Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 Sequence analysis Similarity searches for full length sequences and con- served domains were performed using a combination of standard bioinformatics programs and customized Python scripts. Each assembled transcript was searched against UniProtKB database (Release 15.5, TrEMBL and Swiss-Prot at http://www.uniprot.org) resources [75] using the BLASTX algorithm [76]. Using UniProt/Gene Ontology (GO) crossed tables, candidate GO assign- ments were predicted on the basis of best transcripts matches (E-value <10-05) to the UniProt reference sequences. Categories were assigned on the basis of the biological, functional and molecular annotations avail- able from GO http://www.geneontology.org[46]. Additionally, we constructed and searched (using the BLASTX algorithm) two other fungal sequence data- bases. The BasidiomycotaDB database included Basidio- mycota (excluding Pucciniales) protein sequences from the non-redundant database of NCBI (124,751 sequences), a 6-frame translation of Basidiomycota (excluding Pucciniales) EST sequences from NCBI (287,259 sequences), and gene models from eight gen- ome projects: Coprinus cinereus Okayama 7 (#130) (Broad Institute); Cryptococcus neoformans var. grubii serotype A, strain H99 (Broad Institute); Laccaria bico- lor S238N-H82 (JGI); Malassezia globosa CBS 7966 (Procter and Gamble Co.); Phanerochaete chrysosporium RP78 (JGI); Postia placenta MAD-698 (JGI); Sporobolo- myces roseus IAM 13481 (JGI); and Ustilago maydis 521 (Broad Institute) (a total of 85,025 gene models). The PuccinialesDB database included Pucciniales protein sequences from the non-redundant database of NCBI (390 sequences), a 6-frame translation of Pucciniales EST sequences from NCBI (84,006 sequences), and gene models from P. graminis f. sp. tritici CRL 75-36-700-3 (20,567 gene models). The hmmpfam program (HMMer software; http://hmmer.janelia.org[77] was used to search the PFAM HMM profile database of protein domains [45]. Signal peptide prediction In silico predictions of secreted proteins were carried out using a combination of SignalP 3.0, TargetP 1.1 and TMHMM 2.0 [44,78,79]. The SignalP algorithms incor- porate a cleavage site and signal peptide prediction based on artificial neural networks (NN) and hidden Markov models (HMM). In order to support the SignalP results and exclude proteins with either mitochondrial targeting peptides or transmembrane domains, protein sequences were also entered into different prediction servers. TargetP is a neural networks server that predicts the subcellular localization of eukaryotic proteins based on the presence of any of the N-terminal presequences, either chloroplast transit peptide (for plant predictions), Page 12 of 16 mitochondrial targeting peptide or secretory pathway signal peptide, while TMHMM uses hidden Markov models for the prediction of transmembrane helices. Following predictions, the output files were manipulated to select signal peptides containing sequences using the following criteria: (1) positive SignalP-HMM Sprob score, (2) positive SignalP-NN Smax and D scores, (3) TargetP signal peptide prediction, and (4) no transmem- brane domains. SignalP-HMM Sprob score was selected because of its ability to discriminate between N-terminal signal peptides and N-terminal signal anchors, while SignalP-NN Smax and D scores are the most accurate single scores [80]. Furthermore, because TMHMM may not distinguish signal peptides from transmembrane domains, deduced proteins with a single transmembrane domain within 40 amino acids of the N-terminus were also considered as potential secreted proteins. SAGE analyses The SAGE method was used as initially described in Velculescu et al. [81,82] at the CHUQ Research Centre (CRCHUQ) SAGE Platform, Quebec City, QC, Canada http://www.crchuq.ulaval.ca/plateformes/gpb. Fungal materials from M. larici-populina strain Mlp Berth. 3729 were harvested from both susceptible (P. × jackii) and resistant (P. trichocarpa × P. deltoides ’Boelare’) hosts at two different time points (22 hours and 5 days) after inoculation. Additionally, germinating uredinios- pores and germ tubes were collected 2 hours after inoculation on the susceptible cultivar by painting leaves with 5% cellulose acetate (dissolved in acetone), letting the acetone evaporate, and stripping the cellulose acet- ate film off the leaves. For each of the five treatments, 50 micrograms total RNA were extracted using the RNeasy mini kit (Qiagen, Valencia, CA, USA) and poly (A) RNA isolated with the mRNA Mini kit (Qiagen), annealed with the biotin-50-T18-30 primer, and converted into cDNA using the cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The resulting cDNA were digested with NlaIII (anchoring enzyme), and the 3′ restriction fragments were isolated with streptavidin- coated magnetic beads (Dynal Biotech, Oslo, Norway) before being separated into two populations. Each population was ligated to one of the two annealed linker pairs and washed to remove unligated linkers. The tag beside the most 3′ NIaIII restriction site (CATG) of each transcript was released by digestion with BsmFI (tagging enzyme). The blunting kit from Takara Co. (Otsu, Japan) was used for the blunting and ligation of the two tag populations. The resulting ligation products containing the ditags were amplified by PCR with an initial denaturation step of 1 min at 95°C, followed by 22 cycles of 20 sec at 94°C, 20 sec at 60°C and 20 sec at 72°C with 27 bp primers. The PCR products were then