AoSO protein accumulates at the septal pore in response to various stresses in the filamentous fungus Aspergillus oryzae.

Filamentous ascomycetes form hyphal networks that are compartmentalized by septa which have a perforated pore allowing the passage of cytoplasm and organelles between adjacent hyphal compartments. Thus, the septal pore may play an important role in the organized growth of multicellular organisms. Upon hyphal injury, the septal pore is plugged by a wound-healing organelle, known as the Woronin body, to prevent excessive cytoplasmic leakage. However, the movement of proteins towards the septal pore in response to stress has not been extensively studied in filamentous fungi. In this study, we identified an Aspergillus oryzae protein, AoSO, which is homologous to the Neurospora crassa SO protein that was reported to accumulate at the septal pore in aging hyphae. The DeltaAoso strain showed excessive cytoplasmic leakage upon hyphal injury similar to the Woronin body-deficient strain DeltaAohex1. Cellular localization studies using EGFP showed that AoSO accumulated at the septal pore adjacent to the injured compartment, while it was dispersed throughout the cytoplasm under normal growth conditions. These results indicate that AoSO plays a role in preventing excessive cytoplasmic leakage upon hyphal injury by accumulating at the septal pore. Furthermore, AoSO accumulated at the septal pore in response to various stresses, including low and high temperature, extreme acidic and alkaline pH, and nitrogen and carbon depletion. Physical stress induced by pulse laser treatment on a hyphal region at a distance from the septum caused accumulation of the AoSO protein at the septal pore within only a few minutes. This study presents a novel behavior in which a filamentous fungal protein relocalizes to the septal pore in response to various stresses.

Disruption of the Aopex11-1 gene involved in peroxisome proliferation leads to impaired Woronin body formation in Aspergillus oryzae.

The Woronin body, a unique organelle found in the Pezizomycotina, plugs the septal pore upon hyphal damage to prevent excessive cytoplasmic bleeding. Although it was previously shown that the Woronin body buds out from the peroxisome, the relationship between peroxisomal proliferation/division and Woronin body differentiation has not been extensively investigated. In this report, we examined whether Pex11 required for peroxisomal proliferation participates in Woronin body formation in Aspergillus oryzae. A. oryzae contained two orthologous PEX11 genes that were designated Aopex11-1 and Aopex11-2. Deletion of Aopex11 genes revealed that only the DeltaAopex11-1 strain showed reduced growth and enlarged peroxisomes in the presence of oleic acid as a sole carbon source, indicating a defect in peroxisomal function and proliferation. Disruption of Aopex11-1 gene impaired the Woronin body function, leading to excessive loss of the cytosol upon hyphal injury. Dual localization analysis of the peroxisome and Woronin body protein AoHex1 demonstrated that Woronin bodies fail to fully differentiate from peroxisomes in the DeltaAopex11-1 strain. Furthermore, distribution of AoHex1 was found to be peripheral in the enlarged peroxisome or junctional in dumbbell-shaped peroxisomes. Electron microscopy of the DeltaAopex11-1 strain revealed the presence of Woronin bodies that remained associated with organelles resembling peroxisomes, which was supported from the sucrose gradient centrifugation confirming that the Woronin body protein AoHex1 overlapped with the density-shifted peroxisome in the DeltaAopex11-1 strain. In conclusion, the present study describes the role of Pex11 in Woronin body differentiation for the first time.