Protein methods

2.2.3.1. Protein extraction of Hülle cells from Aspergillus nidulans

In order to extract proteins from enriched Hülle cells the cleistothecia-rolling technique was used as mentioned above. Sexual mycelium of A. nidulans wild-type (FGSC A4 Glasgow, veA⁺) was grown on solid agar plate cultures. Therefore, spores were inoculated on a solid agar plate and the petri dish was sealed with a parafilm to lower the oxygen condition and was covered with an aluminium foil. After three, five and seven days the cleistothecia were transferred to a new petri dish with the help of a syringe. In order to separate Hülle cells from the cleistothecium they were rolled on the solid agar plate and Hülle cells adhered to the surface. Hülle cells were then transferred with the help of the syringe from the solid agar plate to a 1.5 ml collection tube containing 40 µl extraction buffer B ⁺ 300 (100 mM Tris pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM EDTA, 0.1% NP-40, 1 mM DTT, protease inhibitor mix (Roche Pharma AG, Grenzach-Wyhlen, Germany), 0.5 M benzamidine, 100 mM PMSF) with phosphatase inhibitor (100 mM NaF, 50 mM sodium orthovanadate, 50 mM -glycerolphosphate; sterile filtered). All further steps were performed on ice. 270 – 300 cleistothecia were rolled on the solid agar plate and the resulting Hülle cells were used for further protein extractions. Hülle cells were sonicated (60 seconds, 60% of sonoporation power, in between centrifugation steps for 30 seconds at 4°C, procedure repeated 6 times). The tube was centrifuged for 10 min by 13.000 rpm at 4°C and the supernatant was transferred into a new 1.5 ml tube. The cell disruption was verified by observation of cellular debris. Protein extract was then loaded onto 12% SDS-PAGE.

2.2.3.2. Protein extraction of sexual mycelium from Aspergillus nidulans

Sexual mycelium of A. nidulans wild-type FGSC A4, veA⁺ was harvested from the same plate as Hülle cells were collected to be enriched. Sexual mycelium was scraped off the solid agar plate with a spatula and transferred into a 1.5 ml tube. Sexual mycelium was frozen in liquid nitrogen and grinded in a retsch^TM-mill. 300 µl of

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extraction buffer B⁺ 300 (100 mM Tris pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM EDTA, 0.1% NP-40, 1 mM DTT, protease inhibitor mix (Roche Pharma AG, Grenzach- Wyhlen, Germany), 0.5 M benzamidine, 100 mM PMSF) with phosphatase inhibitor (100 mM NaF, 50 mM sodium orthovanadate, 50 mM -glycerolphosphate; sterile filtered) was added and a sonication step as described before followed by centrifugation (10 min, 13.000 rpm, 4 °C) was performed. Protein extraction was performed as described (Bayram et al., 2012). 80 µg of protein were loaded onto a 12% SDS-PAGE. All other steps were performed as mentioned above.

2.2.3.3. Protein extraction of vegetative mycelium from Aspergillus nidulans

In order to cultivate the vegetative mycelium spores were inoculated in 50 ml (250 ml shake flask) minimal medium (1% glucose; 1% AspA; 2 mM MgSO4; and trace elements; pH 6.5; 5.937 X10⁵ sp/ml) for 20 hours (for Hülle cell formation 72 hours) at 37 °C with constant shaking. The vegetative mycelium was filtered to remove the media and washed twice with 0.96% NaCl. 300 mg of vegetative mycelium was transferred into a 2 ml reaction tube. The vegetative mycelium was grinded with a retsch^TM-mill for 2 min by 30 frequency I/s. Proteins were extracted as described (Bayram et al., 2012). 80 µg of total protein extract was loaded onto a 12% SDS-PAGE.

2.2.3.4. Protein extraction of asexual mycelium from Aspergillus nidulans

Protein extraction of an asexual mycelium was performed at the same time points as a sexual mycelium. Harvesting and protein extraction was performed as mentioned above. Asexual mycelium was grown on solid agar plate cultures. Therefore, spore solution was directly inoculated on the solid agar plate. Asexual mycelium was grown in light conditions at 37 °C. The asexual mycelium was scratched off the solid agar plate and proteins were extracted as mentioned above and as described (Bayram et al., 2012).

