Abstract
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.
Highlights
Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production
To observe the fused cells of A. oryzae by the bi-molecular fluorescence complementation (BiFC) system, we constructed three plasmids: pUNANgA for expressing LZA fused with N-terminal half (1-153 aa) of EGFP, pUNACgB for expressing LZB fused with C-terminal half (154–239 aa) of EGFP, and pgDNNgACgB for expressing both the fusion proteins (Fig. S1B)
The BiFC system is a powerful tool to analyse the morphology of fused cells in the filamentous fungal species with considerably low cell fusion efficiency
Summary
Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. 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. Conidial anastomosis and vegetative hyphal fusion in this fungus were observed by light microscopy, and many genes involved in cell fusion including ham genes have been identified and characterised[5,6,7,8,9,10,11]. Cell fusion between genetically distinct strains leads to the formation of heterologous nuclei-containing cells called heterokaryons. Numerous A. oryzae strains are used, depending on different industrial purposes (e.g. manufacturing sake, soy sauce, and miso), there is poor knowledge about their cell fusion ability and heterokaryon incompatibility. Additional techniques are required for further investigating the cell fusion mechanism and genetic incompatibility in A. oryzae
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