Polarity-dependent expression and localization of secretory glucoamylase mRNA in filamentous fungal cells

Live cell imaging of β-tubulin mRNA reveals spatiotemporal expression dynamics in the filamentous fungus Aspergillus oryzae

“…The quantitative data on the number of nuclei and cytoplasmic mRNA suggested that the same amount of btuA mRNA was expressed per nucleus at all sites in the hyphal cells. This mode of expression of btuA mRNA differed from the finding that glaA mRNA, encoding the secretory protein glucoamylase, is transcribed predominantly in nuclei near the hyphal tip and septum 34 . In filamentous fungi, secretory proteins are secreted from the hyphal tips and septa 31 , so it makes sense that glaA mRNA is preferentially expressed in nuclei near such sites.…”

“…Therefore, in order to analyze tubulin mRNA and microtubules simultaneously in A. oryzae , we decided to analyze microtubules with α-tubulin AtuA as described above and visualize β-tubulin mRNA. The MS2 system is often used to visualize mRNA in living cells, and in fact, mRNA for glaA , which encodes glucoamylase, a secreted enzyme, has been visualized and analyzed in A. oryzae 17 , 34 . We introduced 24 copies of the aptamer MS2-binding sites (MBS) downstream of the endogenous btuA (AO090009000281) encoding β-tubulin and generated a btuA -MS2 strain that expresses two copies of EGFP fused to MS2-coat protein (MCP), which binds specifically to MBS (Fig.…”

“…It has been shown that in A. oryzae , a portion of the mRNA for glaA , which encodes the secretory protein glucoamylase, migrates long distances on microtubules in a kinesin motor-dependent manner 34 . Therefore, nocodazole treatment, a microtubule polymerization inhibitor, was first applied to analyze the microtubule dependence of btuA mRNA dynamics.…”

“…Genomic DNA of the wild-type A. oryzae strain RIB40 was used as the template for the DNA cloning. For visualization of microtubules, the plasmid pgPaEGAtuA 34 for expression of EGFP-AtuA was transformed into the NSlD1 strain to create the GaA1 strain. To further visualize nuclei, the plasmid pgPtmCN 34 expressing mCherry-NLS was introduced into the GaA1 strain to create the GaA_CN1 strain.…”

“…In A. oryzae , the localization of each mRNA and translated protein, including α-amylase and actin, was analyzed by single-molecule fluorescence in situ hybridization 32 , 33 . Furthermore, mRNA for glucoamylase (GlaA), one of the major secretory proteins in A. oryzae , was analyzed by the MS2 system, which enables mRNA to be visualized in living cells, revealing that glaA mRNA is transcribed from the nucleus near the hyphal tip and septum, the sites of secretion 34 . However, filamentous fungi, including A. oryzae , are multinuclear and multicellular, and the mechanisms that regulate the spatiotemporal expression of proteins that function intracellularly, such as tubulin, are largely unknown.…”