Disordered Cell Integrity Signaling Caused by Disruption of thekexBGene inAspergillus oryzae

Abstract

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 (⌬kexB), 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 ⌬kexB 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 ⌬kexB cells than in wild-type cells. The ⌬kexB cells also contained higher levels of transcripts for cell wallrelated genes encoding ␤-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 ⌬kexB 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 ⌬kexB cells, concomitantly suppressing the morphological defects. These results suggest that the upregulation of transcription levels of mpkA and cell wall biogenesis genes in the ⌬kexB strain is autoregulated 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.

Genomics ofAspergillus oryzae

“…This suggests that the signal transduction cascade of A. oryzae is the most complex of the four filamentous fungi. In the genus Aspergillus, only the HOG 95,[109][110][111] and cell integrity pathways 112,113) are genetically characterized, and the others remain unclear.…”

“…Fujioka et al isolated the A. nidulans rlmA gene, which encodes a transcription factor with high similarity to yeast Rlm1p, using Rlm1p-specific signatures such as MADS (MCM1, Agamous, Deficients, SRF) box and an Mpk1p-docking region. 113) In A. oryzae, the cell integrity pathway has been analyzed using a morphologic mutant in which the kexB gene, encoding a subtilisin-like processing enzyme similar to S. cerevisiae Kex2p, was disrupted.…”

MpkA-Dependent and -Independent Cell Wall Integrity Signaling in Aspergillus nidulans

“…Since the morphologies of filamentous fungi differ significantly from those of yeasts such as Saccharomyces cerevisiae, cell wall architecture and its biogenesis are thought to reflect differences in types of cell wallrelated genes and their transcriptional regulation through cell signaling in response to external stimuli or stresses as well as to the cell cycle in the two groups of eukaryotes. However, few studies of cell wall biogenesis in filamentous fungi and its regulation via signaling, in particular by transcription factors that control the transcription of cell wall-related genes as the targets of the putative signaling pathways, have been reported previously (5,8,41,64).…”

“…Thus, the functionality of transcription factors must be examined in vivo. Although most genes orthologous to the yeast CWIS genes are conserved in the genomes of the three Aspergillus species (14,39,43) and the signaling pathway seems to maintain cell wall biogenesis in these fungi as described above (5,41), the target genes for transcription factors downstream of the CWIS via MpkA remain unclear except for those identified in a few studies, such as a study of the constitutive autoregulation of mpkA expression in an A. oryzae kexB disruptant (41) and a study of the RlmA-dependent expression of agsA, which encodes ␣-1,3-glucan synthase, in A. niger (8).…”

“…In filamentous fungi, including Aspergillus nidulans, A. oryzae, and Magnaporthe grisea, several genes that encode MAPKs of CWIS have been isolated (5,41,64). In A. nidulans, mpkA, which is a counterpart of the yeast MPK1 (SLT2), has been cloned and characterized previously (5).…”