125NISR RESEARCH GRANT
2003 Young Investigator Research Grant

Functional Characterization of AoHex1 Involved in Woronin
Body Formation in Aspergillus oryzae
Jun-ichi MARUYAMA
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo
Woronin bodies are unique organelles restricted to filamentous ascomycetes and deuteromycetes. They are observed in very close association with the septa. When hyphae are lysed, Woronin bodies appear to plug the septal
pores within a few seconds and prevent excessive loss ofcytoplasm. However, its cellular origins and biochemicalcomposition had remained unknown since the first report ofits discovery by Woronin (1864).
Aspergillus oryzae is one of the favored hosts for commercial enzyme production owing to its safety and ability
to secrete a large amount of proteins into media. Solid-state
culture is a unique culturing system known to provide
higher-level of enzyme production when compared to thesubmerged culture in A. oryzae, in which cellular proteins
are known to disperse into media as a result of hyphal lysis.
While gene transcription and post-translational protein
modification occurring specifically in solid-state culture
were recently investigated, the mechanisms of lysis in solidstate culture are poorly understood at cellular and molecular levels. This study describes the functional relevance of
A. oryzae AoHex1 required for Woronin body formation
during lysis in solid-state culture.
1. Excessive loss of cytoplasm in the Aohex1 disruptant under hypotonic shock
Since after cultivation of mycelia on solid-state culture
the enzymes are extracted by adding water, I designed anobservation system with inverted microscopy for flooding
the colony grown on agar media with water, mimicking enzyme extraction in solid-state culture. Whereas few burst
tips were found before flooding with water, most of the hyphal tips burst and their cytoplasm leaked out of themycelia within several minutes after flooding. Addition of
1 M NaCl solution to the culture did not result in such alysis, indicating that the hyphal tip bursting occurred due tohypotonic shock.
In order to observe the difference between the control
strain and the Aohex1 disruptant with regard to their cytoplasmic status and extent of leakage during hypotonicshock, the second compartments adjacent to lysed apicalcompartments were checked. In the control strain most of
the second compartments adjacent to lysed tips retained thecytoplasm. On the contrary, cytoplasm leaked out of thesecond compartment in most of the lysed hyphae in theAohex1 disruptant. Therefore, it may be noted that AoHex1
was indeed required for preventing extensive loss of cyto-plasm in the second compartment of bursting hyphae underhypotonic shock.
2. Three-dimensional image analysis for visualizing
the plugging of the septal pore by Woronin body using
DsRed2 and EGFP fluorescent proteins
To clearly visualize the septal pore and septal plugging
by Woronin body, dual fluorescence labeling strategy wasadopted. In order to visualize septa, a secretory protein,
RNase T1, from A. oryzae was fused with EGFP and expressed. Previous studies from our laboratory have also detected green fluorescence of RNase T1-EGFP fusion protein at septa. In order to mark septal pores, I utilized confocal microscopy and observed green fluorescence ofRNaseT1-EGFP fusion protein. On the middle plane ofsepta, septal pores were marked as a discontinuity at thecenter of linear green fluorescence of RNase T1-EGFP. In
order to more spatially demonstrate septal pores, three-dimensional images were reconstructed. The septal pores
were successfully marked as dark regions surrounded bygreen fluorescence of RNase T1-EGFP fusion protein, onthe septum. For visualization of Woronin bodies along withsepta, DsRed2-AoHex1 fusion protein was also expressed.
The A. oryzae strain expressing both the two fusion constructs was observed by fluorescence microscopy. Red fluorescence of DsRed2-AoHex1 was detected as small circularspots and some of them were found in the vicinity of septa
labeled by green fluorescence of RNase T1-EGFP fusion
protein. The red fluorescent spots were also observed at thecenter of the green fluorescent septa adjacent to lysed compartments. In order to confirm the localization of Woroninbodies at the septal pores during hyphal lysis, the strain expressing both RNase T1-EGFP and DsRed2-AoHex1 fusion proteins were observed by confocal microscopy. Unlysed compartments showed straight septa and were accompanied with a number of red fluorescent spots of Woroninbodies in their vicinity but not at the septal pores. On thecontrary the septa next to lysed compartments were curved
probably due to cytoplasmic pressure during hyphal lysis
and contained red fluorescent spots of Woronin bodies attheir pores. Three-dimensional reconstruction demonstrated
that the red fluorescence of DsRed2-AoHex1 located at theseptal pore next to the lysed compartment and sometimes
protruded from the septa, strongly supporting the locationof Woronin bodies at septal pores. Hence, dual fluorescentlabeling revealed that the Woronin bodies formed by

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AoHex1 protein plugged the septal pore adjacent to lysedapical compartments upon hypotonic shock.
Conclusion
I established a novel observation system mimicking enzyme extraction in solid-state culture and demonstrated that
A. oryzae Aohex1 gene involved in Woronin body formation prevented excessive loss of cytoplasm during hyphallysis upon hypotonic shock by plugging the septal pore.

This is the first study to investigate environmental responses such as lysis during solid-state culture at a cellularlevel, which could help understand the physiology of A.
oryzae and be beneficial for industrial uses.
References
Maruyama, J., Juvvadi, P. R., Ishi, K., and Kitamoto, K., Aspergillus
oryzae AoHex1 is required for Woronin body formation and septal
plugging during hyphal lysis induced by hypotonic shock. Microbiology(Submitted)