Disordered cell integrity signaling caused by disruption of the kexB gene in Aspergillus oryzae

We isolated the kexB gene, which encodes a subtilisin-like processing enzyme, from a filamentous fungus, Aspergillus oryzae. To examine the physiological role of kexB in A. oryzae, we constructed a kexB disruptant (ΔkexB), which formed shrunken colonies with poor generation of conidia on Czapek-Dox (CD) agar plates and hyperbranched mycelia in CD liquid medium. The phenotypes of the ΔkexB strain were restored under high osmolarity in both solid and liquid culture conditions. We found that transcription of the mpkA gene, which encodes a putative mitogen-activated protein kinase involved in cell integrity signaling, was significantly higher in ΔkexB cells than in wild-type cells. The ΔkexB cells also contained higher levels of transcripts for cell wall-related genes encoding β-1,3- glucanosyltransferase and chitin synthases, which is presumably attributable to cell integrity signaling through the increased gene expression of mpkA. As expected, constitutively increased levels of phosphorylated MpkA were observed in ΔkexB cells on the CD plate culture. High osmotic stress greatly downregulated the increased levels of both transcripts of mpkA and the phosphorylated form of MpkA in ΔkexB cells, concomitantly suppressing the morphological defects. These results suggest that the upregulation of transcription levels of mpkA and cell wall biogenesis genes in the ΔkexB strain is autoregalated by phosphorylated MpkA as the active form through cell integrity signaling. We think that KexB is required for precise proteolytic processing of sensor proteins in the cell integrity pathway or of cell wall-related enzymes under transcriptional control by the pathway and that the KexB defect thus induces disordered cell integrity signaling.