Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 somewhat similar number of genes encoding putative secreted proteins when compared to other libraries, their level of expression, as reflected by the percentage of identified sequences, was quite lower (Additional File 4). Secreted proteins accounted for 25% of ex planta libraries (from 15.3 to 33.0%), while it was only 6.8% for the haustorium-enriched library. Secretory activity among obligate biotrophs thus appears limited and strongly regulated. A strict control of the secretory activity is required to form the interface layers that are observed in biotrophic interactions [59] and this could be a component of the pathogen’s strategy to evade recognition by host factors [19]. Evolutionary constraints of the poplar leaf rust secretome: adaptive evolution None of the effectors described from various fungi are known to have close homologues beyond species or genus boundary [27]. In order to determine if the evolu- tionary model of poplar leaf rust S+ proteins was con- fined to the presence/absence pattern observed above, we generated new BLASTP searches using 6033 (5349 NS/684 S+) predicted ORFs against the translated gene models of P. graminis f. sp. tritici, and plotted the per- centage of identity of each dataset to their closest homologues (Figure 3). Around 40% of each dataset had homologues in the P. graminis f. sp. tritici genome, a proportion that could be explained by the relatively short length of many ORFs, the presence of false ORFs predicted from non-coding regions such as UTRs, and the relative divergence expected between these genera [60]. However, approximately half of NS-predicted ORFs had high identity values (more than 70%) with their clo- sest homologue from the P. graminis f. sp. tritici gene models compared to only 7% of S+-predicted ORFs, thus suggesting that the secretome is under different evolutionary constraints. Again, fast evolution of coding regions due to molecular arms races between pathogens and hosts might explain the high divergence between orthologues or the absence of close homologues in related species. We used reciprocal TBLASTX searches (E-value ≤ 1e- 30) among our taxonomic cDNA libraries to identify orthologues and/or paralogues in different Melampsora species libraries and to classify them into homologous gene groups (HGGs). In order to increase the number of HGGs, we included the gene models from P. graminis f. sp. tritici. We found 369 HGGs consisting of at least three different sequences with a minimum of two Mel- ampsora unisequences retrieved. These HGGs included 1159 Melampsora unisequences and 437 P. graminis f. sp. tritici gene models. HGGs were divided to 283 non- secreted HGGs (76.7%) and 86 secreted HGGs (23.3%) according to their predicted localization. Consistent Page 7 of 16 Figure 3 Accelerated sequence divergence of secreted proteins between predicted ORFs from the Melampsora unisequence dataset and the Puccinia graminis f. sp. tritici gene models. The set of Melampsora predicted ORFs (2319 non-secreted ORFs, represented by white circles; and 267 secreted ORFs, represented by red circles) was compared with translated gene models of Puccinia graminis f. sp. tritici. Each radius ranges from 0 (center) to 100 (outer circle), representing BLASTP percent identity. A BLASTP hit was considered significant when E-value ≤ 1e-4. Position of ORFs along the circumference is random. with the above similarity searches, approximately 45% of secreted HGGs had no P. graminis f. sp. tritici homolo- gues (at E-value ≤ 1e-30), compared with 12% for non- secreted HGGs. Moreover, the proportion of secreted HGGs (23.3%) was twice the proportion of S+ unise- quences (11.4%), and the mean number of Melampsora unisequences per HGG was slightly higher in secreted HGGs (4.3 unisequences/HGG) compared with non- secreted HGGs (2.9 unisequences/HGG). These results could suggest that a larger proportion of allelic forms and/or paralogue families exist among the rust secre- tome, which is consistent with an extensive sequence diversification motivated by the coevolutionary arms race [30]. In order to visualize the evolutionary relationships between Melampsora and other fungi we used SimiTri [61], which plots in two-dimensional space the relative similarities of gene sequences between one group (Melampsora) and three comparators. For each sequence included in an HGG, a BLASTX was performed against three other basidiomycetes for which genome sequences were available: Puccinia (Pucciniomycotina; Pucciniomycetes), the wheat stem rust, which is phylogenetically close to Melampsora and shares a similar biotrophic lifestyle; Sporobolomyces