of Cry3Bb1 with midgut fluid 60 min incubation Proteolytic processing of B.t. corn-toxin Cry3Bb1 with midgut fluid (DS) of third instar WCR larvae

12 Proteolytic processing of Cry34Ab1/Cry35Ab1 with midgut fluid 30 min incubation 30 min incubation Proteolytic processing of the binary B.t. toxin Cry34Ab1/Cry35Ab1 with midgut fluid (DS) of third instar WCR larvae

13 Studies on the processing of B.t. toxins in the midgut of Western corn rootworm third instar larvae Protease activities in the midgut fluid Proteolytic processing of B.t. toxins with midgut fluid Binding of B.t. toxins to receptors of the midgut epithelium (binding analysis)

14 Binding analysis - method Objectives proof of the binding of B.t. toxins to the midgut epithelium characterization of receptors studies on binding site competition between B.t. toxins Steps preparation of intact BBMV s separation with SDS-PAGE transfer to a membrane (western-blot) incubation with the B.t. toxins (ligand-blot) Proof of the binding biotinylation of the B.t. toxins streptavidin-horseradish-peroxidaseconjugat and ECL-system

15 Binding analysis test system

16 Binding analysis Cry3Bb1 vs. Cry34Ab1/Cry35Ab1

17 Acknowledgments Monsanto Cry3Bb1 ARS Patent Culture Collection DOW AgroSciences Cry34Ab1/Cry35Ab1 Rearing Diabrotica larvae, Preparation of midguts Kai Gloyna, Ines Rogotzki BTL Bio-Test Labor GmbH Biochemical work Simon Feiertag JKI Darmstadt