title: Single substitutions to closely related amino acids contribute to the functional diversification of an insect-inducible, positively selected plant cystatin.
abstract: A causal link has been reported between positively selected amino acids in plant cystatins and the inhibitory range of these proteins against insect digestive cysteine (Cys) proteases. Here we assessed the impact of single substitutions to closely related amino acids on the contribution of positive selection to cystatin diversification. Cystatin sequence alignments, while confirming hypervariability, indicated a preference for related amino acids at positively selected sites. For example, the non-polar residues leucine (Leu), isoleucine (Ile) and valine (Val) were shown to predominate at positively selected site 2 in the N-terminal region, unlike selected sites 6 and 10, where polar residues are preferred. The model cystatin SlCYS8 and single variants with Leu, Ile or Val at position 2 were compared with regard to their ability to bind digestive proteases of the coleopteran pest Leptinotarsa decemlineata and to induce compensatory responses in this insect. A functional proteomics procedure to capture target Cys proteases in midgut extracts allowed confirmation of distinct binding profiles for the cystatin variants. A shotgun proteomics procedure to monitor whole Cys protease complements revealed protease family specific compensatory responses in the insect, dependent on the variant ingested. Our data confirm the contribution of closely related amino acids to the functional diversity of positively selected plant cystatins in a broader structure/function context imposing physicochemical constraints to primary structure alterations. They also underline the complexity of protease/inhibitor interactions in plant-insect systems, and the challenges still to be met in order to harness the full potential of ectopically expressed protease inhibitors in crop protection.
authors.affiliation: Département de Phytologie, Université Laval, Québec City, Canada.
authors.forename: Asieh
authors.initials: A
authors.lastname: Rasoolizadeh
authors.affiliation: Département de Phytologie, Université Laval, Québec City, Canada.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Goulet
authors.affiliation: Département de Phytologie, Université Laval, Québec City, Canada.
authors.forename: Frank
authors.initials: F
authors.lastname: Sainsbury
authors.affiliation: Département de Biologie, Université Laval, Québec City, Canada.
authors.forename: Conrad
authors.initials: C
authors.lastname: Cloutier
authors.affiliation: Département de Phytologie, Université Laval, Québec City, Canada.
authors.forename: Dominique
authors.initials: D
authors.lastname: Michaud
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001517:Coleoptera / Q000201:enzymology / Q000502:physiology; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D014644:Genetic Variation; D006790:Host-Parasite Interactions; D007313:Insecta / Q000201:enzymology / Q000502:physiology; D007814:Larva / Q000201:enzymology / Q000502:physiology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000469:parasitology; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000235:genetics* / Q000378:metabolism / Q000469:parasitology; D017386:Sequence Homology, Amino Acid
chemical_list: D000596:Amino Acids; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010940:Plant Proteins
keywords: activity-based functional proteomics; functional diversification; insect digestive proteases; plant cystatins; positive selection