Resistant and susceptible strains of rice differ in oxylipin biosynthesis, suggesting anti-fungal effects of allylic alcohols formed by rice LOXs

This article is available online at http://www.jlr.org array of biological mediators, e.g., eicosanoids and leukotrienes in man and to oxylipins and jasmonates in plants and fungi ( 1–4 ). All LOXs belong to the same gene family and usually contain nonheme iron ( 2, 5 ). LOXs have been implicated in physiological and pathophysiological events, ranging from asthma, cancer, and infl ammation in humans to plant-pathogen interactions. LOXs are therefore targets of drug design and of agricultural interest ( 1, 3, 6, 7 ). The harvests of rice, wheat, and other staple crops can be severely reduced by fungal pathogens, and plant LOXs can be induced and form oxylipins in early defense reactions ( 8 ). Rice is a model organism for studies of fungal infections and for biosynthesis of oxylipins in response to pathogens ( 9–12 ). The rice genome contains 14 genes with LOX homology ( 13 ). Different 13 S -LOXs can be expressed in the leaves in response to the rice blast fungus, Magnaporthe oryzae, and 13 S -LOXs occur in developing seeds together with 9 S -LOX ( 10, 11, 14 ). Resistant and susceptible strains of rice differ in oxylipin biosynthesis, suggesting anti-fungal effects of allylic alcohols formed by rice LOXs ( 15 ). Paradoxically, LOXs also occur in three rice pathogenic fungi: the blast fungus Magnaporthe oryzae (anamorph Pyricularia oryzae ), the stem rot fungus Magnaporthe salvinii (syn. Sclerotium oryzae, anamorph Pyricularia salvinii ), and the root rot and Take-all fungus Gaeumannomyces graminis var. graminis. Mycelia of G. graminis penetrate the rice roots and secrete 13 R -LOX with catalytic manganese lipoxygenase (13 R -MnLOX) ( 16, 17 ). The biological function of Abstract The mycelium of the rice stem pathogen, Magnaporthe salvinii , secreted linoleate 9 S -lipoxygenase (9 S -LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9 Shydroperoxy-octadeca-10 E ,12 Z -dienoic acid (9 SHPODE) to threo 10 (11)-epoxy-9 S -hydroxy-12 Z -octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9 S LOX was expressed in Pichia pastoris. Recombinant 9 S -LOX oxidized 18:2n-6 directly to 9 SHPODE, the end product, and also to two intermediates, 11 Shydroperoxy-9 Z ,12 Z octadecenoic acid (11 SHPODE; 5%) and 13 Rhydroperoxy9 Z ,11 E -octadecadienoic acid (13 RHPODE; 1%). 11 Sand 13 RHPODE were isomerized to 9 SHPODE, probably after oxidation to peroxyl radicals,  -fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10 E ,12,14 Eoctadecatrienoic acid. 9 S -LOX contained catalytic manganese (Mn:protein  0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13 R -LOX with catalytic manganese lipoxygenase (13 RMnLOX) of the Takeall fungus. The Leu350Met mutant of 9 SLOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13 RMnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9 S -LOX with catalytic manganese along with a specifi c EAS. Alterations in the Sloane determinant of 9 S -LOX and 13 R -MnLOX with larger and smaller hydrophobic residues interconverted the regiospecifi c oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion. — Wennman, A., and E. H. Oliw. Secretion of two novel enzymes, manganese 9 S -lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii. J. Lipid Res . 2013. 54: 762–775.