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2.2.3.5. Protein concentration measurement

In order to determine the concentration of proteins a Bradford assay was used (Bradford 1976). Additionally, the concentration of proteins was measured with an NanoDrop ND-1000 photospectrometer (peqlab biotechnologie GmbH, Erlangen, Germany).

2.2.3.6. SDS-polyacrylamide gel electrophoresis

Protein samples were mixed with 4X loading dye (50 mM Tris pH 6.8, 2% SDS, 10%

glycerol, 1% -mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue). The samples were heated for 5 minutes at 95 °C. The protein samples were loaded onto a 12% SDS page according to (Laemmli 1970). SDS-gels were prepared manually and consisted of a lower running gel (1 M Tris pH 8.8, 0.1% SDS, 12% acrylamide (Rotiphorese® Gel 40 37,5:1) (Roth GmbH, 3029.1), 10% ammonium persulfate (APS), 20 µl TEMED) and an upper stacking gel (1 M Tris pH 6.8, 0.1% SDS, 12%

acrylamide¸ 5% ammonium persulfate (APS), 10 µl TEMED). Proteins were separated electrophoretically for 60 minutes at 180-200 V. The PageRuler^TM prestained protein ladder (Thermo Scientific, SM26616) was used as size marker.

2.2.3.7. Colloidal Coomassie staining of proteins

Protein staining was performed according (Neuhoff et al., 1988). Gels were fixed in 40% (v/v) ethanol, 10% (v/v) acetic acid for 60 min, washed two times in water for 10 minutes and stained for 12 hours in coomassie solution (0.1% (w/v) coomassie brilliant blue G250, 5% (w/v) aluminum sulfate-(14-18)-hydrate, 10% (v/v) methanol, 2% (v/v) ortho-phosphoric acid). The lanes were excised from the polyacrylamide gels. The gel pieces were divided into equal lots into six 1.5 ml peptide low binding reaction tubes, destained in v (methanol):v (water) (40:60).

2.2.3.8. In-gel protein digestion with trypsin

The protein lane was excised from the gel and separated into six peptide low binding reaction tubes and tryptic digestion with trypsin was performed as described (Shevchenko et al., 1996). The gel pieces were covered with acetonitrile and shaken

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for 10 minutes at room temperature. Acetonitrile was removed and the gel pieces were dried in a speedvac (savant speedvac concentrator, Thermo Scientific). 150 µl 10 mM DTT (in 100 mM NH4HCO3) was added to the gel pieces. The samples were incubated at 56 °C for 60 minutes. DTT solution was removed and 150 µl 55 mM iodoacetamide (in 100 mM NH4HCO3) was added and the samples were incubated for 45 minutes at room temperature in dark. Iodoacetamide solution was removed and the gel pieces were washed in 150 µl NH4HCO3, shaken for 10 minutes at room temperature. The solution was removed and 150 µl acetonitrile was added. The samples were shaken for 10 minutes at room temperature. The washing steps were repeated once. The gel pieces were dried in a speedvac at 50 °C and covered subsequently with trypsin (SERVA GmbH, Heidelberg, Germany, 37286.01). The trypsin buffer was prepared according to the manufacturer’s instructions. The samples were incubated for 45 min on ice and the remaining digestion buffer was removed. Next 30 µl 25 mM NH4HCO3

pH 8.0 was added and the samples were incubated for 12 hours at 37 °C.

The samples were centrifuged (1 minute by 13.000 rpm) and the supernatant was collected in a 1.5 ml peptide low binding reaction tube. The gel pieces were covered with 20 mM NH4HCO3 and shaken for 10 minutes at room temperature. The samples were centrifuged (1 minute by 13.000 rpm) and the supernatant was collected into the collection tube. Next the gel pieces were covered with 50% acetonitrile / 5% formic acid. After an incubation step of 20 minutes, shaken and at room temperature the samples were centrifuged (1 minute by 13.000 rpm) and the supernatant was collected. This step was repeated twice. The final volume of the combined supernatant was completely dried in a speedvac at 50 °C. The peptides were resupended in 20 µl sample buffer (2% acetonitrile, 0.1% formic acid).

