Nce102 co-localizes with main cytosolic components
of eisosomes, Pil1, and Lsp, and the deletion of Nce102 resulted in an altered number and distribution of eisosomes (Walther et al., 2006). These data suggest that Nce102 is required for normal eisosome and MCC formation. In a recent study, the eisosomal protein homologues (PilA, PilB, and SurG) in Aspergillus nidulans have been characterized (Vangelatos et al., 2010). Detailed analysis of pilA, pilB, or surG deletion strains have confirmed the nonessential role of these proteins in fungal http://www.selleckchem.com/products/azd4547.html growth. Furthermore, the endocytosis process was not affected in these mutants, demonstrating a possible functional divergence of eisosomal proteins in Aspergilli. A similar study on eisosomal proteins of filamentous fungus Ashbya PLX3397 gossypii confirmed the important role of pil1 homologue in polar growth and the nonessential role of Nce102 homologue
in growth and eisosomal stability (Seger et al., 2011). In the present study, we have characterized Nce102 homologue, AfuNce102, in the human pathogen, Aspergillus fumigatus. Our results indicate that this gene is not essential for hyphal growth or pathogenesis, but it is required for normal sporulation. Localization studies using an enhanced green fluorescent protein (EGFP)-tagged AfuNce102 construct demonstrated that AfuNce102 is primarily localized to endoplasmic reticulum with more intensity at the hyphal tip. Edoxaban During the conidiogenesis, the protein localized to conidiophores and conidia. Aspergillus fumigatus strain AF293 and its PyrG derivative were used for the isolation of the Nce102 gene homologue and in gene disruption experiments. Escherchia coli Top10 (Invitrogen) cells were used in DNA recombinant procedures. pGEM-T Easy cloning system (Promega) was used for cloning of PCR products. Plasmid pGEM-GlaA-EGFP, comprised the Aspergillus niger glaA promoter, EGFP sequence, and glaA termination signal, was used for preparation of NCE-GFP-tagged construct. Plasmid pAN7.1 containing
the hygromycin resistance gene as a fungal selection marker was used in co-transformation experiments of NCE-GFP-tagged construct (Punt et al., 1987). Fungal strains were grown and kept on SAB agar or SAB agar medium supplemented with uridine and uracil (UU). Modified Vogel’s medium (Vogel, 1956) was used in isolation of fungal transformants. For phenotypic analysis of strains, radial growth rates were determined by cultivation of fungal spores (104 spores) on center of SAB and modified Vogel’s agar plates at 30, 37, and 42 °C followed by serial measurement of colonies’ diameter for 5 days. Fungal DNA was prepared as previously described (Moller et al., 1992). RNA samples were purified using a commercial kit (Qiagen, RNA easy® Mini kit). Molecular methods including ligation of DNA fragments, transformation of E.