Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N -(3-triethylammoniumpropyl)-4-( p -diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC ( Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was clearly demonstrated, its physiological importance in filamentous fungi still remains largely unaddressed. We generated a strain in which A. oryzae end4 (Ao end4 ), the A. oryzae homolog of Saccharomyces cerevisiae END4 / SLA2 , was expressed from the Ao end4 locus under the control of a regulatable thiA promoter. The growth of this strain was severely impaired, and its hyphal morphology was altered in the Ao end4 -repressed condition. Moreover, in the Ao end4 -repressed condition, neither FM4-64 nor AoUapC-EGFP was internalized, indicating defective endocytosis. Furthermore, the localization of a secretory soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) was abnormal in the Ao end4 -repressed condition. Aberrant accumulation of cell wall components was also observed by calcofluor white staining and transmission electron microscopy analysis, and several genes that encode cell wall-building enzymes were upregulated, indicating that the regulation of cell wall synthesis is abnormal in the Ao end4 -repressed condition, whereas Ao pil1 disruptants do not display the phenotype exhibited in the Ao end4 -repressed condition. Our results strongly suggest that endocytosis is crucial for the hyphal tip growth in filamentous fungi.

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