Novel Fus3- and Ste12-interacting protein FsiA activates cell fusion-related genes in both Ste12-dependent and -independent manners in Ascomycete filamentous fungi.

Filamentous fungal cells, unlike yeasts, fuse during vegetative growth. The orthologs of mitogen-activated protein (MAP) kinase Fus3 and transcription factor Ste12 are commonly involved in the regulation of cell fusion. However, the specific regulatory mechanisms underlying cell fusion in filamentous fungi have not been revealed. In the present study, we identified the novel protein FsiA as an AoFus3- and AoSte12-interacting protein in the filamentous fungus Aspergillus oryzae. The expression of AonosA and cell fusion-related genes decreased upon fsiA deletion and increased with fsiA overexpression, indicating that FsiA is a positive regulator of cell fusion. In addition, the induction of cell fusion-related genes by fsiA overexpression was also observed in the Aoste12 deletion mutant, indicating that FsiA can induce the cell fusion-related genes in an AoSte12-independent manner. Surprisingly, the fsiA and Aoste12 double deletion mutant exhibited higher cell fusion efficiency and increased mRNA levels of the cell fusion-related genes as compared to the fsiA single deletion mutant, which revealed that AoSte12 represses the cell fusion-related genes in the fsiA deletion mutant. Taken together, our data demonstrate that FsiA activates the cell fusion-related genes by suppressing the negative function of AoSte12 as well as by an AoSte12-independent mechanism.

Author Correction: BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

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BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.