title: Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa).
abstract: Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6μg/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality.
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Serbia.
authors.forename: Milica
authors.initials: M
authors.lastname: Popovic
authors.forename: Uros
authors.initials: U
authors.lastname: Andjelkovic
authors.forename: Lidija
authors.initials: L
authors.lastname: Burazer
authors.forename: Buko
authors.initials: B
authors.lastname: Lindner
authors.forename: Arnd
authors.initials: A
authors.lastname: Petersen
authors.forename: Marija
authors.initials: M
authors.lastname: Gavrovic-Jankulovic
mesh_terms: D029042:Actinidia / Q000235:genetics / Q000378:metabolism*; D000528:Alternaria / Q000187:drug effects / Q000254:growth & development; D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology; D020171:Botrytis / Q000187:drug effects / Q000254:growth & development; D002942:Circular Dichroism; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D025282:Mycelium / Q000187:drug effects / Q000254:growth & development; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D020539:Sequence Analysis, Protein
chemical_list: D000935:Antifungal Agents; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D011994:Recombinant Proteins
keywords: Antifungal activity; Cysteine proteinase inhibitor; Kiwifruit; Phytocystatin