34

Noda Institute for Scientific Research GRANT
2012 Research Grant

Molecular investigation of Woronin body function and its
application in Aspergillus oryzae
Jun-ichi Maruyama
Department of Biotechnology, The University of Tokyo

Research aims

Methods

Species of Pezizomycotina (filamentous ascomycetes)
grow via elongation of the hyphal tip to form straight primary hyphae with branches. The hyphae are divided into
distinct cells by the formation of septa; thus, filamentous
fungi are characterized by multicellularity. Cytoplasmic
continuity between adjacent cells through the septal pore isassociated with the risk of cytoplasmic loss in adjacentcells upon hyphal lysis. This risk was clearly demonstrated
by the exposure of the industrial filamentous fungus Aspergillus oryzae grown on agar medium to hypotonic shock,which caused most of the hyphal tips to burst and lose theircytoplasmic constituents (Fig. 1A).1) However, ~80% of thecells immediately adjacent to the lysed tip cells retained
their cytoplasmic constituents,1) allowing them to initiate
regrowth by creating a new hyphal tip.2)
The Woronin body is a unique organelle present in Pezizomycotina species that plugs the septal pore upon hyphallysis and prevents excessive cytoplasmic loss from the cell
adjacent to the lysed cell (Fig. 1B). Previously, we characterized AoHex1 as the major protein of Woronin body in A.
oryzae, and investigated Woronin body differentiation fromthe peroxisome.1,3)
However, how Woronin bodies localize to the vicinity ofthe septum is not known. Recently, the Leashin (LAH) protein was found to tether Woronin bodies to the cell cortex in
Neurospora crassa,4) which surprisingly does not recruit
the organelle to the vicinity of the septum. In this study, weattempted to elucidate the mechanism for tethering Woronin bodies at the septum in A. oryzae.

Gene disruption in A. oryzae
Gene disruption in A. oryzae strains was done by replacement of coding sequences with the adeA marker gene,
and was confirmed by Southern blot analysis.
Transmission electron microscopic analysis
Mycelia grown on DPY agar medium at 30°C for 4 days
were fixed with glutaraldehyde and osmium tetroxide. Samples were observed by transmission electron microscopy(Model JEM-1010; JEOL, Japan).
Hypotonic shock experiment
To induce hyphal tip bursting, 1 ml of water was added
to A. oryzae strains cultured on a thin layer of DPY agar
medium at 30°C for 24 h and observed by differential interference contrast microscopy as described in Maruyama etal.1)
Results
Functional analysis of AoLAH protein
We identified one gene (AO090011000895) encoding aprotein showing similarity to N. crassa LAH in the A. oryzae genome database. According to homology comparison,
the ORF region of the gene was corrected. We subsequently
named this gene Aolah (A. oryzae lah). The Aolah gene waspredicted to encode a single polypeptide of 5,727 aminoacids, which is homologous to two N. crassa LAH proteins(LAH-1 and LAH-2) expressed from the unidirectionallyaligned locus.
In order to examine the function of AoLAH, we disrupted the Aolah gene. Transmission electron microscopic

Fig. 1. Hypotonic shock experiment to induce hyphal tip bursting (A) and septal pore plugging by the Woronin body (B).

		Fig. 2.

Structure and function of the AoLAH protein.

analysis showed that, in the wild-type strain, Woronin bodies appeared as electron-dense spherical structures close tothe septum. However, in the Aolah disruptant, no tethered
Woronin bodies were observed in the vicinity of the septum. There were un-tethered Woronin bodies along the hyphae. It was therefore concluded that AoLAH is required
for Woronin body tethering at the septum in A. oryzae.
To investigate the role of AoLAH in Woronin body function, we performed a hypotonic shock experiment to induce
hyphal tip bursting (Fig. 1A). In contrast to the wild-typestrain that exhibited an approximately 80% ability to retain
cytoplasmic constituents in the second cell, the Aolah disruptant showed a reduced ability with approximately 60%.
This indicates the functional involvement of AoLAH in theseptal plugging ability of the Woronin body.

35

fluorescence observed at the septal pore, which is consistent with the transmission electron microscopic analysis.
These data revealed a role for the AoLAH N-terminal
region in the association with Woronin bodies.
The C-terminal conserved region of AoLAH(AoLAH[4710–5727]) expressed as an EGFP fusion protein was detected close to the septal pore in both the wildtype strain and the Aolah disruptant, indicating that theAoLAH C-terminal region itself is associated with theseptum.
When a fusion of the AoLAH N- and C-terminal regions
lacking the non-conserved middle region was expressed inthe Aolah disruptant, Woronin bodies were located in thevicinity of the septum, although they were closer to the septum. This suggests that the non-conserved middle region ofAoLAH has a role in regulating the distance of Woroninbodies from the septum.
Conclusion
Recently, we reported that the Woronin body reversibly
closes the septal pore during normal growth of A. oryzae, afunction that impedes cytoplasmic continuity between adjacent cells during normal growth and helps maintain hyphal
heterogeneity in mycelia.5) Although wild-type cells show aheterogeneous distribution of gene expression activity, theabsence of Woronin bodies results in uniform activity indifferent cells. Based on our results, investigation of therole of AoLAH in plugging the septal pore during normal
growth is warranted.
References1)

Deletion analysis of AoLAH protein
AoLAH was predicted to be a very large protein withover 5,000 amino acids. To analyze the functional role ofindividual regions, we analyzed the localization of N-terminal and C-terminal regions by expressing each individuallyas EGFP fusion proteins in A. oryzae.
When the N-terminal conserved region of AoLAH(AoLAH[1–2039]) was expressed as an EGFP fusion protein, it was detected on both sides of the septum at a defined distance from the septal pore, which is the localization pattern typical for Woronin bodies in the vicinity of theseptum. Upon hyphal lysis by hypotonic shock, AoLAH[1–2039]-EGFP moved to the septal pore similar to Woroninbodies. In the Aolah-disrupted background, there was noMaruyama, J., Juvvadi, P. R., Ishi, K., Kitamoto K. (2005) Three-dimensional image analysis of plugging at the septal pore by Woroninbody during hypotonic shock inducing hyphal tip bursting in Aspergillus oryzae. Biochem. Biophys. Res. Commun. 331: 1081–1088.
2) Maruyama, J., Kitamoto K. (2013) Expanding functional repertoiresof fungal peroxisomes: contribution to growth and survival
processes. Front. Physiol. 4: 177.
3) Escano, C. S., et al. (2009) Disruption of the Aopex11–1 gene involved in peroxisome proliferation leads to impaired Woronin bodyformation in Aspergillus oryzae. Eukaryot. Cell 8: 296–305.
4) Ng, S. K., Liu, F., Lai, J., Low, W., Jedd, G. (2009) A tether forWoronin body inheritance is associated with evolutionary variationin organelle positioning. PLoS Genet. 5: e1000521.
5) Bleichrodt, R. J., van Veluw, G. J., Recter, B., Maruyama, J., Kitamoto, K., Wösten, H. A. (2012) Hyphal heterogeneity in Aspergillus
oryzae is the result of dynamic closure of septa by Woronin bodies.
Mol. Microbiol. 86: 1334–1344.