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[…] Protein sequence data were obtained from public databases NCBI (www.ncbi.nlm.nih.gov), JGI (www.jgi.doe.gov), Broad institute (www.broadinstitute.org), MIPS (http://mips.gsf.de), INRA (http://mycor.nancy.inra.fr) and from J. Mondego (LGE lab, UNICAMP, Brazil) (, Table A). Dynein heavy chain protein sequences of representatives with split DHC genes (i.e. Agaricomycetes, Dacryomycetes, Wallemiomycetes and Ustilaginomycotina) were concatenated, allowing for simultanous analysis of all samples. The sequences were aligned using the E-INS-I option of MAFFT v6 [] which assumes multiple conserved domains and long gaps. For control of alignment quality and adjustment, BIOEDIT v7.0.9.0 [] was used.Phylogenetic reconstruction was performed at the Bioportal of the University of Oslo, Norway (http://www.bioportal.uio.no) and the CIPRES science gateway [], using MrBayes v3.1.2 [, ], and RAxML v.7.2.6 [, ] for comparison. For Bayesian analysis, the mixed model option with four gamma categories was implemented, allowing for model jumping during the analysis. For all datasets, two runs with each 2,000,000 generations in four chains were performed; sampling every 100 generations, and with a burn-in of 25 percent. Results were evaluated with Tracer v1.5 []. Both analyses had log likelyhood ESS values above 100. For the RAxML Maximum Likelihood analysis, phylogeny was inferred under the PROTGAMMAGTR model with four gamma categories and branch support through 100 bootstrap replicates. For visualisation of phylogenetic trees, FigTree v1.3.1 [] was used. [...] DNA of the wildtype strain 12–43 was isolated []. Flanking regions to dhc1 were amplified with oligonucleotides dhc1AaXbaI and dhc1AbXbaI for the 1585 bp large upstream flank. Analog, the 1699 bp large downstream flank was amplified with the oligonucleotides dhc1Ba and dhc1Bb. The upstream flank was cloned into XbaI restriction sites of the cloning vector pChi which already contained the marker gene uraI. The marker gene ura1, linked to the tef promoter of S. commune [] was cloned in between the two homologuous flanks, using KpnI/ClaI for the 5´flank, ClaI for tef-ura and ClaI/BamHI for cloning of the 3`flank, resulting in vector p∆dhc2 [].The gene cassette of the upstream flank and the marker gene was restricted with BamHI and NotI and cloned into the appropriate restriction sites of the vector pBluescript II SK. The downstream flank was cloned into EcoRI restriction site of pBluescript II SK. This procedure resulted in the deletion vector pΔdhc1. The flanking regions of dhc2 were obtained by cloning the 4229 bp BamHI/ClaI fragment from a sub-library of respective BamHI/ClaI genomic fragments after screening with a probe obtained from clone pD5. For the 5´ area, an 1863 bp fragment amplified with oligonucleotides dhcClaI and dhcKpnI, was cloned in the ClaI/KpnI-restriction sites of the cloning vector. Protoplasts of strain 12–43 were transformed according to Munoz-Rivas et al. []. For transformation, 20 μg plasmid-DNA of pΔdhc1 respectively p∆dhc2 were used. In order to complement for potentially lethal dhc2 knock-out, macerated mycelium of the compatible wildtype strain E6 was mixed with the transfected protoplasts. DNA of the resulting dikaryons was tested for the deletion of dhc2 by PCR. Spores of the positive dikaryon were isolated, plated on selective media and monokaryotic strains with the deleted dhc2 gene were harvested. For dhc1, transfected mycelium was plated to selective media as lethality had not been observed with deletion of dhc2. Transformants were analyzed by PCR for the successful deletion of dhc1.Transcriptome analyses RNA of seven days old, solid cultures of the strains 12–43, E6 and ∆dhc2 was isolated using RNeasy Plant Mini Kit performing an additional DNAse digestion with RNase-Free DNase Set (both Qiagen, Hilden,Germany). RNA-sequencing was performed by LGC Genomics, Berlin (Germany) with mRNA-based cDNA-libraries constructed from sequencing adaptors ligated to cDNA fragments. The transcriptome was sequenced from two biological and technical replicates each.Raw data of RNA sequences were mapped against the genome of S. commune (genome.jgi.doe.gov/Schco2) using the splice junction mapper TopHat (release 1.4.1) []. Htseq (www.heber.emgl.de/users/anders/HTSeq/doc/index.html) was used to calculate the number of reads mapped within each gene (raw counts). Normalized (gene length, library size) expression values (RPKM) for all genes were calculated using the statistical software R. For expression differences, the ratios (fold-change) of mean values (wildtype): mean values(∆dhc2) were determined. Four different statistical tests, DeSeq [], EdgeR [], BaySeq [] and Noiseq [] were used to scan for significantly differentially expressed genes (false discovery rate adjusted p-value cutoff 0.01). A gene was defined to be differentially expressed if it was detected by each method.To validate RNA-sequencing data, quantitative real-time PCR was performed after Erdmann et al., 2012. cDNA synthesis was modified by using the QuantiTect Rev. Transcription Kit (Qiagen, Hilden, Germany). […]

Pipeline specifications

Software tools MAFFT, BioEdit, CIPRES Science Gateway, MrBayes, RAxML, FigTree, TopHat, HTSeq, DESeq, edgeR, baySeq, NOISeq
Databases BioPortal
Applications Phylogenetics, RNA-seq analysis
Organisms Schizophyllum commune