Supplemental Material for Radchenko et al., 2018
2018-07-11T17:22:44Z (GMT) by
<b>Figure S1</b> Generation and verification of ΔSmKin3 strains. (A) Genomic organization of the Smkin3 locus in the wild type and ΔSmkin3 deletion strain. In ΔSmkin3 the Smkin3 gene is substituted by the hygromycin B phosphotransferase coding gene (hph) under control of the trpC promoter. Arrows indicate oligonucleotides used for PCR (Table 3). Thin black bars represent expected fragments in PCR and Southern Blot analysis. Thick gray bars represent probes used for radioactive hybridization. (B) PCR verification of 5’ flank, 3’ flank and Smkin3 gene in the wild type (WT), ΔSmkin3 with a sterile and septation deficient phenotype (ΔSmkin3sos1-) and ΔSmkin3 with a fertile phenotype (ΔSmkin3sos1+). Distilled water (A. dest.) was used as a contamination control. (C) Southern Blot verification of ΔSmkin3 strains. Genomic DNA of WT, the recipient strain used for the homologous recombination of the deletion cassette (Δku70), ΔSmkin3sos1- and ΔSmkin3sos1+ was digested with PvuII. Blots were hybridized with P32-labled probes specific for the Smkin3 gene (left box) and hph (right box).<br><br> <br><br><b>Figure S2</b> Verification of ΔSmkin3/pro11 and ΔSmkin3Δpro11 double mutant strains. (A) Genomic organization of the pro11 locus in the wild type and Δpro11 deletion strain (Bloemendal et al. 2012). PCR and Southern Blot analyses were performed as indicated in Fig. S1. (B) PCR verification of Smkin3 gene and pro11 gene with 5’ and 3’ flanking regions in WT, pro11, ΔSmkin3, Δpro11, ΔSmkin3/pro11 and ΔSmkin3Δpro11 strains. (C) Southern Blot verification of double mutant strains.<br><br> <br><br><b>Figure S3</b> Verification of Δpro22/pro11 and Δpro22Δpro11 double mutant strains. (A) Genomic organization of the pro22 locus in the wild type and Δpro22 deletion strain (Bloemendal et al. 2012). PCR and Southern blot analysis was performed as indicated in Fig. S1 and Fig. S2. (B) PCR verification of pro22 gene and pro11 gene with 5’ and 3’ flanking regions in WT, pro11, Δpro22, Δpro11, Δpro22/pro11 and Δpro22Δpro11 strains. (C) Southern Blot verification of double mutant strains.<br><br> <br><br><b>Figure S4</b> Sequence alignment of conserved STK domains from GCKs from different organisms. Abbreviations and color highlights are the same as in Figure 1. Protein accession numbers: AnMST1, CBF81427.1; AnPOD6, CBF69669.1; AnSEPH, Q5B4Z3.2; AnSEPL, BN001302.1; AtSIK1, NP_849865.1; CeCST-1, CCD65403.1; CeCST-2, NP_001041219.2; CeGCK-1, CCD72152.2; DmGckIII, NP_650596.1; DmHppy, NP_725863.1; DmMsn, NP_001261333.1; DmTao, AGB95548.1; HsMAP4K1, NP_001036065.1; HsMAP4K2, AAH47865.1; HsMAP4K3, AAN75850.1; HsMAP4K4, NP_004825.3; HsMAP4K5, NP_942089.1; HsMAP4K6, XP_006721594.1; HsMST1, AAA83254.1; HsMST2, AAC50386.1; HsMST3, NP_003567.2; HsMST4, AAK38484.1; HsTAO1, NP_065842.1; HsTAO2, NP_057235.2; HsTAO3, NP_001333416.1; HsYSK1, NP_001258907.1; NcCDC-7, XP_961421.2; NcMST-1, XP_011392830.1; NcPOD-6, XP_011393318.1; NcPOD-6, XP_011393316.1; NcSID-1, XP_011394611.1; ScCdc15p, CAA43041.1; ScKic1p, NP_011970.1; ScSps1p, NP_010811.1; SmKIN24, XP_024511144.1; SmKIN3, XP_003343831.1; SmSMAC_04024, XP_003348179.1; SmSMAC_05827, XP_003346290.1; SpCdc7p, NP_596340.1; SpNak1p, NP_596023.1; SpPpk11p, NP_594517.1; SpSid1p, O14305.1; TtCDAI, XP_001026112.1<br><br> <br><br><b>Figure S5</b> Mendelian inheritance of sos1+/sos1- allele. Cross numbers, ascus types, genotype and phenotype designation, as well as color code are the same as in Figure 2. Wtsos1- (S147487) obtained from cross 1 is indicated with the black frame.<br><br> <br><br><b>Figure S6</b> Hyphal fusion in ΔSmkin3sos1- and related strains. Wild type, ΔSmkin3sos1-, complementation strains with native promoter and overexpression promoter constructs and ΔSmkin3/pro11 double mutant were grown on MMS medium covered with cellophane for 2 days and examined for hyphal fusion events within subperipheral colony regions. Black arrows indicate hyphal fusion bridges. Asterisks indicate hyphal contact areas without fusion. Wild type as well as ΔSmkin3::NAkin3 complementation strain is able to undergo normal hyphal fusions. Vegetative mycelium of ΔSmkin3sos1- and ΔSmkin3/pro11 tends to grow in loose overlapping layers and no fusion was observed. Analysis of ΔSmkin3Δpro11 failed, since this strain is unable to grow on cellophane.