Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 R E S E A R C H A R T I C L E Open Access Comparative analysis of secreted protein evolution using expressed sequence tags from four poplar leaf rusts (Melampsora spp.) David L Joly1, Nicolas Feau1,3, Philippe Tanguay1, Richard C Hamelin1,2* Abstract Background: Obligate biotrophs such as rust fungi are believed to establish long-term relationships by modulating plant defenses through a plethora of effector proteins, whose most recognizable feature is the presence of a signal peptide for secretion. Since the phenotypes of these effectors extend to host cells, their genes are expected to be under accelerated evolution stimulated by host-pathogen coevolutionary arms races. Recently, whole genome sequence data has allowed the prediction of secretomes, facilitating the identification of putative effectors. Results: We generated cDNA libraries from four poplar leaf rust pathogens (Melampsora spp.) and used computational approaches to identify and annotate putative secreted proteins with the aim of uncovering new knowledge about the nature and evolution of the rust secretome. While more than half of the predicted secretome members encoded lineage-specific proteins, similarities with experimentally characterized fungal effectors were also identified. A SAGE analysis indicated a strong stage-specific regulation of transcripts encoding secreted proteins. The average sequence identity of putative secreted proteins to their closest orthologs in the wheat stem rust Puccinia graminis f. sp. tritici was dramatically reduced compared with non-secreted ones. A comparative genomics approach based on homologous gene groups unravelled positive selection in putative members of the secretome. Conclusion: We uncovered robust evidence that different evolutionary constraints are acting on the rust secretome when compared to the rest of the genome. These results are consistent with the view that these genes are more likely to exhibit an effector activity and be involved in coevolutionary arms races with host factors. Background Rust fungi or Pucciniales (= Uredinales) represent the largest group of fungal plant pathogens, including more than 7000 species that possess the most complex life cycles in the Kingdom Fungi [1]. Some of these obligate biotrophs have been of long standing concern for agri- culture and forestry while others have emerged in recent epidemics. For instance, poplar leaf rusts belonging to the genus Melampsora are considered as the world’s most important disease of poplars [2]. Selection for dur- able resistance to these pathogens is thus an important challenge for poplar breeders [3]. Although poplar breeding programs have been in place for decades in * Correspondence: richard.hamelin@ubc.ca 1Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du PEPS, P.O. Box 10380, Stn. Sainte-Foy, Québec, QC, G1V 4C7, Canada Europe, clones selected for complete resistance against rust have increasingly succumbed in time to new races of Melampsora larici-populina [4]. Sustainability of newly selected resistance clearly requires a better understanding of the molecular mechanisms involved in Populus- Melampsora interactions. Prokaryotic and eukaryotic plant pathogens have evolved highly advanced strategies to engage their hosts in intimate contacts and deliver suites of effector pro- teins to modulate plants’ innate immunity and enable parasitic colonization [5-12]. Understanding the translo- cation mechanisms of bacterial pathogen effectors inside host cells has been an outstanding breakthrough with the characterization of the type III secretion system. This export apparatus enables a bacterium to manipu- late host cellular processes by injecting effector proteins into the host cytoplasm [7]. Similarly, plant-parasitic © 2010 Joly et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.