After tryptic digestion peptides were desalted with the use of C18-StageTips as described (Rappsilber et al., 2007). Therefore, three C18 disks were punced out from a solid phase extraction disk (3M, Neuss, Germany, 2215) and placed into a 200 µl tip. The C18 material was equilibrated. The C18-StageTips were placed into a 2 ml reaction tube using an adaptor. For equilibration of the C18-StageTips 100 µl methanol 0.1% formic acid was added to the C18 material. After a centrifugation step (2 minutes, 13.000 rpm) 100 µl 70% acetonitrile 0.1% formic acid was added. Then the C18-StageTips were centrifuged and 100 µl 0.1% formic acid was added. This step was repeated. The peptide solution was added onto the C18 material and the C18-StageTips were centrifuged for 5 minutes by 4.000 rpm. This step was repeated. The

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C18-StageTips were washed twice with 100 µl 0.1% formic acid. The peptides were eluted from the C18 material with 70% acetonitrile 0.1% formic acid. The C18-StageTips were centrifuged for 5 minutes at 4.000 rpm. The peptide samples were dried in a speedvac. For LC-MS analysis the peptide samples were resuspended in 20 µl LC-MS sample buffer.

2.2.3.9. Liquid chromatography-mass spectrometry (LC-MS) and data analysis

Peptides were analyzed on a Liquid chromatography (LC) coupled to an Orbitrap Velos Pro™ Hybrid Ion Trap-Orbitrap mass spectrometer (MS) (Thermo Fisher Scientific, Bremen, Germany). Peptides were dissolved in 2% (v/v) acetonitril, 0.1%

(v/v) formic acid and raw data were searched with SEQUEST and Mascot algorithms present in Proteome Discoverer 1.4 (Eng et al., 1994, Koenig et al., 2008). The search parameter for SEQUEST and Mascot algorithm were: (i) precursor ion mass tolerance 10 ppm, (ii) fragment ion mass tolerance 0.6 Da, (iii) maximum of two missed cleavage sites were set, (iv) fixed cysteine static modification by carboxyamidomethylation, (v) variable modification by methionine oxidation. The MS/MS spectra were matched against the A. nidulans genome database (UBMG0112_Anidulans_20120325.fasta).

Results filter settings of Proteome Discoverer 1.4 were set to: (a) high peptide confidence and (b) minimal number of two peptides per protein. The MS/MS data were furthermore analyzed with the Andromeda search engine operating in MaxQuant 1.5.1.0 software with the program’s default parameters using the same genome database as mentioned above (Tyanova et al., 2016).

Identified proteins of enriched Hülle cells from solid agar plate cultures were analyzed within three different time points. For each time point three biological replicates were considered. The proteome of sexual mycelium was analyzed from the same solid agar plates from where Hülle cells were enriched. The proteome of sexual mycelium was also analyzed within three different time points. The same conditions were applied to analyze the proteome of asexual mycelium. For each time point three biological replicates were considered. Only proteins identified in two or more biological replicates and with two or more peptides per protein were considered for the analysis. For visualization of the analysis the accession numbers of the proteins were inserted into

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a Venn diagram (Oliveros 2016). The proteome of a vegetative mycelium in submerged liquid cultures was analyzed by three biological replicates. Only proteins identified in two or more biological replicates and with two or more peptides per protein were considered for the analysis. For peptide quantification Lys4 was defined as medium peptide labels and Lys8 as heavy peptide labels. The peptide median ratio distribution was determined using Proteome Discoverer 1.4. In order to correct experimental bias, the protein median was normalized. Only peptide ratios that were determined similar in two or more biological replicates were considered for the analysis. Additionally, normalized ratios were analyzed and processed using the Perseus 1.5.0.15 software. A t-test (p value 0.05) was performed to statistically compare the log2 SILAC ratios across three biological replicates.The log2 SILAC ratio was determined for the strains laeA (AGB1074) in comparison to laeA (AGB1092).

