Phosphatidylcholine levels regulate hyphal elongation and differentiation in the filamentous fungus Aspergillus oryzae

Filamentous fungi are eukaryotic microorganisms that differentiate into diverse cellular forms. Recent research demonstrated that phospholipid homeostasis is crucial for the morphogenesis of filamentous fungi. However, phospholipids involved in the morphological regulation are yet to be systematically analyzed. In this study, we artificially controlled the amount of phosphatidylcholine (PC), a primary membrane lipid in many eukaryotes, in a filamentous fungus Aspergillus oryzae, by deleting the genes involved in PC synthesis or by repressing their expression. Under the condition where only a small amount of PC was synthesized, A. oryzae hardly formed aerial hyphae, the basic structures for asexual development. In contrast, hyphae were formed on the surface or in the interior of agar media (we collectively called substrate hyphae) under the same conditions. Furthermore, we demonstrated that supplying sufficient choline to the media led to the formation of aerial hyphae from the substrate hyphae. We suggested that acyl chains in PC were shorter in the substrate hyphae than in the aerial hyphae by utilizing the strain in which intracellular PC levels were controlled. Our findings suggested that the PC levels regulate hyphal elongation and differentiation processes in A. oryzae and that phospholipid composition varied depending on the hyphal types.

The ∆pemA colonies at 5, 20, 100, and 1,000 µM Cho were also shown in Fig. 2C.The conidia of the wild-type, tA-pemA, or tA-pemB were inoculated on a CD medium containing thiamine at different concentrations and incubated at 30°C for 96 h.Top and side views of the colonies that were grown on each concentration of thiamine.The wild-type and tA-pemA colonies at 0.01, 0.1, and 1 µM thiamine were also shown in Fig. 5E.

Fig
Fig. S2.5'-RACE analyses of pemA and pemB, and domain organizations of PemA and PemB.(A) Transcription start sites are indicated by right-pointing arrows.The number at the right of the arrow indicates the number of reads to the total number of reads.The "1" indicates the first position of the estimated start codon.Introns are shown in italic lowercase letters.(B) The deduced domain organizations of S. cerevisiae Pem1, A. oryzae PemA, S. cerevisiae Pem2, and A. oryzae PemB by InterPro (https://www.ebi.ac.uk/interpro/).

Fig. S3 .
Fig. S3.Changes in DCW of each strain cultured in the CD liquid medium.The conidia of the wild-type, ∆pemA, or ∆pemB strain were inoculated into the CD liquid medium containing 1 mM Etn (−) or that containing 1 mM Etn and 1 mM Cho (+ Cho) and incubated at 30°C.The bars, the dots, and the error bars indicate the mean (n = 3), individual data, and the standard error, respectively.Statistically significant differences among DCWs under the same culture conditions and the same strains are indicated by different letters (P < 0.05; Tukey's HSD test).Statistically significant differences among DCWs under the same culture conditions and at the same incubation time are indicated by asterisks (***P < 0.001; Dunnett's test).

Fig. S4 .
Fig. S4.Changes in the transcript levels during the formation of aerial hyphae and conidiophores.Total RNA was extracted from the colonies, and the mRNA levels of genes related to PC synthesis were quantified.The fold changes were calculated compared to mycelia incubated for 24 h.The bars, the dots, and the error bars indicate the mean (n = 3), individual data, and the standard error, respectively.Statistically significant differences among fold changes of the same genes are indicated by different letters (P < 0.05; Tukey's HSD test).

Fig. S5 .
Fig. S5.Growth of the ∆pemA or ∆pemB strain at different concentrations of Cho.(A, B) The conidia of the ∆pemA (A) and ∆pemB (B) were inoculated on a CD medium containing 1 mM Etn and Cho at different concentrations.Then, they were incubated at 30°C for 96 h.Top

Fig. S6 .
Fig. S6.Hyphal morphology of the deletion mutant of pemA or pemB on the media containing different concentrations of Cho.(A) The conidia of the wild-type, ∆pemA, or ∆pemB strain were inoculated on the CD medium containing 1 mM Etn and 5, 10, 20, or 50 µM Cho and incubated at 30°C for 96 h.The hyphae at the edge of the colonies were observed.(B) The aerial hyphae and substrate hyphae were observed.

Fig. S7 .
Fig. S7.Validity of phospholipid analysis in medium-transferring experiments (related to Fig. 4C).(A) The conidia of the wild-type, ∆pemA, or ∆pemB were inoculated on a CD medium containing 1 mM Etn and 5 µM Cho (LC) or 1,000 µM Cho (HC) covered with a cellophane sheet and incubated at 30°C for 72 h.Phospholipids were extracted from mycelia harvested from the margin of the colony with a width of 5 mm and subjected to lipidome analysis.The obtained data (colony margin) and the data of 5 µM Cho and 1,000 µM Cho derived from Fig. 3A (entire colony) were plotted; dots indicate the mean and error bars represent S.E.The values shown indicate the correlation coefficient in each strain.(B) Mycelia of the wild-type strain were harvested as described in (A) after the transfer in Fig. 4A and B. Phospholipids were extracted from these samples and subjected to lipidome analysis.The mean values are depicted as bars (n = 4), while dots indicate individual data points.The error bars denote the standard error.Statistically significant differences are indicated by asterisks (**P < 0.01, *P < 0.05; Tukey-Kramer post-hoc test).

Fig. S9 .
Fig. S9.Comparison of amino acid sequences and putative structures of A. oryzae PemA and A. niger Cho2.(A) Amino acid sequence alignments of A. oryzae PemA and A. niger Cho2.Regions 1 and 2 indicate the amino acid sequences do not present in Cho2.Region 3 indicates the amino acid sequence do not present in PemA.(B) The predicted structures of A. oryzae PemA (cyan) and A. niger Cho2 (brown) by AlphaFold2.Regions 1-3 correspond to the regions shown in (A).(C) The deduced domain organizations of A. niger Cho2 by InterPro.

Table S1 .
Genes involved in the Kennedy pathway in S. cerevisiae and A. oryzae