title: Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.
abstract: Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides.
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, Ljubljana 1000, Slovenia.
authors.forename: Ida
authors.initials: I
authors.lastname: Šmid
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, Ljubljana 1000, Slovenia.
authors.forename: Ana
authors.initials: A
authors.lastname: Rotter
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, Ljubljana 1000, Slovenia; Department of Biotechnology, Jožef Stefan Institute, Jamova cesta 39, Ljubljana 1000, Slovenia.
authors.forename: Kristina
authors.initials: K
authors.lastname: Gruden
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova cesta 39, Ljubljana 1000, Slovenia.
authors.forename: Jože
authors.initials: J
authors.lastname: Brzin
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, Ljubljana 1000, Slovenia.
authors.forename: Meti
authors.initials: M
authors.lastname: Buh Gašparič
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova cesta 39, Ljubljana 1000, Slovenia; Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, Ljubljana 1000, Slovenia.
authors.forename: Janko
authors.initials: J
authors.lastname: Kos
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, Ljubljana 1000, Slovenia.
authors.forename: Jana
authors.initials: J
authors.lastname: Žel
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova cesta 39, Ljubljana 1000, Slovenia. Electronic address: jerica.sabotic@ijs.si.
authors.forename: Jerica
authors.initials: J
authors.lastname: Sabotič
mesh_terms: D000818:Animals; D001517:Coleoptera / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004064:Digestive System / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D005260:Female; D005656:Fungal Proteins / Q000235:genetics / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D007306:Insecticides / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects / Q000502:physiology; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000494:pharmacology; D011198:Solanum tuberosum / Q000235:genetics*
chemical_list: C411656:Clitocypin protein, Clitocybe nebularis; D015853:Cysteine Proteinase Inhibitors; D005656:Fungal Proteins; D007306:Insecticides; D011994:Recombinant Proteins
keywords: Bioinsecticide; Crop protection; Cysteine protease; Intestain; Protease inhibitor; Transgenic potato