Page 3 of 16 Table 2 Similarity of Melampsora unisequences to sequences from UniProtKB, BasidiomycotaDB and PuccinialesDB Library non-secreted proteins (NS). For 248 candidates, i.e. 36% of the S+ dataset, significant similarity was found in the UniProtKB database, mostly among putative effectors or proteolytic and carbohydrate-degrading enzymes from other fungal species (Table 2). Less than a quarter of the unisequences had similarity to one or more PFAM domain predictions or GO term assignments. The most abundantly represented PFAM domains in the poplar leaf rusts secretome are presented in Additional File 1, and GO classifications are depicted in Figure 1. Host infection by biotrophic fungi is believed to involve the secretion of effectors that suppress plant defenses and alter cellular metabolism to fulfill the requirements of the invading pathogen [47]. Among the candidates that had significant similarity to known sequences, homologies with potential effectors pre- viously identified in the haustoria of other rust species were observed. Interestingly, 22 candidates had similari- ties with eight “haustorially expressed secreted proteins” (HESPs) from Melampsora lini identified using a similar bioinformatics approach. Some of these HESPs were shown to co-segregate with known avirulence genes and are proven HR elicitors in flax, such as the avirulence protein AvrM [26]. The percentage of identity between flax rust and poplar leaf rust HESPs differed greatly. For instance, HESP-379 had close homologues in Melamp- sora spp. (identity: 93%), while AvrM appeared less con- served (identity: 26%). Other candidates had similarities with Rust Transferred Protein (RTP1) from Uromyces fabae, a small secreted protein that is specifically expressed in broad bean rust haustoria and translocated into host cells. This protein accumulates within the cytoplasm of the infected host cell and in the host nucleus, suggesting a role in influencing host gene expression [13]. Homologies with other proteins thought to contribute to pathogenesis were unravelled, with 19 unisequences having similarity to CFEM domain % with homologues in BasidiomycotaDB % with homologues in PuccinialesDB NS 21.5 54.1 54.7 38.3 49.3 S+ 13.9 50.0 45.8 23.2 37.2 NS 29.6 62.4 64.9 47.4 59.6 S+ 23.6 56.5 56.5 35.4 54.7 Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 algorithms (SignalP, TargetP and TMHMM), yielding 405 sequences encoding putative proteins with predicted secretory signal peptides (# Putative S, Table 1). A num- ber of sources exist that lead to false prediction, i.e. selecting individual proteins that should not be included in the secretome. For example, mitochondrial localiza- tion sequences and N-terminal transmembrane anchors are frequently interpreted as signal peptides [44]. Even though no computational method seems fully accurate, the prediction algorithms included in our approach should have excluded such false positives. The assign- ment of a protein to the secretome is also totally depen- dent on having the full-length open reading frame (ORF). An inaccurate ab initio gene prediction or an incomplete ORF could lack an additional sequence encoding a transmembrane domain or a signal peptide. For this reason, we conducted reciprocal blast between libraries to reduce the number of false assignments due to partial or mispredicted ORFs. Most reassignments were false negatives with truncated N termini, thus increasing the number of putative secreted members to 689 (# Putative S+, Table 1). Functional annotation of the poplar leaf rust secretome: novel proteins and the identification of orthologues This S+ dataset was used as search query in BLASTX on the non-redundant UniProtKB database to find sequences with significant matches. Furthermore, to develop an understanding of how protein secretion by poplar leaf rusts might be related to specialized func- tions or processes, we used the PFAM [45] and Gene Ontology (GO) [46] databases to determine whether any class of proteins was more likely to be found in the Mel- ampsora secreted proteins. In addition, we used the Gene Ontology methodology within a statistical framework to determine whether any GO terms were significantly enriched in S+ proteins when compared to % with homologues in UniProtKB NSa S+b M. larici-populina haustoria M. larici-populina ex planta material M. medusae f. sp. deltoidae ex planta material M. medusae f. sp. tremuloidae ex planta material M. occidentalis ex planta material 22.6 54.0 56.2 36.7 47.4 13.9 54.3 50.4 23.6 43.9 BasidiomycotaDB included Basidiomycota (excluding Pucciniales) EST sequence 6-frame translations and protein sequences from the non-redundant database of NCBI and gene models from Coprinus cinereus Okayama 7 (#130), Cryptococcus neoformans var. grubii serotype A, strain H99, Laccaria bicolor S238N-H82, Malassezia globosa CBS 7966, Phanerochaete chrysosporium RP78, Postia placenta MAD-698, Sporobolomyces roseus IAM 13481 and Ustilago maydis 521. PuccinialesDB included Pucciniales EST sequence 6-frame translations and protein sequences from the non-redundant database of NCBI and gene models from P. graminis f. sp. tritici CRL 75-36-700-3. BLASTX hits were considered significant when E-value ≤ 1e-4. aNS: Final set of unisequences not predicted to encode putative secreted proteins. bS+: Final set of unisequences encoding putative secreted proteins, following reassignments based on reciprocal BLAST.