âsakA partial resistance to fludioxonil is not mediated by SrkA. 1x104 spores from strains CLK43 (WT), TOL1 (âsakA), CRJ2 (âsrkA) and CRJ5 (âsakA âsrkA) ...
FIG. S1. The srkA gene encodes a protein member of CAMK serine-‐threonine protein kinase superfamily. Protein alignment of A. nidulans SrkA, S. pombe Srk1, S. cerevisiae Rck2, C. albicans Rck2 and homologs from N. crassa, A. oryzae, A. flavus and A. fumigatus. Consensus gray bars indicate identity. All proteins share a kinase domain and conserved motifs from the CAMK superfamily. A conserved glycine loop, a potential phosphorylation site and a MAP kinase interaction domain are present at the –C terminus. Alignment was performed using Unipro UGene software with muscle algorithm. A putative mitochondrial targeting sequence (PTM) was identified only in SrkA homologs from filamentous fungi, using MitoProt software, which uses the MTLS algorithm (Mitochondrial-‐Targeting-‐ Sequence-‐Localization). The predicted cleavage site for SrkA is ARL∧VT.
FIG. S2. Deletion of the srkA gene. (A) The srkA ORF was replaced by the AfpyrG gene, used as a selective marker. The indicated cassette was generated by double joint PCR and used to transform strain 1155. (B) PCR products flanking the srkA ORF or the AfpyrG replacing cassette were generated using primers 5’ srkA For and 3’ srkA Rev, then purified and digested with EcoRI. WT restriction pattern corresponds to bands of 2.2, 2.4 Kb and 401 bp, while, in while ∆sakA strains EcoRI pattern corresponds to bands of 4.3 Kb and 401 bp. Numbers above each row correspond to different transformants.
FIG. S3. Sexual derepression in ∆sakA and ∆srkA mutants in a veA+ background. (A) veA+ strains CRJ9 (WT), CRJ6 (∆sakA), CRJ7 (∆srkA) and CRJ8 (∆sakA ∆srkA) were induced to undergo sexual development as reported previously (1). Pictures were taken from confluent cultures after 5 days of induction. (B) The total number of cleistothecia per fixed area was counted under a dissection microscope and used to calculate cleistothecia per cm2 as reported (1). Data are mean values from three independent experiments; bars indicate standard deviation. Circles in (A) highlight cleistothecia.
FIG. S4. In contrast to ∆sakA mutants, ∆srkA strains are not sensitive to calcofluor. 1x104 spores from strains CLK43 (WT), TOL1 (∆sakA) and CRJ2 (∆srkA) and CRJ5 (∆sakA ∆srkA) were inoculated on minimal media plus supplements with or without the indicated concentration of calcofluor.
FIG. S5. ∆sakA partial resistance to fludioxonil is not mediated by SrkA. 1x104 spores from strains CLK43 (WT), TOL1 (∆sakA), CRJ2 (∆srkA) and CRJ5 (∆sakA ∆srkA) were inoculated on minimal medium plus supplements without (upper panel) or with the fungicide fludioxonil (lower panel).
FIG. S6. GFP or S-‐tag tagging of SrkA does not affect its function in repressing sexual development. Strains 1155 (WT), TRJ4 (∆srkA) TRJ1 (SrkA::GFP) and TRJ2 (SrkA::S-‐tag) were induced to undergo sexual development. Pictures were taken under a dissection microscope after 10 days of induction. Arrows point some of the masses of Hülle cells associated with the development of fruiting bodies or cleistothecia.
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FIG. S7. Detection of SrkA::S-‐tag by Western blot. A DNA fragment, based in the AfpyrG gene as genetic marker, was generated double joint PCR to label SrkA at its C-‐terminus. This gene replacement module was used to transform strain 1155 by protoplast fusion. The resultant transformants were grown in liquid media at 37 C with shaking during 12 hours. Mycelial samples were frozen and processed for protein extraction. 30 μg of protein from each transformant were used for immunoblot detection using an anti-‐S-‐tag antibody. Untagged strain 1155 (WT) was used as control.
