Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 html will reveal further information on the whole reper- toire of secreted proteins in this pathogen. Comparative genomics studies with other biotrophs should elucidate molecular mechanisms underlying common strategies to infect plants. The identification of host targets will pro- vide further insight into the evolutionary forces that shaped the rust secretome, a key step facilitated by the availability of the poplar genome sequence [70] and transcript profiling of poplar-rust interactions [55,71,72]. This pathosystem clearly represents an unprecedented opportunity to understand the particularities of host- pathogen interactions. Methods Rust and plant material cDNA libraries were constructed from ex planta mate- rial (resting and germinating urediniospores, germ tubes, etc.) of four different Melampsora taxa. Fungal materials from isolates of the North American subspe- cies M. medusae f. sp. deltoidae and the Eurasian spe- cies M. larici-populina were harvested from naturally infected leaves of eastern cottonwood (Populus del- toides) and hybrid poplar (P. balsamifera × P. maximo- wiczii) clones, respectively. Melampsora medusae f. sp. tremuloidae and M. occidentalis mono-uredinial cultures were used to inoculate fresh leaves of P. tremuloides and P. trichocarpa, respectively. Inoculated leaves were maintained for 13 days in a growth chamber at 60% humidity, 19°C and 16 h photoperiod. In addition, we generated one additional haustorium-enriched library (biotrophic stage) for the M. larici-populina species. Haustoria were isolated by affinity chromatography as described by Hahn and Mendgen [73]. A mixture of plant leaf and fungal tissues was collected 6 days after inoculation of a rust-susceptible P. × jackii clone 1014 with the rust strain Mlp Berth. 3729. A 100-μm pore size nylon mesh was used to remove the bulk of the plant cell material from the crude preparation, which was then passed through an 11-μm pore size nylon mesh to remove intact plant cells. An affinity column was prepared by covalently attaching concanavalin A (Pharmacia Biotech) to cyanogen bromide-activated Sepharose 6 MB (Pharmacia Biotech) as described in the manufacturer’s protocol. Samples of purified haustoria colored using Calcofluor white (50 μM final concentra- tion) were then examined using a fluorescence micro- scope under UV filters. cDNA libraries and DNA sequencing Haustoria samples were pelleted by centrifugation at 20,800 g, washed twice with sterile distilled water, and maintained at -80°C until total RNA extraction. Fungal material was directly frozen in liquid nitrogen and ground using the Mixer Mill MM 300 with 2 mm Page 11 of 16 tungsten carbide beads. All the following manipulations were performed according to the manufacturer’s instruc- tions. Total RNA was extracted from ex planta material and purified haustoria using the RNeasy mini kit (Qia- gen, Valencia, CA, USA) and the Absolutely RNA® mini- prep kit (Stratagene, La Jolla, CA, USA), respectively. PolyA RNA was purified using biotylinated oligo-dT/ streptavidin-coated magnetic beads (Dynabeads® Oligo (dT)25; Dynal Biotech, Oslo, Norway). Haustorium- enriched M. larici-populina as well as M. medusae f. sp. tremuloidae and M. occidentalis ex planta cDNA libraries were generated using the SMART™ cDNA library construction kit (Clontech, Mountain View, CA, USA). Melampsora larici-populina and M. medusae f. sp. deltoidae ex planta cDNA libraries were constructed using the pBlueScript II XR cDNA library construction kit (Stratagene) according to the manufacturer’s instruc- tions. Following the column sepharose chromatography step included in the protocol, only size fractions above 500 bp were retained for ligation in the SfiI-digested, dephosphorylated pDNR-LIB vector. Plasmid ligations were transformed by electroporation into E. coli Electro- MAXTM DH10BTM Cells (Invitrogen, Carlsbad, CA, USA). After library amplification and tittering, individual colonies were transferred onto 384-well microtiter plates containing LB medium with 30 μg/ml chloramphenicol for PCR amplification and sequencing. cDNA inserts were amplified according to the manufacturer’s PCR protocol and then sequenced with the M13 forward pri- mer (5′-GTAAAACGACGGCCAGT-3′) using the Big Dye Terminator Cycle Sequencing Kit v1.1 on an ABI 3730xl sequencer (Applied Biosystems) at the CHUQ Research Centre (CRCHUQ) Sequencing and Genotyp- ing Platform, Quebec City, QC, Canada. Sequences were deposited at NCBI under accession numbers GW672673 to GW687576. cDNA assembly and ORF prediction Raw sequences were trimmed and cleaned using the Phred software [74], which resulted in the identifica- tion and removal of poor quality regions (quality cut- off of 20). Cross-match was then used to mask vector sequence in each read (minimum match of 10, mini- mum score of 20). The extent of redundancy for each library was ascertained using the Phrap software (Phil Green, http://www.phrap.org), which was also used to compile the unisequence set (minimum match of 50, minimum score of 100). In order to identify and remove plant sequences in the ESTs, unisequences were used in BLAST comparisons against the Populus trichocarpa genome and predicted gene models. Initial ORF prediction for Melampsora spp. was generated with the bestORF algorithm (Softberry) using Ustilago parameters.