title: A new F131V mutation in Chlamydomonas phytoene desaturase locates a cluster of norflurazon resistance mutations near the FAD-binding site in 3D protein models.
abstract: The green alga Chlamydomonas reinhardtii provides a tractable genetic model to study herbicide mode of action using forward genetics. The herbicide norflurazon inhibits phytoene desaturase, which is required for carotenoid synthesis. Locating amino acid substitutions in mutant phytoene desaturases conferring norflurazon resistance provides a genetic approach to map the herbicide binding site. We isolated a UV-induced mutant able to grow in very high concentrations of norflurazon (150 µM). The phytoene desaturase gene in the mutant strain contained the first resistance mutation to be localised to the dinucleotide-binding Rossmann-likedomain. A highly conserved phenylalanine amino acid at position 131 of the 564 amino acid precursor protein was changed to a valine in the mutant protein. F131, and two other amino acids whose substitution confers norflurazon resistance in homologous phytoene desaturase proteins, map to distant regions in the primary sequence of the C. reinhardtii protein (V472, L505) but in tertiary models these residues cluster together to a region close to the predicted FAD binding site. The mutant gene allowed direct 5 µM norflurazon based selection of transformants, which were tolerant to other bleaching herbicides including fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wild type cells. Norflurazon resistance and beflubutamid sensitivity allow either positive or negative selection against transformants expressing the mutant phytoene desaturase gene.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Julio V
authors.initials: JV
authors.lastname: Suarez
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: Banks
authors.affiliation: Syngenta, Bracknell, Berkshire, United Kingdom.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Thomas
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Anil
authors.initials: A
authors.lastname: Day
mesh_terms: D000577:Amides / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D053209:Herbicide Resistance / Q000235:genetics; D006540:Herbicides / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D011724:Pyridazines / Q000494:pharmacology*
chemical_list: D000577:Amides; D006540:Herbicides; D010940:Plant Proteins; D011724:Pyridazines; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C022398:phytoene dehydrogenase; C008677:norflurazone; C587871:2-(4-fluoro-3-(trifluoromethyl)phenoxy)-N-(phenylmethyl)butanamide