Early endosome motility mediates α-amylase production and cell differentiation in Aspergillus oryzae

Abstract

Recent research in filamentous fungi has revealed that the motility of an endocytic organelle early endosome (EE) has a versatile role in many physiological functions. Here, to further examine the motility of EEs in the industrially important fungus Aspergillus oryzae, we visualized these organelles via the Rab5 homolog AoRab5 and identified AoHok1, a putative linker protein between an EE and a motor protein. The Aohok1 disruptant showed retarded mycelial growth and no EE motility, in addition to an apical accumulation of EEs and peroxisomes. We further demonstrated that the Aohok1 disruptant exhibited less sensitivity to osmotic and cell wall stresses. Analyses on the protein secretory pathway in ΔAohok1 cells showed that, although distribution of the endoplasmic reticulum and Golgi was not affected, formation of the apical secretory vesicle cluster Spitzenkörper was impaired, probably resulting in the observed reduction of the A. oryzae major secretory protein α-amylase. Moreover, we revealed that the transcript level of α-amylase-encoding gene amyB was significantly reduced in the Aohok1 disruptant. Furthermore, we observed perturbed conidial and sclerotial formations, indicating a defect in cell differentiation, in the Aohok1 disruptant. Collectively, our results suggest that EE motility is crucial for α-amylase production and cell differentiation in A. oryzae.

Single-Molecule FISH Reveals Subcellular Localization of α-Amylase and Actin mRNAs in the Filamentous Fungus Aspergillus oryzae

“…Since amyB mRNAs were not observed at the tip regions of hyphae cultured in both minimal and complete media, they may be immediately targeted to the ER for translation before being released to diffuse through the cytoplasm. Indeed, in A. oryzae, the ER is localized throughout the hyphal cells and normally surrounds the nuclei (Kuratsu et al, 2007;Togo et al, 2017;Morita et al, 2020). Thus, such preferential localization of amyB mRNAs might result in the efficient translation of large amounts of α-amylase for In contrast, actA mRNAs are mainly localized to the apical region, where actin cables and patches preferentially reside.…”

Membrane Traffic in Aspergillus oryzae and Related Filamentous Fungi

“…Endocytosed proteins are first transported to early endosome (EE), then to late endosome (LE) and finally to the vacuole for degradation ( Figure 2 B). To characterize these endocytic organelles, SNARE and small GTPase Rab proteins have been employed in A. oryzae [ 53 , 99 ]. In mammalian cells, EEs are maturated to LEs by conversion of Rab5 to Rab7, the molecular mechanisms of which are thought to be conserved in filamentous fungi [ 100 , 101 ].…”

“…In mammalian cells, EEs are maturated to LEs by conversion of Rab5 to Rab7, the molecular mechanisms of which are thought to be conserved in filamentous fungi [ 100 , 101 ]. In filamentous fungi, Rab5-positive EEs are highly motile and move through the cell; by contrast, Rab7-positive LEs are mostly static and in general observed adjacent to vacuoles [ 99 , 101 , 102 ]. EEs motility is a hallmark of endocytic organelles in filamentous fungi and its underlying molecular mechanisms are described in the following section.…”

“…EEs visualized with EGFP-tagged AoRab5, the Rab5 homolog in A. oryzae , exhibit constant motility through the cell, not restricted when endocytosis is induced [ 99 ]. This observation raised the possibility that EEs have other physiological roles than endocytic function.…”

“…Moreover, long-range EE motility has a role in signal transduction in the process of plant infection [ 121 ]. To elucidate further physiological roles of EE motility, the deletion mutant of Aohok1 , the Hook ortholog in A. oryzae , was investigated [ 99 ]. In the Aohok1 disruptant, levels of transcripts and secreted proteins of α-amylase are reduced, suggesting that EE motility has roles in gene expression and secretion for α-amylase in A. oryzae .…”