Increase in number of nuclei and cell volume in Aspergillus species.

(A) The nuclear distribution in the tip cells was categorized into Classes I–III. The ratio of hyphae in each class was measured in Aspergillus species at 24, 48, and 72 hours of growth (n=50). Data for A. oryzae RIB40 and A. nidulans are reproduced from a previous study (33). (B) Colony morphology after 3 days of culture on the minimal medium. The nuclear distribution in the hyphae at the colony periphery stained with SYBR Green. Scale bars: hyphae, 20 μm; colonies, 1 cm. (C) 3D images of hyphae without increased nuclei (left) and with increased nuclei (right) in A. oryzae RIB40 from Movie 1. Each nucleus is indicated with different colors by Imaris soft. Scale bar: 5 μm. (D) Comparison of nuclear size in Class I hyphae (2.4 μm) and Class III hyphae (1.6 μm), indicated by white lines. (E) Box plots of nucleus diameters in hyphae without increased nuclei and those with increased nuclei (n=35, ** p < 0.01, t-test). (F) Box plots of hyphal width at the colony periphery grown for 72 hours (n=10–14, ** p< 0.01, * p< 0.05, t-test). Strains with increased nuclei are marked in red, and those without are marked in blue. (G) Correlation between nuclear number and cell volume of hyphae at 100 μm from the tips in A. oryzae RIB40, RIB128, RIB915, and A. flavus.

Thick hyphae with increased nuclei emerge by branching.

(A) Time-lapse image sequence of A. oryzae RIB40 expressing H2B-GFP showing the emergence of thick hyphae with increased nuclei by branching from Movie 2. Scale bar: 20 μm. Elapsed time is indicated in min. (B) Image sequence of successive nuclear division within the newly emerged branched hypha from Movie 3. Scale bar: 10 μm. Elapsed time is indicated in min. (C) Time course of nuclear number per hypha. Nuclear numbers were counted with the start time of branching set as 0, every hour for 5 to 10 h. Thick; hyphal width >7 μm, Thin; hyphal width <5 μm. (D) Branching from hyphae with increased nuclei generates both thick hyphae with increased nuclei (left) and thin hyphae without increased nuclei (right). Scale bars: 20 μm. (E) Image sequence of mycelial growth showing hyphae with increased nuclei (green) and without increased nuclei (white) from Movie 4. Scale bars: 200 μm. Elapsed time is indicated in h. (F) Line histogram of hyphal width at the colony periphery at 20, 40, and 60 h, calculated from Movie 4 (n=40). (G) Correlation between hyphal width and maximum elongation rate calculated from Movie 4 (n=45). The maximum elongation rate is determined from elongation rates measured every hour over 10 hours period. (H) TEM images of A. oryzae RIB40 hyphae. Hyphal diameter and cell wall thickness are indicated white and black text respectively. Scale bar: 1 μm. (I) Correlation plots of hyphal width and cell wall thickness based on TEM images. Thick hyphae (>7 μm) are marked in red, and thin hyphae (<7 μm) are marked in blue. (J) 3D surface images of A. oryzae RIB40 hyphal tips in the thick hypha (upper) and the thin hypha (lower) constructed using AFM. (K) Surface roughness of cell walls in thick (red) and thin (blue) hyphae along the arrows in J.

Correlation between number of nuclei and enzyme secretion.

