All obtained predicted proteins were analyzed with the TMHMM, ConPred II and HMMTOP algorithms [70–72] to test for the typical 7-transmembrane domain topology. For those few proteins exhibiting less than seven transmembrane domains, the respective encoding gene and flanking regions were retrieved from the genome database and examined manually. Wrongly predicted

intron-exon boundaries were mainly found and manually corrected resulting in the detection of the missing transmembrane domains. Protein alignments and phylogenetic analysis The classification system of Lafon et al. [1], which classifies fungal GPCRs into nine classes according to their sequence similarity, was buy GS-1101 applied to all detected putative GPCRs of Trichoderma. In addition, members of the three additional classes identified in Verticillium spp. [36], and the GPR11 protein of Phytophtora sojae[35] were used to identify

and classify respective members of T. atroviride, T. virens and T. reesei. Multiple sequence alignments of the identified putative GPCR-like proteins and phylogenetic trees with a neighbor-joining approach were generated using ClustalX [73]. A bootstrap with 1000 repetitions was included. Cultivations and RT-qPCR analysis T. atroviride strain P1 (ATCC 74058; teleomorph Hypocrea atroviridis), T. virens strain IMI 206040 (teleomorph Hypocrea virens), Selleckchem RG7112 and T. reesei strain QM6a (ATCC13631; teleomorph Hypocrea jecorina) were used in this study. The fungi were cultivated at 28°C on either complete medium (PDA, PDB) or minimal medium (MM, containing [g/l]: MgSO4 · 7H2O 1, KH2PO4 10, (NH4)2SO4 6, tri-sodium citrate 3, FeSO4 · 7H2O 0.005, ZnSO4 · 2H2O 0.0014, CoCl2 · 6H2O 0.002, MnSO4 · 6H2O Cetuximab ic50 0.0017, glucose 10) on plates and in liquid culture, respectively. Plate confrontation assays were performed by cultivating Trichoderma together with Rhizoctonia solani on PDA plates covered with a cellophane membrane at 28°C. After direct contact between the two fungi, mycelium

of Trichoderma was harvested from the confrontation zone. For RNA isolation, 30 mg fungal mycelium was grinded in liquid nitrogen and RNA isolated using the peqGOLD TriFast Solution (PeqLab, Erlangen, Germany) according to the manufacturer´s instructions. For cDNA synthesis the Revert Aid H Minus First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) was used according to the manufacturer´s instructions with a combination of an oligo(dT)18 and a random hexamer primer. The sequences for the respective primer pairs for cDNA amplification of the reference gene sar1 and the genes encoding the putative receptors of class VIII identified in the Trichoderma genomes are given in Additional file 3.