title: Prohibitin, an essential protein for Colorado potato beetle larval viability, is relevant to Bacillus thuringiensis Cry3Aa toxicity.
abstract: Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.
authors.affiliation: Departamento de Genética, Facultad de Ciencias Biológicas, Universidad de Valencia, Dr. Moliner 50, Burjassot, 46100 Valencia, Spain.
authors.forename: Camila
authors.initials: C
authors.lastname: Ochoa-Campuzano
authors.forename: Amparo C
authors.initials: AC
authors.lastname: Martínez-Ramírez
authors.forename: Estefanía
authors.initials: E
authors.lastname: Contreras
authors.forename: Carolina
authors.initials: C
authors.lastname: Rausell
authors.forename: M Dolores
authors.initials: MD
authors.lastname: Real
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000633:toxicity*; D001517:Coleoptera / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000633:toxicity*; D007814:Larva / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D000091402:Prohibitins; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011198:Solanum tuberosum / Q000469:parasitology*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D000091402:Prohibitins; D012097:Repressor Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Bacillus thuringiensis; Colorado potato beetle; Cry3Aa; Prohibitin; RNAi