Characterisation of fatty acid synthase beta subunit dehydratase (FASI) in lipid biosynthesis pathway of Magnaporthe oryzae

Abstract

Lipid biosynthesis pathway produces glycerol, which is essential in fuelling the turgor pressure necessary for conidiogenesis and penetration of host by M. oryzae. Lipid biosynthesis pathways are required for pathogenicity and virulence in many fungi, but the connection between fungal pathogenicity and lipid biosynthesis pathway is not fully understood. Here, we report on an upstream component of lipid biosynthesis, fatty acid synthase beta subunit dehydratase (FAS1) gene, which has been reported to play a role in fatty acid metabolism in yeast and bacteria. Firstly, FAS1 was structurally studied and showed presence of a long α-helix “HotDog” domain surrounded by a bent β-sheet “bun”, essential for fatty acid metabolism in organisms. Majority of the subfamilies of HotDog were categorised into two groups; dehydratase and thioesterase. These two groups were found in ascomycetes, basidiomycetes, Gram-positive and negative bacteria. Then, FAS1 was silenced through homologous double crossover to study its role in lipid biosynthesis of Magnaporthe oryzae strain S6. The resulting Δfas1-1 and Δfas1-2 were analysed morphologically, biochemically and at the molecular level to determine functional variations from wildtype. FAS1 is able to utilise 1 to 19 C fatty acids as a sole carbon source and is required for lipid biosynthesis and pathogenesis. The vegetative growth of Δfas1-1 and Δfas1-2 mutants is significantly different (p<0.05) from that of the wildtype and the highest drop was observed in oleic acid at 46% and 14%, respectively. The mycelial dry weight of Δfas1-1 and Δfas1-2 mutants on all media dropped more than 50% compared to wildtype. There was a ~10-fold reduction on oatmeal media and a ~5-folds reduction on PDA media observed in conidiation assessment assays of FAS1 although both media were known to trigger sporulation. The mutants showed disruption in terms of sporulation and morphology. Compared to the wildtype strain, Δfas1-1 and Δfas1-2 mutants formed mycelium that was mildly pigmented. Further, biochemical and gene expression (PEX6 and ICL1) studies revealed that fatty acid degradation pathway may have been interrupted by FAS1 deletion. According to the sandwich enzyme immunoassay, FAS1 activity was not observed in mutants growing on glucose (1-6 C fatty acid) or olive oil (7-19 C fatty acid). This indicates a possible interruption of the functional peroxisomal pathway in the mutants. FAS protein concentration was not detected on media supplemented by short chain (1-6 C) and medium chain fatty acid (7-19 C) in both mutants and these results suggests that the Δfas1 mutants may lack functional peroxisomes and therefore may be defective in β-oxidation of fatty acids which explains the reduced or nonpresence of lipid deposits in the spores. Taken together, FAS1 is an important gene for development of mycelium structure in terms of cell wall components, lipid metabolism and mobilisation of lipid. Results also implicate FAS1 in sporulation, conidium morphology and melanin biosynthesis. Therefore, the deletion of FAS1 may have resulted in the interruption of the machinery related to the fungal pathogenicity.