fig. s2. growth assay of wild type and - eukaryotic...

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Fig.S1. (A) Complementation of oshC-deletion and oshD-deletion. Colonies of wild type, oshC-deletion

transformed with oshC-coding plasmid, and oshC-deletion strains (upper). Colonies of wild type,

oshD-deletion transformed with oshD-coding plasmid, and oshD-deletion strains (lower). The strains

were incubated for 3 days at 37 °C. (B) Colony diameters of the wild type and strains expressing GFP-

Osh grown on the minimal media with glucose or glycerol plates for 3 days at 37 °C. Error bars

represent the standard deviation (n=3). (C) Colony diameters of the wild type and osh-deletion strains

grown on the minimal media with glucose or glycerol plates for 3 days at 37 °C. Error bars represent

the standard deviation (n=3). (D) Number of spore production per square centimeter of wild type, GFP-

OshE and oshE-deletion strains. Inoculation of spores were done in 5 ml topagar with spore density of

2.5 x 105 spores and incubated on the minimal media with glucose or glycerol for 3 days at 37°C.

Error bars represent the standard deviation (n=3).

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Fig. S2. Growth assay of wild type and osh-deletion strains on the minimal media glucose

plates at different temperatures. (A) The 2.5 x 104 spores from strains were inoculated on

plates for 3 days at 28 °C, 37 °C and 42 °C. (B) Colony diameter of wild type and osh-

deletion strains were indicated by the bar graph. Error bars represent the standard deviation

(n=3). Black, dark grey and light grey asterisks represent statistically significant (p < 0.01).

The significant colonies from 28 °C marked with black asterisks, the 37 °C significant

colonies marked with light grey asterisks and the dark grey asterisks marked the 42 °C

significant colonies.

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Fig. S3. Calcofluor white (CFW) growth assay with different concentrations. (A) Solutions

with 2.5 x 104 spores from wild type and oshA-E deletion strains were inoculated on plates

with different CFW concentrations ranged from 0-100 µM for 3 days at 37 °C. (B) Colony

diameter of the strains were denounced by the bar graph. Error bars represent the standard

deviation (n=3).

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Fig.S4. Relative expression levels of the osh genes in A. nidulans and A. fumigatus. (A)

Quantitative Real-time PCR was made from total RNA of oshE deletion strain, which was

isolated after different time points to show gene expression during development. Error bars

represent the standard deviation (n=3). Asterisk represent statistically significant (p < 0.01).

(B) The localization of GFP-OshE was analysed during conidiophore development. GFP-

OshE localized to several dots through the stalk of conidiophore and inside of metula,

whereas no clear localization pattern was observed at phialide. (C) Quantification of oshA-E

gene expression during asexual stage. Af293 (WT) strain was cultured in liquid YGMM for

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18h, and the mycelia were transferred onto YGMM plate (set as 0h of asexual stage) and

were incubated at 37°C. RNA samples were extracted from the mycelia harvested at 0h, 6h,

12h, and 24h. The relative expression rates were quantified by real-time RT PCR analysis.

Each value represents the expression rate relative to that of WT at 0h. (D) Expression levels

of Afosh genes under hypoxic condition. From a dataset of RNA-sequencing analysis

(GSE30579) deposited by Dr. Cramer’s group, RPKM values of Afosh genes at 0, 12, 24,

and 36h after transfer to hypoxic condition were extracted and plotted on y-axis.

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Table S1. A. nidulans strains used in this study

Strain Genotype Reference or source

TN02A3 pyrG89; argB2; ΔnkuA::argB; pyroA4 (1)

SNT100* paba1; teaR(p)::gfp::teaR::pyr-4; teaA(p)::mrfp1::teaA::pyr-4 (2)

SNB2 pyrG89; argB2; ΔnkuA::argB; pyroA4;

[alcA(p)::gfp::oshA::pyr-4] This study


[alcA(p)::gfp::oshB::pyr-4] This study


[alcA(p)::gfp::oshC::pyr-4] This study


[alcA(p)::gfp::oshD::pyr-4] This study


[alcA(p)::gfp::oshE::pyr-4] This study


[alcA(p)::oshE::gfp::pyr-4] This study


[ΔoshB:: pyrG] This study


[ΔoshC::pyroA] This study


[ΔoshD::pyroA] This study


[ΔoshE::pyrG] This study

SHH2 pyrG89; argB2; ΔnkuA::argB; pyroA4;

