S1 File - Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex

Table A, Plasmids used for this study. Table B, Primers used for this study. Fig A, RT-PCR analysis of Smkin3 and Smkin24. (A) Schematic illustration of the genes Smkin3 and Smkin24. Introns are indicated as grey boxes, positions of primers used for analysis of intron splicing are indicated by arrows. (B) Results of the RT-PCR analysis. Shown are the obtained amplicons for respective primer pairs from cDNA and gDNA. (C) Schematic illustration of Smkin3 and Smkin24 transcripts identified by cDNA sequencing. Fig B, Alignment of aa sequences encoded by alternatively spliced Smkin24 transcripts. Smkin24 without introns results in a protein of 882 aa and thus representing the largest protein. Expression of Smkin24 with remaining intron I but removed intron II-IV results in a protein comprising aa 94–882 of the protein derived from Smkin24 without introns. Smkin24 with spliced intron I-III but remaining intron IV results in a protein comprising aa 1–729. Fig C, Identity of aligned amino-acid sequences in pair-wise comparison. Fig D, Controls of yeast two-hybrid assay AD-PRO11 and AD-SmMOB3 with empty vector pGBKT7. Shown are serial dilutions of diploid yeast strains obtained after mating and spread on SD medium lacking tryptophan (trp) and leucine (leu) or trp, leu and adenine (ade) to test the interaction of both proteins. The left picture displays a control, which ensures presence of both plasmids in the diploid strain; trp and leu prototrophy are regained by genes present on the plasmid. The right picture displays the interaction assay; ade prototrophy is not obtained by negative interaction between the GAL4 binding domain (BD) and proteins PRO11 and SmMOB3 fused GAL4 activation domain (AD). Fig E, Co-IP of FLAG-SmKIN3 and HA-PRO11 without crosslinker added. As a positive control a cell extract of a strain expressing the respective protein in suitable amounts (cell extract) is shown in the left lane; to exclude unspecific antibody binding, cell extracts of the wt was used as second negative control for Western blot analysis (right lane). The co-IP without crosslinker added are shown in the middle lane. (A) FLAG-SmKIN3 detected by FLAG antibody (anti-FLAG) (B) HA-PRO11 by anti-HA antibody (anti-HA). Fig F, Generation of the ΔSmkin3 deletion strain. (A) Schematic illustration of the Smkin3 locus before and after homologous integration of the deletion cassette. Primers used for verification of the deletion strain are indicated by arrows. Sizes of PCR fragments and the probe used for Southern hybridization are given. (B) Verification of the respective deletion using PCR. Sizes of amplicons and positions of the primers as indicated in (A). (C) Integration of the deletion cassette was verified by Southern hybridization [46]. Positions of the respective probes are indicated in (A). ΔSmkin3 was verified using a hygromycin specific probe that only binds within the deletion cassette. Fig G, Generation of the ΔSmkin24 deletion strain. (A) Schematic illustration of the Smkin24 locus before and after homologous integration of the deletion cassette. Primers used for verification of the deletion strain are indicated by arrows. Sizes of PCR fragments and the probe used for Southern hybridization are given. (B) Verification of the respective deletion using PCR. Sizes of amplicons and positions of the primers as indicated in (A). (C) Integration of the deletion cassette was verified by Southern hybridization [46]. Positions of the respective probes are indicated in (A). ΔSmkin24 was verified using a probe binding at the 3’ region of Smkin24. The successful integration is represented by a band shift. Fig H, Macroscopic and microscopic analysis of the sexual development of wt, complemented ΔSmkin3 (ΔSmkin3+), complemented ΔSmkin24 (ΔSmkin24+) and partially complemented ΔSmkin3/ΔSmkin24. The wt strain produces ascogonia after 3 days which develop to unpigmented protoperithecia at day 4 and pigmented protoperithecia at day 5. After 7 days mature perithecia with asci and ascospores are formed. Similar to the wt ΔSmkin3+ and ΔSmkin24+ and ΔSmkin3/ΔSmkin24 + Smkin24 completed the lifecycle within 7 days and produced mature ascospores. Development of ΔSmkin3/ΔSmkin24 + Smkin3 is arrested at stage of late protoperithecia formation, similar to ΔSmkin24. Scale bars as indicated. Fig I, Microscopic investigation of hyphal fusion in wt, ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24. ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 are capable of hyphal fusion. Hyphal fusion events are highlighted with circles. Pictures of hyphal fusion events were taken at subperiphal regions 10 mm behind the growth front. Hyphal fusion was investigated 2–3 days past inoculation. (PDF)