(A) Secreted protein per biomass in strains with increased nuclei (red) and without increased nuclei (blue) after 4 days of culture in minimal medium with 1% maltose (mean ± S.E., n=3, ** p < 0.01, t-test). (B) Time course of α-amylase activity per biomass in A. oryzae RIB40 and RIB915 cultured in minimal medium with 1% maltose (mean ± S.E., n=3). (C) Measurement of α-amylase activity in a single hypha using a microfluidic device. A fluorescent substrate increases in fluorescence upon hydrolysis by α-amylase. ROIs for thick hypha are marked in red, and thin hypha in blue. Scale bar: 10 μm. (D) Temporal changes in fluorescence intensity measured in individual flow channels, as described in C. (E) Box plots of hyphal width in colonies grown in minimal medium with or without 1% yeast extract (n=10– 18, ** p < 0.01, * p < 0.05, t-test). Conditions with increased nuclei are marked in red, and those without in blue. (F) Secreted protein per biomass in minimal medium with or without 1% yeast extract (mean ± S.E., n=3, ** p < 0.01, t-test). (G) Ratio of class I-III hyphae in A. oryzae RIB915 colonies grown in minimal medium supplemented with 1% yeast extract or 0.1% individual amino acids (n=50). (H) Correlation between the ratio of Class III hyphae and secreted protein per biomass in the 0.1% amino acid-supplemented medium. (I) Images of hyphae and nuclear distribution in A. oryzae RIB40 grown on minimal medium with or without 0.5 ng/ml rapamycin. Scale bars: 10 μm. (J) Ratio of class I-III hyphae in A. oryzae RIB40 cultured on minimal medium containing 0, 0.5, 5, or 100 ng/ml rapamycin (n=50). (K) Box plots of hyphal width in the colonies cultured under the conditions in J (n=11-18, ** p < 0.01, t-test).

Transcriptome analyses in hyphae with increased nuclei.

(A) Heatmap of gene expression in A. oryzae RIB40, RIB915, and A. nidulans grown in the minimal medium with or without 1% yeast extract. The top 500 genes with the largest variation in expression are shown. (B) Venn diagram of genes upregulated more than four-fold in the medium with yeast extract. (C) GO process analysis of genes uniquely upregulated in A. oryzae RIB915 grown in the medium with yeast extract. (D) Image sequence of cutting and collecting the targeted hypha by using laser microdissection. Scale bar: 100 μm. (E) Heatmap of gene expression in thick (n=3) and thin (n=2) hyphae dissected from A. oryzae RIB40. The top 300 genes with the largest expression differences are shown. (F) GO process analysis of genes upregulated more than four-fold in thick hyphae compared to thin hyphae. (G) Venn diagram of 449 genes upregulated in RIB915 with yeast extract and 558 genes upregulated in thick hyphae of RIB40. (H) GO annotations of 13 genes from the 21 shared genes in G. Genes related to cell wall processes are shown in green, cell membrane in blue, Ca²⁺ transport in yellow, and rRNA processing in orange. (I) Images of hyphae of RIB40 and ΔmsyA strains after 3 min of low osmotic stress. Scale bar: 200 μm. (J) Ratio of hyphal tip lysis under the hypoosmotic shock (mean ± S.E., n=50, ** p < 0.01, t-test).

Comparison of industrial bred strains in A. oryzae, T. reesei and P. chrysogenum.

(A) Phylogenetic clade A-F of A. oryzae TK strains. The strains with increased nuclei are indicated in red. (B) Colonies and hyphae stained with SYBR Green of strains in clades C, F, and G grown for 3 days on the minimal medium. Scale bar: 20 μm. (C) Sequence differences in rseA gene among strains in clades C, F, and G. UTR regions are marked in pink, exons in purple, and nucleotide differences in white. (D) Colonies and hyphae stained with SYBR Green of RIB40 and the ΔrseA strain grown for 3 days on the minimal medium. Scale bar: 10 μm. (E) Colonies and hyphae stained with SYBR Green of RIB915 expressing rseA gene from RIB40 and TK-47 strains with increased nuclei, and TK-41 strain without increased nuclei. Scale bar: 20 μm. (F) Hyphal width of RIB915 and strains in E (n=14– 23). Strains with rseA from strains with increased nuclei are marked in red, and those without are marked in blue. (G) Hyphal images stained with SYBR Green of T. reesei (QM9414) and P. chrysogenum (IFO4688) grown for 3 days on the minimal medium with or without 1% yeast extract. Scale bar: 20 μm. (H) Ratio of class I-III hyphae in the T. reesei and P. chrysogenum control and bred strains cultured on the minimal medium with or without 1% yeast extract for 3 days (n=50). (I) CMCase activity per biomass of T. reesei industrial and control strains under the conditions in G (mean ± S.E., n=3, ** p < 0.01, t-test). (J) Protease activity per biomass of P. chrysogenum industrial and control strains under the conditions in G (mean ± S.E., n=3, ** p < 0.01, t-test).