[sec63::mcherry::pyrG] This study

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SNB12 (SNB9 crossed to SNT100) ΔoshC::pyroA;

teaR(p)::gfp::teaR::pyr-4; teaA(p)::mrfp1::teaA::pyr-4 This study

SNB13 (SNB10 crossed to SNT100) ΔoshD::pyroA;


SNB14 (SNB11 crossed to SNT100) ΔoshE::pyrG;


SNB15 (SNB3 crossed to SHH2) alcA(p)::gfp::oshB:pyr-4;

sec63::mcherry::pyrG This study

SNB16 (SNB6 crossed to SHH2) alcA(p)::gfp::oshE::pyr-4;

sec63:mcherry:pyrG This study


[oshB(p)::gfp::oshB::pyr-4] This study


[oshE(p)::gfp::oshE::pyr-4] This study


[ΔoshA::pyroA] This study


[oshA(p)::gfp::oshA::pyr-4] This study


[oshC(p)-oshC-oshC UTR::pyr-4] This study


[oshD(p)-oshD-oshD UTR::pyr-4] This study

SNB23 SNB2 crossed to SSH35 (alcA(p)::gfp::oshA, pyr-4; alcA(p)::mrfp1-tglB, pyroA) argB2; ΔnkuA::argB

This study

* The presence/absence of nkuA is not confirmed.

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Table S2. Plasmids used in this study

Plasmid Description Reference or

source

pCMB17apx alcA(p)-GFP, for N-terminal tagging of GFP to proteins of

interest; contains Neurospora crassa pyr-4 (3)

pJET1.2/blunt Cloning vector Fermentas

pRM83 myoV::mCherry::pyrG, for C-terminal tagging of mCherry-

pyrG to proteins of interest

Mank et al.,

unpublished

pNB1 alcA(p)-gfp-oshA::pyr-4 This study

pNB2 alcA(p)-gfp-oshB::pyr-4 This study

pNB3 alcA(p)-gfp-oshC::pyr-4 This study

pNB5 alcA(p)-gfp-oshD::pyr-4 This study

pNB6 alcA(p)-gfp-oshE::pyr-4 This study

pNB7 alcA(p)-oshE-gfp::pyr-4 This study

pNB8 oshB(p)-gfp-oshB::pyr-4 This study

pNB9 oshE(p)-gfp-oshE::pyr-4 This study

pNB10 oshA(p)-gfp-oshA::pyr-4 This study

pNB11 oshC(p)-oshC-oshC UTR::pyr4 This study

pNB12 oshD(p)-oshD-oshD UTR::pyr4 This study

pHH2 pJet::Sec63::mcherry::pyrG This study

pHH3 pJet::ΔoshA::pyroA This study

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Table S3. Primers used in this study

Name sequence

OshA-FW 5'-GGCGCGCCcATGTCGGAGAAGGTTGAAGG-3'

OshA-Rev 5'-GAAGGGAAAGCTGCCACAGTTAATTAA-3'

OshB-FW 5'-ACAGGCGCGCCCATGGCAGCCATGGAAGAGC-3'

OshB-Rev 5'-CAGAGCAAACCAATGGCGTATTAATTAA-3'

OshC-FW 5'-GGCGCGCCtATGTCTTCCAAGGATACTGC-3'

OshC-Rev 5'-GCAGGAGAAATATGTCATCTTAATTAA-3'

OshD-FW 5'-GGCGCGCCtATGTCCGCTGACTGGAGCT-3'

OshD-Rev 5'-TCCTACGATTCCTGAACCGTTTAATTAA-3'

OshE-FW 5'-GGCGCGCCtATGTCTTCCACATTAACGCC-3'

OshE-Rev 5'-AATCTTCATCAACATTAGCAAGTTAATTAA-3'

GFP-FW 5'-GGCGCGCCTGGCGCACTGCTTATGAGTAAAGGAGAAGAACTTT-3'

