The small GTP-binding proteins AgRho2 and AgRho5 regulate tip-branching, maintenance of the growth axis and actin-ring-integrity in the filamentous fungus Ashbya gossypii.

GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.

A network involving Rho-type GTPases, a paxillin and a formin homologue regulates spore length and spore wall integrity in the filamentous fungus Ashbya gossypii.

Fungi produce spores that allow for their dispersal and survival under harsh environmental conditions. These spores can have an astonishing variety of shapes and sizes. Using the highly polar, needle-shaped spores of the ascomycete Ashbya gossypii as a model, we demonstrated that spores produced by this organism are not simple continuous structures but rather consist of three different segments that correlate with the accumulation of different materials: a rigid tip segment, a more fragile main spore-compartment and a solid tail segment. Little is currently known about the regulatory mechanisms that control the formation of the characteristic spore morphologies. We tested a variety of mutant strains for their spore phenotypes, including spore size, shape and wall defects. The mutants that we identified as displaying such phenotypes are all known for their roles in the regulation of hyphal tip growth, including the formin protein AgBni1, the homologous Rho-type GTPases AgRho1a and AgRho1b and the scaffold protein AgPxl1. Our observations suggest that these proteins form a signalling network controlling spore length by regulating the formation of actin structures.

A Bnr-like formin links actin to the spindle pole body during sporulation in the filamentous fungus Ashbya gossypii.

Formin proteins are nucleators of actin filaments and regulators of the microtubule cytoskeleton. As such, they play important roles in the development of yeast and other fungi. We show here that AgBnr2, a homologue of the ScBnr1 formin from the filamentous fungus Ashbya gossypii, localizes to the spindle pole body (SPB), the fungal analogue of the centrosome of metazoans. This protein plays an important role in the development of the typical needle-shaped spores of A. gossypii, as suggested by several findings. First, downregulation of AgBNR2 causes defects in sporangium formation and a decrease in the total spore number. Second, a fusion of AgBNR2 to GFP that is driven by the native AgBNR2 promoter is only visible in sporangia. Third, AgBnr2 interacts with a AgSpo21, a sporulation-specific component of the SPB. Furthermore, we provide evidence that AgBnr2 might nucleate actin cables, which are connected to SPBs during sporulation. Our findings add to our understanding of fungal sporulation, particularly the formation of spores with a complex, elongated morphology, and provide novel insights into formin function.

Selection of STOP-free sequences from random mutagenesis for 'loss of interaction' two-hybrid studies.

The investigation of protein-protein interactions is an essential part of biological research. To obtain a deeper insight into regulatory protein networks, the identification of the components, domains and especially single residues that are involved in these interactions is helpful. A widespread and attractive genetic tool for investigation of protein-protein interactions is the yeast two-hybrid system. This method enables large-scale screens and its application is cheap and relatively simple. For identification of the amino acids in a protein sequence that are essential for interaction with a specific partner, yeast two-hybrid assays can be combined with random mutagenesis of the sequence of interest. A common problem with such an experiment is the generation of stop codons within the mutagenized fragments, leading to the isolation of many false positives when screening for loss of interaction using the two-hybrid method. To overcome this problem, we modified the yeast two-hybrid system to allow selection for sequences without stop codons. To achieve this, we fused the ScURA3 marker-gene in frame to the mutagenized fragments. We show here that this marker is fully functional when fused to a two-hybrid construct with a nuclear localization signal, such as a Gal4 activation domain and a prey protein, thus allowing selection of stop-free sequences on media without uracil. Using the Rho-binding domain from a Bni1-like formin and different Rho-type GTPases from Ashbya gossypii as examples, we further show that our system can be used to screen large numbers of transformants for loss of protein-protein interactions in combination with random mutagenesis.