Table 3 Characteristics of the Melampsora homologous gene groups (HGGs) predicted to be under positive selection (at least one pairwise ω = dN/dS > 1.2) HGG NS/Sa Length E-value PFAM Cys ωb BLASTX E-value BLASTX Fungi UniProtKB Puccinia 205 254 1278 28 92 729 747 4191 5606 5610 5617 5624 6067 NS NS NS S S S S S S S S S S 115 No hit 200-202 No hit 173-175 XP_001211022 conserved hypothetical protein [Aspergillus terreus] 161 No hit 271-276c (227)d AAS45284 proline-rich antigen [Chrysosporium lucknowense] 269 EAT81533 hypothetical protein [Phaeosphaeria nodorum] 163 XP_757360 hypothetical protein [Ustilago maydis] 129 No hit 119-123e No hit 98 No hit 168-169f No hit 176 No hit 285 XP_758577 hypothetical protein [Ustilago maydis] 3e-07 4e-09 7e-11 1e-04 6e-06 No hit PGTT03363 PGTT11073 No hit PGTT01207 PGTT12331 PGTT02151 No hit No hit No hit No hit No hit PGTT13234 1e-10 1e-34 2e-13 7e-61 2e-14 5e-21 - CFEM - - - - - - - - - - - Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 BLASTX and PFAM hits were considered significant when E-value ≤ 1e-4 and 1e-5, respectively. aNS: non-secreted; S: secreted. bHighest calculated dN/dS ratio among all pairwise comparisons within each HGG. cLength is 271 (M. medusae f. sp. deltoidae, M. medusae f. sp. tremuloidae and M. occidentalis), and 276 (M. larici-populina); an 86 amino acids insertion and a 5 amino acids minisatellite are also present in a M. medusae f. sp. deltoidae unisequence (length: 357) and a M. larici-populina unisequence (length: 281), respectively. dA 44 amino acids deletion (including 4 Cys residues) is present in a M. medusae f. sp. deltoidae unisequence. eLength is 119 (M. medusae f. sp. tremuloidae) and 123 (M. occidentalis). fA 12 amino acids minisatellite is present in a M. occidentalis unisequence (length: 181). gAdditional Cys residues are present before the signal peptide cleavage site. were absent from Sporobolomyces, one secreted HGG (92) was absent from Ustilago, one secreted HGG (729) was present only in Puccinia, and the remaining HGGs (6 secreted and 2 non-secreted HGGs) had no hit. Two other groups of sequences identified using FEL statistics [68] had corresponding HGGs plotted on the line between Puccinia and Ustilago (absent from Sporobolo- myces) (data not shown). Conclusion Database searches with sequences of small secreted pro- teins from fungi commonly do not yield homologues or known protein domains, the only recognizable features being the presence of a signal peptide for secretion and, in many cases, an even number of cysteine residues. Despite these commonalities, effectors appear to be evo- lutionarily diverse and highly variable in their distribu- tion, showing very limited phylogenetic distributions possibly due to accelerated evolution stimulated by plant-pathogen arms races [27]. In a straightforward in silico approach, we generated a first overview of the secretome from poplar leaf rusts belonging to the genus Melampsora, unravelling an unknown and diversifying set of genes. The identification of positive selection in putative secreted proteins reported here suggests that these genes are likely to encode candidate effectors implicated in host-pathogen interactions. Such informa- tion should be used to augment other selection criteria (such as gene expression data) for prioritizing candidate effector genes for functional studies. Two intriguing properties of rust fungi are their host specificity and their need for host alternation. Even though host specifi- city is probably controlled at several levels, examples from the flax rust fungus suggest that the secretion of effectors plays a prominent role [26]. Their intimate interactions with host factors expose them to very strong selective pressures resulting in their rapid evolu- tionary turnover. However, poplar leaf rust effectors not only cope with the poplar defense machinery, they also face another phylogenetically diverse host plant, which differs between species, from other dicots to monocots and even gymnosperms. Is this particularity responsible for a greater sequence diversification? Or is it responsi- ble for a broader arsenal of effectors in poplar leaf rusts? The recent completion of the M. larici-populina genome http://genome.jgi-psf.org/Mellp1/Mellp1.home. Page 10 of 16 ND ND ND 0 6 (2)d 8 4 4 6 6g 0g 8 10g 2.68 2.59 1.88 1.38 1.43 1.32 1.95 1.57 1.76 4.31 1.23 1.93 1.27