Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 nematodes have developed diverse relationships to obtain nutrients from their host plants. Perhaps their most evolutionary sophisticated adaptations are effector proteins encoded by parasitism genes expressed in oeso- phageal gland cells and secreted through a protrusible feeding spear, called a stylet [5,6]. However, still little is known about such translocation machineries in filamen- tous pathogens (mainly fungi and oomycetes), although another specialized biotrophic infection cell called the haustorium is thought to be involved [13-15]. The haus- torium invaginates host cells and makes near-direct con- tact with the host plasma membrane, where it plays a crucial role in nutrient acquisition. This structure is a regulatory hub involved in the manipulation of host metabolism and the suppression of host defenses, which allows the establishment of a successful biotrophic rela- tionship [16-19]. The concomitance of haustoria forma- tion with the induction of a programmed cell death response termed the hypersensitive response (HR) sug- gests a significant role for this structure in delivering effector proteins into the infected host cell [14,20]. Key insights have emerged from the recent identifica- tion of filamentous pathogen effectors with avirulence activity inducing plant defense responses and HR [21-26]. Most of the avirulence genes identified encode small proteins with N-terminal signal peptides that direct them through the endoplasmic reticulum secre- tory pathway [14,27]. While effector genes reside in pathogen genomes, their products essentially generate phenotypes that extend to host cells and tissues, and are hence likely to be the direct target of the never-ending coevolutionary conflict between host and pathogen [28,29,36]. In fact, avirulence proteins recognition by plant resistance proteins imposes selection against effec- tor function, and pathogen effector proteins probably overcome resistance through diversification of the genes encoding them [30]. For instance, several avirulence genes or their plant counterparts display molecular hall- marks of positive selection [21,22,25,30-38]. Recently, the availability of filamentous plant pathogen genome sequences facilitated the cataloguing of whole secre- tomes using computational analyses, thus allowing the identification of putative effectors [39-43]. Indeed, Tyler Page 2 of 16 et al. [41] provided evidence that secreted proteins have been subject to accelerated evolution by contrasting the genome sequences of Phytophthora ramorum and Phy- tophthora sojae. Here we provide an overview of the expressed secre- tome of poplar leaf rusts belonging to the genus Mel- ampsora. We constructed cDNA libraries for four related poplar leaf rust pathogens with different host specificities to test for the signature of selection in these rust secretomes and used computational tools to anno- tate putative secreted proteins. A comparative genomics approach based on homologous gene groups (HGGs) was undertaken to evaluate the extent to which secre- tome members are under different evolutionary con- straints. We describe adaptive evolution (positive selection) in genes encoding secreted proteins of poplar leaf rusts, in agreement with the idea that these genes are expected to display an effector activity and be involved in the escalating and reciprocal coevolutionary arms race with host resistance factors. Results and Discussion Defining the poplar leaf rust secretome We constructed cDNA libraries from ex planta material of four different Melampsora taxa with different host specificities: the Eurasian M. larici-populina, the North American M. occidentalis, and two formae speciales of the North American M. medusae, M. medusae f. sp. del- toidae and M. medusae f. sp. tremuloidae (Table 1; see Methods). This allowed the comparison of expressed sequence tags (ESTs) with their putative orthologues and made it possible to incorporate evolutionary infor- mation into our analyses. In order to extend our dataset with candidate effectors expressed in and secreted from haustoria, we generated an additional haustorium- enriched library (biotrophic stage) of M. larici-populina. In total, 14,904 clones were sequenced in this study, which represented 6,044 unique sequences (unise- quences, i.e. all contigs and singletons). Clone sequences are available under GenBank accession numbers GW672673 to GW687576. The identification of tran- scripts coding for secreted proteins was carried out using an in silico analysis including a series of prediction Table 1 Summary of Melampsora cDNA libraries characteristics Species/Natural host M. larici-populina/Populus nigra M. larici-populina/P. nigra M. medusae f. sp. deltoidae/P. deltoides M. medusae f. sp. tremuloidae/P. tremuloides M. occidentalis/P. trichocarpa Material Haustoria Ex planta Ex planta Ex planta Ex planta 1148 787 937 1547 1625 aS: Initial predicted set of unisequences encoding putative secreted proteins. bS+: Final set of unisequences encoding putative secreted proteins, following reassignments based on reciprocal BLAST. Unisequences Contigs Singletons Putative Sa (%) Putative S+b (%) 427 266 452 571 493 721 521 485 976 1132 54 (4.7) 51 (6.5) 95 (10.1) 98 (6.3) 107 (6.6) 72 (6.3) 92 (11.7) 131 (14.0) 246 (15.9) 148 (9.1)