Therese Oskarsson:
Filamentous growth in Eremothecium fungi - Molecular characterization of the Ashbya gossypii ARF3 module

Date: 13-03-2014    Supervisor: Steen Holmberg

The fungal kingdom encompasses a diverse group of organisms some of which have a great impact on human lives, either as domesticated benefactors or as human and crop pathogens. Using the filamentous fungus Ashbya gossypii and its close relative Eremothecium cymbalariae as model organisms, this thesis deals with some of the aspects of hyphal growth, which is an important virulence factor for pathogenic fungi infecting both humans and plants.

Hyphal establishment through continuous polar growth is a complex process, requiring the careful coordination of a large subset of proteins involved in polarity establishment and maintenance, cytoskeleton dynamics and intracellular transport. The first part of this thesis addresses the A. gossypii Arf3 small GTPase and its GEF- and GAP regulators; Yel1 and Gts1, which has been implicated in polar growth in a wide range of organisms. We could demonstrate that manipulations of the regulatory AgGts1 rendered A. gossypii strains with severe actin localization, endocytic and morphological phenotypes, presumably due to unregulated AgArf3 activity. As a homolog of the extensively more studied mammalian ARF6, we hypothesize that the continuous AgArf3 activity causes an abnormal accumulation of PI(4,5)P2 in the plasma membrane, which in turn can be linked to the slow endocytic uptake of FM4-64, the sub-apical actin localization and subsequently the atypical mycelial morphology observed in Aggts1 mutants. Furthermore, we demonstrated that in addition to the AgArf3 GAP-regulatory activity of AgGts1, the protein could have additional actin organizing properties.

In the second and third part, this thesis addresses the use of A. gossypii and its relative E. cymbalariae as model organisms for filamentous growth. A series of assays analyzed the capability of Eremothecium genus fungi to invade and colonize both plant- and insect hosts. We found that neither A. gossypii nor E. cymbalariae are able to penetrate any host tissue, and although A. gossypii is classified as a plant pathogen it is strictly dependent on its insect vectors for infection. In addition, we optimized a series of molecular tools for E. cymbalariae to enable a faster and more efficient approach for genetic comparisons between Eremothecium genus fungi.