After an overnight incubation, zoospores and cysts were collected. Germinating cysts were collected after vortexing the zoospore/cyst suspension and incubation at 24 °C for 4–5 h. The RTG-2 cell line is a continuous cell line obtained from ATCC (ATCC CCL-55). It was derived from rainbow trout (Oncorhynchus mykiss) gonadal tissue (Wolf & Quimby, 1962) and was maintained at 24 °C in 75-cm2 cell culture flasks (Nunc) in 25 mL Leibovitz’s L-15 medium (Gibco) supplemented with 10% foetal bovine serum (BioSera), 200 U mL−1 penicillin and

200 μg mL−1 streptomycin (Fisher). Flasks with confluent cell growth were inoculated weekly after splitting cells by washing three times with Hank’s balanced salt solution (Gibco) at room temperature and treating the cells with 5 mL 0.5 g L−1 trypsin–EDTA (Invitrogen) until the cells were detached from the flasks. A fresh medium Pembrolizumab mouse was added, and after gentle shaking, the cells were distributed into three to five flasks, each containing approximately 30 mL of cell suspension, or 2–4 mL was added to each well of six-well plates Raf targets (Nunc), where the wells contained an autoclaved glass coverslip. RNA was isolated from the preinfection stages of S. parasitica strain CBS223.65, including zoospores, cysts and germinating cysts, at Vertis Biotechnology AG (Germany), using a Trizol-based extraction. From total RNA, polyA+ was prepared and cDNA was synthesized according to the Vertis Biotechnology

Anacetrapib AG standard protocol for full-length enriched cDNA using an oligo(dT)-NotI primer for first-strand synthesis. Before cloning, the cDNA was amplified with 13 cycles of PCR. For directional cloning, cDNA was subjected to a limited exonuclease treatment to generate EcoRI overhangs at the 5′ end, and was subsequently digested with NotI. Size-fractioned cDNA fractions >0.5 kb were ligated into EcoRI and NotI digested pcDNA3.1 (Invitrogen) and subsequently transformed via electroporation into T1 phage-resistant TransforMax™ EC100™-T1R electrocompetent cells (Epicentre Biotechnologies). The transformants were stored in 15% v/v glycerol at −80 °C. End-sequencing was performed on plasmid

DNA isolated from 1000 clones of the cDNA library by a single pass sequence from the 5′ end with a primer specific for the pcDNA3.1 vector by GATC Biotech (Cambridge, UK) using an ABI3730 system. The EST sequences were trimmed to remove vector sequence and validated using seqclean (http://compbio.dfci.harvard.edu/tgi/software/), and subsequently, contigs were assembled using cap3 (http://pbil.univ-lyon1.fr/cap3.php). Screening for secreted proteins was performed by signalp (http://www.cbs.dtu.dk/services/SignalP/) analysis using both hidden markov models and neural networks programs, and subsequently, the sequences were screened by word searches for the presence of an RxLR motif. blastp analyses were performed at the NCBI website (http://blast.ncbi.nlm.