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Investigating the role of lipid mobilisation and metabolism in the rice blast fungus Magnaporthe oryzae
Yusof, Mohd Termizi Bin
Thesis or dissertation
University of Exeter
The rice blast fungus Magnaporthe oryzae infects plants by developing a specialised infection structure known as an appressorium. In M. oryzae the appressorium is a melanin-pigmented cell with a reinforced cell wall, allowing the cell to generate enormous internal turgor to enable penetration of the plant tissue by a narrow penetration hypha. Previously it has been shown that mobilisation of lipid droplets to the nascent appressorium is essential for successful plant infection. In this thesis, I describe a series of studies that have identified and characterised genes associated with infection-associated lipid metabolism in M. oryzae, including the role of fatty acid β-oxidation, acetyl-CoA transport and metabolism and regulation of lipid body breakdown. First, I report identification of FAR1 and FAR2, which encode putative Zn2-Cys6 binuclear proteins that appear to act as transcriptional regulators of lipid metabolism. Deletion mutants of M. oryzae FAR1 and FAR2 were deficient in growth on long chain fatty acids. In addition Δfar1 mutants were unable to grow on acetate as a sole carbon source. FAR1 and FAR2 affect the expression of genes involved in fatty acid β-oxidation, acetyl-CoA translocation, peroxisomal biogenesis, the glyoxylate cycle and acetyl-CoA synthesis. Next, I functionally characterized the CAR1, CAR2, CAR3 and CAR4 genes, which encode enzymes involved in carnitine biosynthesis, which is required for translocation of acetyl-CoA between mitochondria, peroxisomes and the cytoplasm. Only a sub-set of carnitine biosynthetic enzymes was necessary for growth on fatty acids and lipids by M. oryzae, but redundancy was also apparent in carnitine biosynthesis, because CAR1, CAR2, CAR3 and CAR4 were dispensable for pathogenicity, while the carnitine acetyltranferase, PTH2, is essential for rice blast disease. To investigate the role of the appressorium acetyl-CoA pool in more detail, I functionally characterized the acetyl-CoA synthetase gene, ACS2 and ACS3, and CRC1, which encodes the mitochondrial carnitine carrier, both of which are highly expressed during appressorium development and appear to play a role in appressorium physiology. Finally, to understand the onset of lipid droplet degradation in more detail, I characterised a putative perilipin, encoded by CAP20, which localizes specifically to the periphery of lipid droplets. Perilipins are known to play roles in lipid droplet mobilisation and lipase accessibility. Consistent with this idea, M. oryzae mutants lacking CAP20, were severely affected in fungal virulence due to impaired appressorium function. When considered together, the results presented in this thesis suggest that lipid body mobilisation and acetyl-CoA metabolism are fundamental processes required for appressoria to function correctly and cause rice blast disease.
PhD in Biological Sciences