GFP-Rev 5'-GGCATGGATGAACTATACAAATGATTAATTAA-3'

OshA(p)-FW 5'-GCTGGTGTGGAGAGAGTTATAC-3'

OshA(p)-Rev 5'-CACGAGGTACCATAGGTGAG-3'

OshB(p)-FW 5'-GAATTCGCGGGAAACGACGCTGAGGG-3'

OshB(p)-Rev 5'-CGCTTAGCGACAAATTGACGGCCGGTACC-3'

OshE(p)-FW 5'-GAATTCCACCCGCACTGCGAGGAGAC-3'

OshE(p)-Rev 5'-CTCGGAAGCACCAAAATAATTATGTCTTCCGGTACC-3'

Sec63-LB-Fw 5'-CTGATCCAAGCTGTTGCGCCG-3'

Sec63-LB-Rev 5'-CCAATACCGAAACCGATGTTGACGACGGAGCTGGTGCAGGCGCTG-3'

Sec63-RB-FW 5'-ATCAGTGCCTCCTCTCAGACAGGGCCTGTCCAGAGTTTTCATTGG-3'

Sec63-RB-Rev 5'-GGATTCGGTTGCGAGAGCTGG-3'

Sec63-nLB-FW2 5'-CACCTCTGTTACAACTCCCTCAC-3'

Sec63-nRB-Rev2 5'-GCAACAGTAGAGATAGGGCTGCG-3'

GFP-GA-Linker FW 5'-GGAGCTGGTGCAGGCGCTG-3'

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pyrG-cas-R 5'-CTGTCTGAGAGGAGGCACTGAT-3'

oshA-FW-LB 5‘-CTGGTGCTGGTGTGGAGAGAG-3‘

oshA-Rev-LB 5‘-GTTTCATCCTACCGAGGTTATGCTCACtcaggcctagatggccacca-3‘

oshA-FW-RB 5‘-ggcctgagtggcctGACTACGTCTGTGCGAGCTGC-3‘

oshA-Rev-RB 5‘-GGAGTCGCCGTACACATAGTGAG-3‘

No.39 (ΔoshB-Fw) 5‘-GTAACGCCAGGGTTTTCCCAGTCACGACGCTATACTAAGGCTAACCCGC-3‘

No.39 (ΔoshB-Rev) 5‘-GCGGATAACAATTTCACACAGGAAACAGCAAGTCTACCACACTGGACCT-3‘

No.40 (ΔoshE-Fw) 5‘-GTAACGCCAGGGTTTTCCCAGTCACGACGCTACGGGGTGCTGTTTATAG-3‘

No. 40 (ΔoshE-Rev) 5‘-GCGGATAACAATTTCACACAGGAAACAGCGTTAGCGGAGAAACTAGCTG-3‘

oshC-LB-del-Fw 5'-TCAGTGAGCTAATGCCTCC-3'

oshC-LB-del-Rev 5‘-ggtggccatctaggccGTTTATGGTTGCTCACCCC-3‘

oshC-RB-del-Fw 5‘-ataggcctgagtggccAGCACAGGATATCAGTGGG-3‘

oshC-RB-del-Rev 5‘-TTTGAATAAAAGTTCATGGCGCCC-3‘

oshD-LB-del-Fw 5‘-ACCCGTTCGAGTACGAAGG-3‘

oshD-LB-del-Rev 5’-ggtggccatctaggccAGTGGGAATGGGAAGACCG-3’

oshD-RB-del-Fw 5‘-ataggcctgagtggccATGATTTTCATTCATCACTATGG-3‘

oshD-RB-del-Rev 5‘-GGACGTTGTAGGTGCCTGT-3‘

Sfi-linker-FW 5'-TCGAGGCCTAGATGGCCACCATGG-3'

Sfi-linker-rev 5'-AGCTCGGCCACTCAGGCCTATTAATG-3'

OshC Prom FW 5’-cgctCCTAGGGGTTCCCATTCCTATTTGC-3‘

OshC 3UTR Rev 5’-GATAATGCTTTACTGGTGCAGGGCGCGCCtaat-3‘

OshD Prom FW 5’-cgctCCTAGGCTGGCGACAGACGATTTG-3’

