ABSTRACTThe University of ManchesterName: Smija Mariam KurianDegree title: Doctor of PhilosophyResearch title: Live-cell imaging of the early stages of colony development in Fusarium oxysporum in vitro and ex vivo during infection of a human corneal modelDate: May 2016Abstract: Fusarium oxysporum is a major fungal plant pathogen and emerging human pathogen. It has been hypothesised that conidial anastomosis tube (CAT) fusion may facilitate horizontal gene/chromosome transfer that could result in the acquisition of new genetic traits in fungi lacking sexual reproduction. However, we know little about the mechanistic basis of CAT fusion in fungi lacking sexual stages such as F. oxysporum. In the first part of my research the optimal culture conditions were determined for subsequent studies of CAT fusion in this fungus. CAT fusion was optimal in 1% potato dextrose broth supplemented with one of a diverse range of chemicals inoculated with microconidia of 1 x 106 spores/ml at 22-25 °C and pH 5.5-6.3. Cell adhesion facilitated by the chemical supplement was required for CAT fusion. ~ 40% CAT fusion was routinely observed with these conditions at 12 h post incubation. In the second part of my research live-cell imaging was used to characterize the process of CAT fusion and differentiate between CATs and germ tubes. In particular, the composition of the CAT cell wall surface was shown to be different from that of germ tubes. Nuclei, mitochondria, vacuoles and lipid droplets were also shown to move between germlings following CAT fusion. In the third part of my research quantitative analysis of the influence of various Ca2+ modulators on CAT fusion was done and evidence obtained showed that Ca2+ signalling is important during CAT fusion and involves the uptake of Ca2+ from the external environment by the Cch1 Ca2+ channel, and the involvement of the primary intracellular Ca2+ receptor, calmodulin, and the mitochondrial Ca2+ uniporter. In the final part of my thesis, the morphogenesis of the fungus was analysed during infection of an ex vivo human corneal model. The stages of infection that were characterised were: spore adhesion; bipolar germination; hyphal extension, branching, fusion and penetration into the corneal tissue; and sporulation involving the formation of microconidia and intercalary chlamydospores within the host tissue. Image analysis techniques were developed for the quantification and analysis of branching angles, total hyphal length, number of hyphal branches and hyphal growth units within the infected ex vivo corneal tissue. Using the fusion mutant, ∆fso, CAT fusion was shown not to be required for infection.