Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 species metabolic process (GO:0006800) and carbohy- drate metabolic process (GO:0005975). Apart from puta- tive effectors, members of the secretome had homology to a battery of glycoside hydrolases and subtilisin-like serine proteases that likely contribute to the penetration of the plant cuticle and cell wall [47]. In fact, glycosyl hydrolase 16 (PF00722) was the second most repre- sented PFAM domain in the secretome, followed by domains typically found in proteolytic enzymes (Pepti- dase_S8 [PF00082], Subtilisin_N [PF05922] and Asp [PF00026]) (Additional File 1). A class of secreted pro- teins exhibiting the ability to neutralize reactive oxygen species (ROS), and including Mn and Cu/Zn superoxide dismutases, was also uncovered by this survey. This finding was not surprising as it is known that rust fungi prevent a variety of non-specific defense responses in invaded cells, thus allowing the establishment of the long-term biotrophic relationship between rust fungi and living host cells [51]. Such host responses frequently involve the production of ROS, whose detoxification is essential for the establishment of the pathogen. A Mn superoxide dismutase homologue had previously been reported in the haustorial stage of Puccinia triticina [52] and was differentially-expressed in Uromyces appendicu- latus germlings during early appressorium development [53]. Concordant with these observations, previous stu- dies have demonstrated the upregulation of several host genes encoding enzymes of the redox regulation path- ways during Populus-Melampsora interactions [54,55]. Another group of interesting secreted proteins identified here that may be critical for evading host recognition or protecting fungal cell wall from hydrolysis by host enzymes were chitin deacetylases, which have already been described in libraries from Phakopsora pachyrhizi [56] and P. triticina [52]. The conversion of chitin into chitosan by de-N-acetylation not only protects fungal infection structures from hydrolytic attack by chitinases present in the host tissue, it also prevents the release of chitin oligomers responsible for the triggering of resis- tance reactions [57]. This functional annotation established the secretome’s ability to perform diverse roles in pathogenicity and interactions with host cells. However, for the major part of the secretome, no GO or PFAM domains could be assigned. For 441 candidates, i.e. 64% of the S+ dataset, no significant similarity was found to known proteins in the UniProtKB database (E-value > 1e-4) (Table 2). To ascertain that the large number of unmatched proteins identified in this study was not due to the paucity of Pucciniales (or even Basidiomycota) sequences in inter- national databases [52], we constructed two specific databases (BasidiomycotaDB and PuccinialesDB). BLASTX searches of the complete dataset of Melamp- sora unisequences (S+ and NS) were performed against Page 5 of 16 these databases. The percentage of unisequences with homologues was similar between BasidiomycotaDB and UniProtKB, indicating that the latter is not deficient in Basidiomycota sequences (Table 2). As previously observed with BLASTX searches, the percentage of S+ unisequences with homologues in both Basidiomyco- taDB and PuccinialesDB was generally lower compared to NS. These results are consistent with the view that secreted effector proteins that subvert host-cell structure and functions often show very limited phylogenetic dis- tribution and no obvious conserved motifs, being less evolutionarily conserved. Furthermore, the percentage of homologues for the haustorium-enriched library was surprisingly low considering that the whole gene set of another rust, Puccinia graminis f. sp. tritici, was included in the database. However, these results were comparable to observations made on the haustorial secretome of other rust fungi [26,58]. Most of the pro- teins secreted from the haustorium could be under rapid evolution and strong diversification due to host selective pressures and, therefore, be species-specific. Taken together, these results strongly indicate that a large portion of the secretome (and especially the haus- torial secretome) of Melampsora may have virulence functions or be involved in host-pathogen interactions. Expression profiles of the poplar leaf rust secretome: stage-specificity Most abundantly represented members of the secretome had no evident homologues in the UniProtKB database (Additional File 2), or even in PuccinialesDB. However, some highly expressed candidates showed similarities to sequences suspected to be involved in pathogenicity or host-pathogen interactions (HESPs and CFEM-contain- ing proteins). To obtain an accurate overview regarding the expression profiles of the secretome, we associated S + members to 15-bp tags from a serial analysis of gene expression (SAGE). We analyzed expression patterns at three time points during the infection process of M. lar- ici-populina: twice during the pre-biotrophic stage (2 and 22 hours after inoculation), and once during the biotrophic stage (5 days after inoculation) (Feau et al., unpublished). The last two time points were also investi- gated on both susceptible and resistant hosts, i.e. during compatible and incompatible interactions. A quarter (182 unisequences) of the S+ members was confidently associated to a SAGE tag. An average linkage hierarchi- cal clustering of SAGE patterns revealed strong stage specificities of the secretome (Figure 2), similar to observations made on U. fabae genes initially identified using the yeast signal sequence trap [58]. Genes expressed during pre-biotrophic growth were clearly turned off at later biotrophic stages, while others were induced only during the biotrophic stage. Global