The establishment of cell polarity in filamentous fungi is critical for the control of many cellular and developmental processes, including polarized hyphal growth, intracellular movement of organelles, protein secretion, cell wall biosynthesis and the development of conidiophores. In yeasts, but also in other eukaryotic cells, Rho-related GTPases (Rho, Rac and Cdc42) are involved in controlling and organizing actin cytoskeleton and thereby controlling polarized cell growth. To assess the roles of the Rac1 and CDC forty-two homologs in Aspergillus niger (RacA and CftA), we have made racA and cftA deletion mutants. Deletion mutants were efficiently obtained using the A. niger kusA mutant strain (Meyer et al., 2007). We also attempted to construct a D racA, D cftA double mutant. However, the double knockout is most probably lethal as primary transformants containing both the racA and the cftA deletion were all heterokaryotic and spores derived from these transformants were never viable. The synthetic lethality between D racA and D cftA suggests an overlapping function of the two GTPases. The phenotypic consequences of the loss of racA or cftA have been studied in detail. Deletion of racA resulted in abnormal germination and branching patterns, and in reduced conidiation. Deletion of cftA resulted in slower germination and a reduction in hyphal growth rate. The racA deletion phenotype was more severe than that of cftA. The localization of actin was examined using immunofluorescence in the wild type and the racA and cftA mutant strains. The localization of actin in a D racA showed condensation at the extreme apex of the hyphal tip, while in both the D cftA and wild type strains a gradient of actin patches towards the tip was observed. Overexpression of the dominant active form of RacA (RacAG12V) resulted in the loss of polarized hyphal growth and swollen germtubes. The polarized actin cytoskeleton in the swollen hyphae induced by RacAG12V overexpression is completely lost. To further assess the role of RacA and CftA in polar growth and their involvement in the organisation of the actin cytoskeleton, GFP-RacA and GFP-CftA fusion constructs will be made.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)