The following threshholds were set. In the range of a log2 SILAC ratio between - 5.0 and - 0.5 the protein quantity of identified proteins was down-regulated. In the range of a log2 SILAC ratio between + 0.5 and + 5.0 the protein quantity of identified proteins was up-regulated. In the range between a -0.5 and + 0.5 log2 SILAC ratio the protein quantity of identified proteins was unchanged. Workflows to process the proteomic data are illustrated in supplementary table 1 and 2.

2.2.3.10. Functional annotation of proteins

Functional annotations of identified proteins were performed using the basic local alignment search tool Blast2GO (http://www.Blast2GO.com/b2ghome). BlastP was used to blast against the NCBInr database (NCBI: National Center for Biotechnology Information) with a blast expect value of 1 x 10^-3. The number of blast hits were set to 20 and the cutoff value for the GO terms was set to 20 (Conesa et al., 2005).

2.2.3.11. Fluorescence microscopy of fusion proteins

In order to visualize the localization of fusion proteins (fused to GFP) fluorescence microscopy was used. Hülle cells were enriched in 40 µl of dH2O using the cleistothecia-rolling technique. After centrifugation (2 minutes at 13.000 rpm) the supernatant was removed. The cellular pellet was resuspended in 5 µl dH2O and transferred to an object slide. The cells were observed under a reflected-light

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microscope (Zeiss Axiolab - Zeiss AG, Jena, Germany) and an Olympus SZX12-ILLB2-200 binocular (Olympus GmbH, Hamburg, Germany). Fluorescence photograph pictures were taken with a confocal light microscope (Zeiss Axiolab - Zeiss AG, Jena, Germany) equipped with a QUAN-TEM: S12SC (Photometrics, Tucson, Arizona, United States of America) digital camera and the SlideBook 6 (Intelligent Imaging Innovations GmbH, Göttingen, Germany) software package. For GFP fusion protein visualization, the following parameters were used; DIC filter 200 ms; GFP filter 300 ms.

2.2.3.12. Immunoblotting

Proteins were extracted from different types of mycelia as described above. For immunoblotting experiments, proteins were first separated using a polyarcrylamide gel electrophoresis and transferred onto a nitrocellulose membrane as previously described (Jöhnk et al., 2016, Schinke et al., 2016). Protein concentrations were determined as described above and as reference for equally loaded proteins amount 0.2% Ponceau S (Sigma- Aldrich, Copenhagen, Denmark), 3% TCA was used.

Blocking was performed in 5% milk powder (skimmed milk powder (Sucofin, 562570765347)). Membranes were incubated for 12 hours with -GFP antibody (dilution: 1:1000, sc-9996, Santa Cruz Biotechnology Inc., Dallas, Texas, United States of America and Heidelberg, Germany). The nitrocellulose membranes were washed three times in 150 ml 1X TBST (Tris buffered saline with tween 20) for 10 minutes at room temperature. Membranes were incubated for 60 minutes using an -mouse secondary antibody (dilution 1:2000, G21234, Invitrogen AG, Carlsbad, California, United States of America). The nitrocellulose membranes were washed three times in 150 ml 1X TBST (Tris buffered saline with tween 20). For peroxidase reaction 100 µl 2.5 mM luminol, 44 µl 400 µM paracoumarat, 100 mM Tris pH 8.5 added to 9 ml dH2O and 6.15 µl 30% H2O2 to 9 ml dH2O. The nitrocellulose membranes were incubated with both solutions for 2 minutes, shaken and in darkness. Detection was performed on a Fusion-SL7 (Vilber Lourmat, Eberhardzell, Germany) system.

Western hybridization experiments were performed with three biological replicates and samples were loaded twice on the 12% SDS-PAGE.

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