The Genome and Development-Dependent Transcriptomes of Pyronema confluens: A Window into Fungal Evolution
A. Comparative analysis of RPKM values for all orthologous genes for P. confluens and S. macrospora (with the exception of genes without read counts in one or more conditions). B. Quantitative real time PCR analysis of the predicted transcription factor gene PCON_06721 in P. confluens. Expression was analyzed in two independent biological replicates for the four conditions LL, DD, LLK and DDK (light and darkness in surface culture and submerged culture). Sexual development is only possible in condition LL. Expression ratios and standard deviations were calculated to address the question if the gene is differentially regulated during sexual development (i.e. in LL/DD and in LL/LLK), or regulated by light (i.e. in LL/DD and LLK/DDK) or regulated by surface versus submerged growth (i.e. LL/LLK and DD/DDK). PCON_06721 is upregulated during sexual development, but also slightly upregulated by light. RNA-seq results are given for comparison. C. Complementation of the Sordaria macrospora mutant Δpro44 with the P. confluens ortholog PCON_06721. The mutant was transformed with plasmid pFA50 that carries the PCON_06721 ORF under control of the respective gpd and trpC promoter and terminator sequences of Aspergillus nidulans. The figure shows a side view (longitudinal section) of the region comprising the agar/air interface from cultures of the wild type, the sterile mutant Δpro44 and a complemented transformant (Δpro44::PCON_06721_constitutive). The small inserts show photographs of Petri dish sections. The S. macrospora wild type forms mature perithecia at the agar/air interface, whereas the mutant only forms protoperithecia that are submerged in the agar. Complemented transformants produce mature perithecia at the agar/air interface like the wild type. Strains were grown on corn meal agar; photographs were taken after 8 d; scale bar indicates 1 mm, and 1 cm in the inserted photographs.