Table S1. Proteins identified as associated with SakA and / or SrkA ID Putative function and protein name Stress
No stress
SakA
SrkA
SakA
SrkA
Cell cycle / Signal transduction
AN4483
Protein serine / threonine kinase (SrkA)
18
28
20
28
AN1017
Putative mitogen-‐activated protein kinase (MAPK) SakA
15
12
18
11
AN6982
PtpA Putative phosphotyrosine-‐specific protein phosphatase MpkC Putative mitogen activated protein kinase (MAPK)
6
17
3
5
3
2
AN4501
Protein with a conserved CDC48, cell division protein N-‐ terminal domain ArtA Putative 14-‐3-‐3 protein
2
AN5744
Putative 14-‐3-‐3-‐like protein
2
AN10593
2
5
Has domain(s) with predicted phosphoserine phosphatase activity
Histone / DNA damage response
AN0734
H4.1 Histone H4.1
3
3
AN3468
H2A.X Histone H2A
2
2
AN2765
HhoA Putative histone H1
2
2
2
AN3469
H2B Histone H2B; core histone protein
2
Protein folding / Signal transduction
AN8269
Hsp90 90 kilodalton heat shock protein
7
3
4
2
AN6089
Putative 60 kilodalton heat shock protein
3
3
AN2062
BipA Putative ER-‐resident chaperone of the HSP70 family
2
4
3
Protein biosynthesis / mRNA stability
AN4802
2
3
3
AN8704
60S ribosomal protein L21; ortholog of S. cerevisiae Rpl21Ap 60S ribosomal protein L24a
2
3
3
AN6202
Rpl3 Putative ribosomal protein L3
2
3
AN10416
Putative 60s ribosomal protein
2
AN1964
3
2
2
AN5520
Ortholog of S. cerevisiae RPS6B and RPS6A; palA-‐ dependent expression Ortholog(s) have structural constituent of ribosome activity Has domain(s) with predicted role in ribosome biogenesis
2
2
3
AN6083
Ortholog(s) have role in ribosomal large subunit assembly
2
3
3
3
AN7003
5
2
2
AN4475
Has domain(s) with predicted structural constituent of ribosome activity Has domain(s) with predicted structural constituent of ribosome activity Ortholog(s) have role in ribosomal large subunit assembly
2
AN4916
Ortholog(s) have role in ribosome biogenesis
2
AN7107
Has domain(s) with predicted structural constituent of ribosome activity
2
2
AN4668 AN7254
AN10681
AN10740
ID
Putative function and protein name
AN2980
AN6679
Ortholog(s) have structural constituent of ribosome activity Ortholog(s) have structural constituent of ribosome activity Ortholog(s) have structural constituent of ribosome activity Ortholog(s) have structural constituent of ribosome activity Ortholog(s) have SSU rRNA binding
AN2734
Stress
No stress
SakA
SrkA
SakA
SrkA
2
2
2
2
2
2
2
2
2
Ortholog(s) have LSU rRNA binding activity
2
3
2
AN4000
FabM Protein with similarity to poly(A)-‐binding proteins
2
AN6202
Rpl3 Putative ribosomal protein L3
3
AN4222
2
2
2
2
2
2
AN3172
Has domain(s) with predicted structural constituent of ribosome activity Has domain(s) with predicted structural constituent of ribosome activity Ortholog(s) have structural constituent of ribosome activity Ortholog(s) have RNA binding activity and role in ribosomal large subunit assembly Putative 40s ribosomal protein S26 ortholog of S. cerevisiae Rps26Bp Ortholog(s) have structural constituent of ribosome activity Ortholog of S. cerevisiae RPS0A and RPS0B
2
AN2932
Putative eukaryotic initiation factor 4A
2
AN5931
Putative ATP-‐dependent RNA helicase
4
Energy metabolism / Mitochondrial function
AN0554
AldA Aldehyde dehydrogenase
9
10
3
AN2435
AclA Putative ATP citrate synthase
4
3
AN2316
Putative cytochrome c oxidase subunit
3
3
2
AN8273
Putative ubiquinol-‐cytochrome-‐c reductase subunit
2
2
AN9403
PdhC Putative pyruvate dehydrogenase (lipoamide)
2
4
AN8979
2
2
AN6717
AlcA Alcohol dehydrogenase with a role in two-‐carbon compound metabolism MdhA Putative mitochondrial malate dehydrogenase
2
AN4888
PdcA Putative pyruvate decarboxylase
3
AN8275
CitA Mitochondrial citrate synthase
2
AN1534
Putative F1F0-‐ATPase complex subunit
2
AN9340
2
2
AN6287
TreA Alpha,alpha-‐trehalase with a role in trehalose hydrolysis Putative F1F0-‐ATPase complex subunit
2
AN10296
Ortholog(s) have fumarate reductase (NADH)
2
AN2526
Putative ketol-‐acid reductoisomerase
2
AN7594
DUF636 domain-‐containing protein
2
AN2275 AN0843 AN9465
AN7003 AN2980 AN8856 AN5715 AN9465
ID
Putative function and protein name
AN5746
Stress
No stress SakA SrkA
SakA
SrkA
AcuN Putative phosphopyruvate hydratase
2
Nitrogen metabolism
AN1007
NiiA Putative nitrite reductase
3
2
AN4376
GdhA Putative NADP-‐linked glutamate dehydrogenase
3
5
4
Fatty acid metabolism
AN9407
FasA Fatty acid synthase, alpha subunit
3
2
3
AN9408
FasB Fatty acid synthase, beta subunit
2
Unclassified
AN10103
Has domain(s) with predicted DNA binding activity
3
3
3
2
AN4463
Ortholog(s) have structural molecular activity
2
AN8870
Expression increased in salt-‐adapted strains
2
2
AN3804
Ortholog(s) have IgE binding activity
2
2
AN4865
Has domain(s) with predicted nucleic acid binding
2
2
AN7725
PyroA Protein required for biosynthesis of pyridoxine
2
2
AN0745
Putative nucleolar protein
2
2
AN0870
2
2
3
AN1551
Putative transporter with a predicted role in small molecule transport BtgE Putative beta-‐glucosidase
2
3
AN8953
AgdB Putative alpha-‐glucosidase
2
2
AN10202
Has domain(s) with predicted ATP binding activity
2
4
AN3226
PkfC aspernidine A secondary metabolism gene cluster member
4
Numbers below SakA and SrkA columns correspond to the number of peptides from each protein identified in those samples.
