AgSPA2 and AgBOI control landmarks of filamentous growth in the filamentous ascomycete Ashbya gossypii

The morphological requirements for the development of a fungal mycelium include the emergence of germ tubes, a permanent hyphal tip extension, lateral and apical branching and septation. The basis for each of these events, also termed landmarks of fi lamentous growth, is polarised growth. Thus polarised growth is essential for hyphal and mycelial morphogenesis. We wanted to investigate genes in the fi lamentous ascomycete Ashbya gossypii that are involved in polarised growth and control landmarks of fi lamentous growth. In "Chapter 1" and "Chapter 2" I described the identifi cation of two genes that are involved in polarised growth and I highlighted their roles in landmarks of fi lamentous growth. At the end of the fi rst two chapters I draw a refi ned model of the developmental pattern in A.gossypii with the insight gained from these fi rst two chapters. In "Chapter 3" and "Chapter 4" I described two novel tools that were established for the functional analysis of the genes investigated in the fi rst two chapters. These two methods were the construction of a versatile module for C-terminal GFP fusion and an integration module based on the AgADE2 marker. Chapter 1 We wanted to identify genes in A.gossypii that are important for hyphal morphogenesis. As polarised growth is crucial for morphogenesis in A.gossypii and S.cerevisiae we hypothesised that a similar set of genes is required for polarised growth in A.gossypii and S.cerevisiae; however, differences in orthologous proteins might have an impact on the regulation of polarised growth and thus guide the process of fi lamentation in A.gossypii and budding in S.cerevisiae. We screened for A.gossypii proteins that have an orthologue in S.cerevisiae implicated in polarised growth and that display a signifi cant difference in the primary structure compared to the S.cerevisiae orthologue. This revealed AgSpa2p, a homologue of ScSpa2p. AgSpa2p is more than twice as long as ScSpa2p due to an extended internal domain without signifi cant homology to ScSpa2p. AgSpa2p localises permanently to hyphal tips and transiently to sites of septation. We constructed two AgSPA2 mutants, a partial deletion of the internal domain alone and a complete deletion. We could show that these mutant strains display alterations in the branching frequency and in the hyphal tip growth speed potential. The branching frequency and the hyphal tip growth speed potential are two factors that have an impact on the hyphal tip growth speed. The two mutants behave opposing in respect of each of these factors and the two factors have a competing effect on the hyphal tip growth speed. We suggest that AgSpa2p is required to balance these two factors to achieve an effi cient hyphal tip growth speed and the extended internal domain in AgSpa2p plays an important role in this process. Chapter 2 We screened an existing A.gossypii knock out library for strains that show defects in landmarks of fi lamentous growth. We identifi ed a strain that frequently displayed spherically enlarged hyphal tips. The deleted gene in that strain was identifi ed as AgBOI. AgBoip represents an orthologue of the redundant proteins in S.cerevisiae ScBoi1p and ScBoi2p, which are implicated in polarised growth. We could show that AgBoip is required for the emergence of germ tubes and for the initiation of lateral branches. Moreover AgBoip is required for permanent hyphal tip extension, as polarisation of hyphal tips is not permanently maintained in AgboiΔ strains. We could show that prior to a spherical enlargement of hyphal tips in AgboiΔ the polarisation marker AgSpa2p delocalises from the tip. Tips can be repolarised which goes in parallel with a relocalisation of AgSpa2p. In spherically enlarged tips the actin cytoskeleton is also depolarised. AgBoip itself localised to sites of polarisation similar to cortical actin patches. We suggest that AgBoip is required for establishment of cell polarity to initiate germ tubes and lateral branches and for maintenance of cell polarity to allow a permanent hyphal tip extension. Chapter 3 The green fl uorescent protein (GFP) is of extraordinary value in molecular biology. It allows the visualisation of proteins in organisms and together with advanced microscopy techniques it enables dynamic studies in living cells. We constructed a versatile module for PCR based C-terminal GFP-fusion and established microscopy techniques for dynamic GFP studies in A.gossypii. The studies described in "Chapter 1" and "Chapter 2" would not have been possible without the development of this module. Chapter 4 The functional analysis of genes often requires the expression of truncated alleles, e.g. reporter modules as the GFP, tagged proteins for biochemical analyses, mutant alleles or overexpression constructs. As it was of importance in this work to express certain GFP fusion proteins as second copies we constructed an integration module on the basis of the AgADE2 marker. The mode of action of the integration module is the reconstitution of a truncated AgADE2 ORF thereby co-integrating the fragment of interest.