OshD 3UTR Rev 5’-CATGTGCCCCAATAAGTATATGGGCGCGCCtaat-3’

H2B FW-qRT 5'-CTGCCGAGAAGAAGCCTAGCAC-3'

H2B Rev-qRT 5’-GAAGAGTAGGTCTCCTTCCTGGTC-3’

oshA FW-qRT 5'-CGAAGAAAAGCAGCGTGCTAAGCG-3'

oshB Rev-qRT 5'-CACGAGAAACCCTTCGCCAGC-3'

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oshC FW-qRT 5'-GTTCTTGGAGCTCGCGAGAAAGG-3'

oshC Rev-qRT 5'-GGAGAAGGCCTTGGTGTCTCAAAC-3'

oshD FW-qRT 5'-AGCCAACAGGCAATCCGGAGAAC-3'

oshD Rev-qRT 5'-TGAGGTTGATCAGTACATGGACGC-3'

oshE FW-qRT 5'-AAAGAACGAGGAGTGGAAGCCGC-3'

oshE Rev-qRT 5'-GATGAAAGCGAGATCAAAGGCGCC-3'

cyp51A-FW 5’-GAGTAGCCCGTATTTACCCTTCGG-3‘

cyp51A-Rev 5’-CTCGTCTATGTTCATGGGGCCTATG-3‘

cyp51B-FW 5’-GACACTGTGTCCGAGTCGGAC-3‘

cyp51B-Rev 5’-GAAGCGCAACGTCACAACCAAAGC-3‘

ptrA-F 5'-GGGGATCTGACAGACGGGCAATTG-3’

ptrA-R 5’-CTATCATGGGGTGACGATGAGCCG-3’

srbA-U-F 5’-GTAAAACGACGGCCAGTGTTCCAATGGTGTCAGATACAGATAC-3'

srbA-U-R(ptrA) 5’-GCCCGTCTGTCAGATCCCCATCAGATGTACTGAGAATAGTCTTG-3'

srbA-D-F(ptrA) 5’-CGGCTCATCGTCACCCCATGATAGTGATGATGACGCTACTGCCAGAAGC-3'

srbA-D-R 5’-CAGGTCGACTCTAGAGATCCGAATGCCACGTCAGGGAAGAGC-3'

RT-oshA-F 5’-GACGGAAAGGGGCGAAGGACACAAACG-3'

RT-oshA-R 5’-CTCAAGCGAACCACTTTCACGACCGG-3'

RT-oshB-F 5’-GGCTGCCATGGAGGAGTTGGAGATCC-3'

RT-oshB-R 5’-GTTGAGGGATTTCTTGTGCGGCTGG-3'

RT-oshC-F 5’-GAGCTCCAGTAAGGATCCCCAGTCTGCC-3'

RT-oshC-R 5’-GAAGGGAGGAGCGGTTAATGAGG-3'

RT-oshD-F 5’-GTCCGCTGACTGGGGCTCCTTTTTCCATC-3'

RT-oshD-R 5’-CACATTGCCCTGGTCGGGCTCAACAACC-3'

RT-oshE-F 5’-GTCTCCTGTTGCAGCTACAGCGACTCCCG-3'

RT-oshE-R 5’-CGCACGGATGCGATGTCTGCGACGCC-3'

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References in Supplemental Tables.

1. Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, Murray SL, Hynes

MJ, Osmani SA, Oakley BR. 2006. A versatile and efficient gene-targeting

system for Aspergillus nidulans. Genetics 172:1557-1566.

2. Takeshita N, Diallinas G, Fischer R. 2012. The role of flotillin FloA and

stomatin StoA in the maintenance of apical sterol-rich membrane domains and

polarity in the filamentous fungus Aspergillus nidulans. Mol Microbiol 83:1136-

1152.

3. Takeshita N, Higashitsuji Y, Konzack S, Fischer R. 2008. Apical sterol-rich

membranes are essential for localizing cell end markers that determine growth

directionality in the filamentous fungus Aspergillus nidulans. Mol Biol Cell

19:339-351.

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