Hyphal differentiation in the fungal mycelium

Abstract

The aim of this PhD Thesis was to study whether hyphae differentiate within a fungal colony. I have shown that this is indeed the case in Aspergillus niger, a fungus used for large scale production of industrial proteins. Future research will aim at the mechanisms underlying differentiation, which could be used to improve the A. niger cell factory. Differentiation with respect to expression of genes encoding secreted proteins was studied using GFP as a reporter. GFP fluorescence resulting from glaA expression was primarily observed at the periphery of colonies that had been transferred from a repressing to an inducing medium. In contrast, aguA expression was shown to be more evenly distributed. The spatial expression of glaA and aguA correlates with the expression of the genes encoding their activators; amyR and xlnR, respectively. The fluorescence of individual hyphae at the periphery of induced colonies was studied using confocal microscopy. Although all hyphae exhibited green fluorescence large differences in the intensity of the signal between hyphae were observed. Statistical analysis showed that the distribution of fluorescence intensities deviated from normality after inducing glaA or aguA. Furthermore, it was inferred that the fluorescence distributions are composed of a weighted mixture of two normal components. Hence, it is concluded that two types of hyphae exist at the periphery of colonies; namely, those hyphae that express glaA or aguA at a high level and those that express these genes at a low level. This is a remarkable finding considering the fact that the high and low expressers experience identical environmental conditions. In liquid shaken cultures fluorescence intensity between hyphae at the outer zone of formed pellets was quite variable. However, statistical analysis did not confirm the existence of hyphal differentiation in the case of glaA and aguA. Yet, the faeA promoter was differentially expressed. It thus seems that differential expression of promoters depends on the growth condition. My Thesis also describes that cytosolic proteins can stream through hyphae and can even be translocated from the vegetative mycelium into the reproductive structure called the conidiophore. Both the vegetative mycelium and the conidiophores were fluorescent when a cytosolic GFP was expressed from the gpdA or glaA promoter, but when a nuclear targeting signal was fused to GFP only nuclei of the vegetative hyphae turned fluorescent. Quantification of fluorescence of cytosolic GFP indicates that the composition of the conidiospore depends, at least partially, on the expression profile of the vegetative mycelium that underlies the conidiophore. These results show that gene expression should not only be studied in the conidiophore but also in the feeding vegetative mycelium to understand development of the reproductive structure. Finally, my Thesis describes the development of a fluorescent reporter system, which facilitates research aiming at hyphal differentiation in the mushroom forming fungus S. commune.