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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.

The interaction between Lycopersicon esculentum and the biotrophic fungal pathogen Cladosporium fulvum is a model for studying the molecular basis of plantpathogen interactions. Cf genes confer resistance against specific races of the fungus through recognition of proteins encoded by fungal avirulence (Avr) genes. Cf genes present in the near-isogenic lines Cf2, Cf4, Cf5 and Cf9 have been shown to encode Type I extracellular leucine-rich repeat (LRR) glycoproteins anchored in the plasma membrane. The objective of this research was to isolate and characterise a novel locus for resistance to C. fulvum. The Cf1l line contains one characterised Cf gene (Cf-4), and one other gene that was designated Cf-11. Previous reports indicated Cf- 11 may represent a novel resistance specificity. Results presented in this thesis demonstrate that Cf-11 is unlinked to known Cf gene loci, and thus represents a new locus in tomato for resistance to C. fulvum. Markers linked in cis and in trans to Cf- 11 were identified by AFLP analysis of various bulked segregant pools. Cf-11 was mapped to an interval on chromosome l1 close to the reported map location of Cf-3. A high-resolution genetic map of the target locus was constructed to facilitate cloning of Cf-I1, and eventually, to provide further insight into the tomato-C. fulvum interaction. To test the hypothesis that Cf-11 and Cf-3 might form part of a complex locus, differences in their resistance specificities were investigated using different C. fislvum races, but these analyses proved inconclusive. However, a detailed analysis of the resistance to C. fulvum race 4 infection revealed clear differences between Cf- 3 and Cf-11, consistent with the hypothesis that they represent distinct resistance genes. Cf-11 arose in a breeding experiment in 1972 but its precise origin is not known. The low level of DNA sequence polymorphism between Cf1l and Cif lines suggests Cf-11 arose from an L. esculentum variety, and possibly a line that contained Cf-3.

L'objectif global était d'évaluer la diversité génétique d'une collection de variétés de pommes de terre indigènes originaires de l'île de Chiloé, pour caractériser la résistance de ces variétés au mildiou (Phytophthora infestans) et pour en savoir davantage sur l'état de conservation in-situ. Les sujets suivants a été élaboré: a) la conservation in situ de Solanum indigènes à l'île la grande de Chiloé et son impact sur la diversité, b) l'évaluation de la diversité morphologique des pommes de terre indigènes que nous avons recueillis c) l'évaluation de la diversité génétique par marqueurs microsatellites et AFLP, d) la caractérisation du terrain et des résistances in-vitro au mildiou. Sur la base des résultats des enquêtes, nous pouvons conclure que le maintien in situ de la diversité de pommes de terre indigènes n' est pas bien conservé, en raison des forts changes sociaux et économiques sur l'île de Chiloé. Il y a un processus clair de remplacer les variétés indigènes pour les cultivars commerciaux en réponse aux conditions du marché. Les variétés natives sont présents dans 80,5% des fermes de Chiloé, mais la diversité a été considérablement réduite. Les résultats de la diversité morphologique ont montré la formation de groupes sous le nom populaire et des attributs locaux affectés par les agriculteurs eux-mêmes. L'évaluation moléculaire de la collection (SSRs et AFLP) révèle un haut degré de diversité génétique. Les deux marqueurs ont été uniformes dans la classification Solanum fernandezianum comme le génotype plus éloigné. La SSR a permis l'estimation de la teneur des informations polymorphes pour sept loci dont les valeurs se situaient entre 0,63 à 0,89. Les deux types de marqueurs, n'a pas fourni les mêmes groupes parmi les accessions. L'étude SSR basé a montré une faible différenciation entre les pommes de terre indigènes et l'amélioration des variétés cultivées. L'analyse de la diversité sur la base des AFLP n'était pas incompatible avec ce résultat parce que, malgré contenant un seul cultivar (Désirée), le cultivar a été regroupée avec les variétés indigènes. En ce qui concerne la résistance au mildiou, la plupart des accessions sont dans les rangs modérément résistante à modérément sensible. / The overall objective was to assess the genetic diversity of a collection of native potato varieties originating from the island of Chiloe, to characterize the resistance of these varieties to late blight (Phytophthora infestans) and to inquire about the status of in-situ conservation. The following specific topics was developed: a) conservation in-situ of native Solanum within the great isle of Chiloe and its impact on diversity, b) evaluation of the morphological diversity of the native potatoes that we have collected c) assessment of genetic diversity of the collection by the mean of microsatellite and AFLP markers, d) characterization of field and in-vitro resistances to late blight. Based on the results of the surveys, we can conclude that in-situ maintenance of native potato diversity i is not well preserved, due to strong social and economic changes on the island of Chiloe. There is a clear process of replacing native varieties for commercial cultivars in response to market conditions. Native varieties are present in 80.5% of the farms of Chiloe, but the diversity of varieties has been significantly reduced. The results morphological diversity showed the formation of groups under the popular name and local attributes assigned by the farmers themselves. Molecular evaluation of the collection (SSRs and AFLP) reveals a high degree of genetic diversity. Both markers were consistent in classifying Solanum fernandezianum as the more distant genotype compared to all the others. The SSRs allowed the estimation of the polymorphic information content for seven loci which values ranged between 0.63 to 0.89. Both types of markers, did not provide the same groupings among the accessions. The SSR-based study showed a low differentiation between native potatoes and improved cultivated varieties. The analysis of diversity based on the AFLP was not inconsistent with this result because despite containing only one cultivar (Desiree), the cultivar was grouped with the native varieties. In relation to resistance to late blight, most accessions of native potato fall into the ranks moderately resistant to moderately susceptible.

Bibliography: leaves 169-185 / viii, 185 leaves, [1] leaf of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Pathology, 1987