Table S2. SakA and / or SrkA-‐interacting proteins previously reported as regulated by farnesol or menadione ID AN7254 AN2435 AN4376 AN9340 AN5746 AN8273 AN8979 AN0745 AN4802 AN5931 AN3226 AN7594 AN3172 AN5715 AN0554 AN6717 AN5746 AN4376 AN7594
Putative function and protein name Farnesol increased Protein with a conserved CDC48, cell division protein N-‐terminal domain AclA Putative ATP citrate synthase
Reference Wartenberg D, et al. (2012) Wartenberg D, et al. (2012) GdhA Putative NADP-‐linked glutamate dehydrogenase Wartenberg D, et al. (2012) TreA Alpha,alpha-‐trehalase with a role in trehalose hydrolysis Savoldi M, et al. (2008) Putative phosphopyruvate hydratase Wartenberg D, et al. (2012) Farnesol decreased Putative ubiquinol-‐cytochrome-‐c reductase subunit Wartenberg D, et al. (2012) AlcA Alcohol dehydrogenase with a role in two-‐carbon compound Wartenberg D, metabolism et al. (2012) Putative nucleolar protein Savoldi M, et al. (2008) 60S ribosomal protein L21; ortholog of S. cerevisiae Rpl21Ap Savoldi M, et al. (2008) Putative ATP-‐dependent RNA helicase Savoldi M, et al. (2008) PkfC Has domain protein; aspernidine A secondary metabolism Wartenberg D, gene cluster member et al. (2012) DUF636 domain-‐containing protein Wartenberg D, et al. (2012) Ortholog of S. cerevisiae RPS0A and RPS0B Savoldi M, et al. (2008) Putative 40s ribosomal protein S26 Savoldi M, et al. (2008) Menadione stress repressed AldA Aldehyde dehydrogenase Pusztahelyi T, et al. (2011) MdhA Putative mitochondrial malate dehydrogenase Pusztahelyi T, et al. (2011) Putative phosphopyruvate hydratase Pusztahelyi T, et al. (2011) Menadione stress induced GdhA Putative NADP-‐linked glutamate dehydrogenase Pusztahelyi T, et al. (2011) DUF636 domain-‐containing protein Pusztahelyi T,
REFERENCES 1.
Kawasaki L, Sanchez O, Shiozaki K, Aguirre J. 2002. SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans. Mol Microbiol 45:1153-‐1163.
2.
Wartenberg D, Vodisch M, Kniemeyer O, Albrecht-‐Eckardt D, Scherlach K, Winkler R, Weide M, Brakhage AA. 2012. Proteome analysis of the farnesol-‐ induced stress response in Aspergillus nidulans-‐-‐The role of a putative dehydrin. J Proteomics 75:4038-‐4049.
3.
Savoldi M, Malavazi I, Soriani FM, Capellaro JL, Kitamoto K, da Silva Ferreira ME, Goldman MH, Goldman GH. 2008. Farnesol induces the transcriptional accumulation of the Aspergillus nidulans Apoptosis-‐Inducing Factor (AIF)-‐like mitochondrial oxidoreductase. Mol Microbiol 70:44-‐59.
4.
Pusztahelyi T, Klement E, Szajli E, Klem J, Miskei M, Karanyi Z, Emri T, Kovacs S, Orosz G, Kovacs KL, Medzihradszky KF, Prade RA, Pocsi I. 2011. Comparison of transcriptional and translational changes caused by long-‐term menadione exposure in Aspergillus nidulans. Fungal Genet Biol 48:92-‐103.
