title: The insecticidal activity of recombinant garlic lectins towards aphids.
abstract: The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at < or =48 h exposure; similar timescale to starvation) was only apparent at the highest lectin concentrations tested (2.0 mg ml(-)1), but dose-dependent chronic toxicity (mortality at >3d exposure) was observed over the concentration range 0.125-2.0 mg ml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding.
authors.affiliation: Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK.
authors.forename: Elaine
authors.initials: E
authors.lastname: Fitches
authors.forename: Duncan
authors.initials: D
authors.lastname: Wiles
authors.forename: Angela E
authors.initials: AE
authors.lastname: Douglas
authors.forename: Gareth
authors.initials: G
authors.lastname: Hinchliffe
authors.forename: Neil
authors.initials: N
authors.lastname: Audsley
authors.forename: John A
authors.initials: JA
authors.lastname: Gatehouse
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids; D018826:CD13 Antigens / Q000378:metabolism; D002850:Chromatography, Gel; D005737:Garlic / Q000737:chemistry* / Q000235:genetics; D041981:Gastrointestinal Tract / Q000378:metabolism; D007306:Insecticides / Q000302:isolation & purification / Q000378:metabolism*; D037102:Lectins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013393:Sucrase / Q000378:metabolism; D018675:Toxicity Tests
chemical_list: D007306:Insecticides; D037102:Lectins; D008024:Ligands; D011994:Recombinant Proteins; D013393:Sucrase; D018826:CD13 Antigens