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Item Transcriptional regulation during appressorium formation and function in Glomerella cingulata : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology at Massey University, Palmerston North, New Zealand(Massey University, 2006) Tong, XingZhangGlomerella cingulata, anamorph Colletotrichum gloeosporioides, causes bitter rot in apples and fruit rot in other subtropical fruits. In response to environmental cues such as contact with the host, Glomerella cingulata forms a special structure called an appressorium, which accumulates glycerol and thereby generates a sufficiently high turgor pressure to push an infection peg into the host tissue. It is known that the cAMP and MAPK signalling transduction pathways control appressorium formation and function in Colletotrichum species and other appressorium-forming fungi. This process is accompanied by a global change in gene expression. Little is known of transcriptional regulation during this process. The aim of this project was to study the transcriptional regulation of appressorium formation and function in G. cingulata. The G cingulata SAP gene had previously been shown to be expressed as a longer transcript during the early stage of appressorium differentiation. It was considered possible that the transcription factors that regulated expression of the longer transcript may be also involved in the regulation of appressorium differentiation. Identification of the transcription factor involved may help to understand the mechanisms that regulate appressorium differentiation. The plan was to use the yeast one-hybrid system to isolate the transcription factor. This required identification of the promoter regions responsible for expression of the longer SAP transcript. Therefore, the G. cingulata SAP promoter was characterized by mapping the transcription start point. Three transcription start points were determined by RLM-RACE. To further characterise the promoters, SAP-GFP reporter plasmids were constructed and transformed into G. cingulata. Even though a reasonable level of GFP expression was observed in RT-PCR experiments, however, no differences in fluorescence intensity were seen between the wild type and GFP reporter transformants. Therefore, no further attempts to study the sap promoter were made. The candidate gene approach was chosen as an alternative way to study the transcriptional regulation of appressorium formation and function in G. cingulata. The G. cingulata StuA gene was cloned using degenerate PCR, single specific primer PCR, subgenomic library screening and plasmid rescue from a disruption mutant. Targeted gene deletion of the G. cingulata StuA gene was successful. Deletion mutants display many phenotypic changes. Complementation mutants were constructed to confirm the function of this gene. A full length copy of this gene together with a second selection marker was reintroduced into the deletion mutant and the wild type phenotype was restored. Deletion mutants form appressoria at the normal rate and with unaltered morphology. In comparison with the wild type, these appressoria did not generate high turgor pressure as shown by a cytorrhysis assay. This resulted in a defect in appressorium penetration of onion epidermal cells. Nor were these mutants able to invade unwounded apples. Therefore, the G. cingulata StuA gene is required for appressorium function. In addition, deletion mutants displayed stunted aerial hyphae, "wettable" mycelium, reduced conidia production, and a defect in conidiophore and perithecium formation. These results suggested that the G. cingulata StuA gene has multiple roles in fungal development.Item Studies on the host-parasite relationships of Phytophthora cactorum (Leb. and Cohn) Schroet. and apple trees : a thesis presented in partial fulfilment of the requirements for the degree of Doctorate of Philosophy in Plant Pathology at Massey University(Massey University, 1986) Kelliher, Kevin JohnThe present work was undertaken to gain a greater understanding of the nature and location of resistance mechanisms present in apple cultivars to Phytophthora cactorum . Artifical inoculation methods of evaluating resistance to this pathogen were also studied. The amount and type of sporangial germination in the presence of root exudates was not related to the susceptibility of the apple cultivar involved. The greatest accumulation of zoospores occurred at root tip regions and wound sites (the sites of maximal root exudation). The numbers of zoospores which encysted at root tip regions was not related to cultivar resistance and the large variation suggested that the quantity and/or quality of exudation varies considerably from root to root. The germination of zoospore cysts was markedly enhanced by root exudates of all cultivars tested. Ninety-five percent of the cysts germinated on the root surface while only 80% germinated on cellophane membranes laid over roots. Very few germ tubes formed appressoria upon membranes. More than 95% of the germ tubes grew towards the root and over 98% of them formed appressoria on the root surface. Appressorial formation appeared to be induced by specific surface configurations such as breaks in the outer wall and junctions between cells, on the root surface. The severity of P. cactorum infection of the seedling unsuberised apple roots (cv. 'COP' and 'GS') was dependent upon the initial inoculum loading, up to a saturation point i.e. extensive infection; a larger inoculum loading was required to reach this point with the more resistant 'GS' cultivar. The resistant cv. 'M 793' tended to have a lower amount of infection than the susceptible 'MM 106' at corresponding levels of inoculum. However, in general, mechanisms of resistance did not appear to be operating external to apple tissue. Electron microscopy studies showed that the process of infection was similar in susceptible (MM 106) and resistant (M 793) cultivars. Hyphae grew both intracellularly and intercellularly within root cortical tissue. Penetration through cells appeared to be both mechanical and enzymatic, although penetration by mechanical pressure alone may sometimes occur. Cellular deterioration was frequently obvious 2-4 cells in advance of the mycelium. The histochemical studies showed that in vivo, wall degrading enzymes produced by P. cactorum appeared to have a significant role in assisting fungal growth through host tissue but were relatively insignificant in its destruction . The lack of apparent structural wound associated responses (e.g. papilla formation) formed in cells of either tested cultivar in response to P. cactorum infection indicates that the mechanisms of resistance of the more resistant cultivars are primarily physiological. Investigations of internal resistance were hampered by the lack of a fully reliable method of assessing resistance. The most convenient system, inoculation of excised twigs, was further characterised revealing that the type of tissue tested (cortex, phloem-cambium or unlignified xylem) and the basal-distal location of the sampled piece of the shoot needs to he specified for valid comparisons with other workers and correlations with other criteria. A technique of assessing levels of infection by oospore numbers in unsuberised roots is described and would possibly be a better method for determining a cultivar's resistance to the root rot and perhaps the crown rot forms of the disease. Endogenous levels of nutritional compounds: total nitrogen, soluble sugars and starch and of phenolic compounds did not appear to determine the rate of apple tissue colonisation by P. cactorum. Phloridzin, the major phenolic of apple tissue, was toxic to P. cactorum in vitro yet mean pathogen growth rates of over 10 mm per day were measured within host tissue indicating that the hyphae are not physically encountering inhibitory amounts of phloridzin in vivo.