Live cell fluorescence imaging for early expression and localization of RIB1 and RIB3 genes in Ashbya gossypii

Abstract

Ashbya gossypii is a riboflavin overproducing filamentous fungus. RIB1 and RIB3 genes encode GTP-cyclohydrolase II (GCH II) and DHBP synthase, respectively, the two rate limiting enzymes of the riboflavin biosynthetic pathway. The genes encoding yeast enhanced green fluorescent protein (yEGFP) and mCherry were fused with RIB1 and RIB3 genes, under their native promoters by PCR-based gene tagging method for their early in vivo expression and cellular localization in A. gossypii. In the integrative transformants, the fusion proteins were expressed as cytoplasmic proteins from the germ bubble stage, in all the cells throughout the hypha. This was evident from the observation that mCherry fusion proteins were seen outside the vacuoles in the cytoplasm. The older matured cells of 14 h hyphae developed large vacuoles which showed green autofluorescence due to riboflavin. It is concluded that RIB1 and RIB3 genes are constitutively expressed in all the cells of this multicellular multinucleate fungus.

Keywords: Ashbya gossypii; Fluorescent imaging; Gene expression; RIB1 and RIB3 genes.