In filamentous fungi, the existence of endocytosis had been elusive mainly because of lack of reliable indicators of endocytosis. Recently, however, we and others have shown that endocytic markers, fluorescent dye FM4-64 and AoUapC (uric acid-xanthine permease) fused with EGFP, are internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was made clear, its physiological roles largely remain unaddressed in filamentous fungi. Thus, we have examined the physiological function of endocytosis in the filamentous fungus Aspergillus oryzae. We cloned Aoend4, the A. oryzae homolog of Saccharomyces cerevisiae END4/SLA2 (synthetic lethal with ABP1), a gene related to endocytosis. End4p/Sla2p functions as an adaptor connecting actin cytoskeleton and invaginated plasma membrane. The Aoend4 disruptant was not obtained probably because Aoend4 is an essential gene for hyphal growth. Thus, we generated a strain, named TE4, in which Aoend4 was expressed under the control of thiA promoter from the Aoend4 locus. The growth of this strain was severely impaired when thiamine was added to the culture medium to shut off the expression of Aoend4. Furthermore, abnormal accumulation of the cell wall was observed by Calcofluor White staining and TEM analysis, indicating that the cell wall became thicker when the expression of Aoend4 was repressed. We next generated a strain named TESn1 which expresses EGFP-AoSnc1, a v-SNARE required for secretion fused with EGFP, in the conditional Aoend4 background. In TESn1, aberrant invagination-like structures labelled by EGFP-AoSnc1 were observed under Aoend4-repressed condition. These results suggest that endocytosis in filamentous fungi functions in recycling of cell wall-building enzymes and components required for the apical growth to the tip region.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)