title: Gene deletion of 7,8-linoleate diol synthase of the rice blast fungus: studies on pathogenicity, stereochemistry, and oxygenation mechanisms.
abstract: Linoleate diol synthases (LDS) are heme enzymes, which oxygenate 18:2n-6 sequentially to (8R)-hydroperoxylinoleic acid ((8R)-HPODE) and to (5S,8R)-dihydroxy-, (7S,8S)-dihydroxy-, or (8R,11S)-dihydroxylinoleic acids (DiHODE). The genome of the rice blast fungus, Magnaporthe oryzae, contains two genes with homology to LDS. M. oryzae oxidized 18:2n-6 to (8R)-HPODE and to (7S,8S)-DiHODE, (6S,8R)-DiHODE, and (8R,11S)-HODE. Small amounts of 10-hydroxy-(8E,12Z)-octadecadienoic acid and traces of 5,8-DiHODE were also detected by liquid chromatography-mass spectrometry. The contribution of the 7,8-LDS gene to M. oryzae pathogenicity was evaluated by replacement of the catalytic domain with hygromycin and green fluorescent protein variant (SGFP) cassettes. This genetically modified strain Delta7,8-LDS infected rice leaves and roots and formed appressoria and conidia as the native fungus. The Delta7,8-LDS mutant had lost the capacity to biosynthesize all the metabolites except small amounts of 8-hydroxylinoleic acid. Studies with stereospecifically deuterated linoleic acids showed that (8R)-HPODE was formed by abstraction of the pro-S hydrogen at C-8 and antarafacial oxygenation, whereas (7S,8S)-DiHODE and (8R,11S)-DiHODE were formed from (8R)-HPODE by suprafacial hydrogen abstraction and oxygenation at C-7 and C-11, respectively. A mac1 suppressor mutant (Delta mac1 sum1-99) of M. oryzae, which shows cAMP-independent protein kinase A activity, oxygenated 18:2n-6 to increased amounts of (10R)-HPODE and (5S,8R)-DiHODE. Expression of the 7,8-LDS gene but not of the second homologue was detected in the suppressor mutant. This suggests that PKA-mediated signaling pathway regulates the dioxygenase and hydroperoxide isomerase activities of M. oryzae.
authors.affiliation: Section of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Centre, SE-751 24 Uppsala, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Jernerén
authors.forename: Ane
authors.initials: A
authors.lastname: Sesma
authors.forename: Marina
authors.initials: M
authors.lastname: Franceschetti
authors.forename: Marina
authors.initials: M
authors.lastname: Francheschetti
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal / Q000502:physiology*; D008041:Linoleic Acids / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000382:microbiology*; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology*; D020037:Sordariales / Q000201:enzymology* / Q000472:pathogenicity*
chemical_list: D005656:Fungal Proteins; D008041:Linoleic Acids; D010105:Oxygenases; C122014:linoleate diol synthase