, 2006). Plant pathogenic oomycetes appear to have evolved a protein translocation system similar to malaria, which involves secreted proteins possessing an RxLR motif located after the signal peptide sequence (Bhattacharjee et al., 2006; Birch et al., 2006; Haldar et al., 2006; Whisson et al., 2007; Dou et al., 2008b). It was found that the RxLR motif is required for translocating these proteins into the host cells of infected plants (Whisson et al., 2007; Dou et al., 2008a). Bioinformatic analysis has shown that over 500 putative RxLR effectors are found in the potato pathogenic oomycete Phytophthora
infestans, and similarly, hundreds more in other plant pathogenic oomycetes (Whisson et al., 2007; Haas et al., 2009; Tyler, 2009). Antiinfection Compound Library cell assay It was demonstrated that the oomycete RxLR motif is functional in Plasmodium, where it can direct an RxLR–GFP fusion protein from the parasite into the host erythrocyte (Bhattacharjee et al., 2006). The PEXEL motif is also functional in P. infestans as it is able to translocate an avirulent chimaeric PEXEL-PiAvr3 protein into PiAvr3-recognizing potato plants (Grouffaud et al., 2008). Replacement of the N-terminal region of the effector protein PsAvr1b with a PEXEL motif selleck chemicals containing leader sequences of three Plasmodium effectors resulted in the translocation of chimaeric
PsAvr1b into the Bacterial neuraminidase soybean cytoplasm (Dou et al., 2008a). Before detailed molecular interaction studies between Saprolegnia and fish can be performed, it is essential to develop a suitable infection model. The ami-momi treatment
established, which involves shaking fish in a net for approximately 2 min to remove part of the mucosal layer and subsequently challenging with Saprolegnia zoospores (Hatai & Hoshiai, 1993), is a good method to characterize the virulence of S. parasitica strains (Stueland et al., 2005). However, it is not a suitable model to study the early cellular and molecular infection mechanisms and events. In order to study these in more detail, the development of a fish cell-line infection assay is necessary. Here, we describe the identification and molecular characterization of a putative effector protein, SpHtp1, containing an RxLR motif. Microscopic studies of a Saprolegnia–fish interaction using an in vitro system involving a rainbow trout cell line showed that SpHtp1 is translocated into the fish cells, also when applied exogenously. A Saprolegnia parasitica isolate CBS223.65, obtained from the Centraal Bureau voor Schimmelcultures (CBS), the Netherlands, was grown on potato dextrose agar (Fluka) for 5 days at 18 °C, before inoculation in pea broth (125 g L−1 frozen peas, autoclaved, filtered through cheese cloth, volume adjusted to 1 L and autoclaved again) and incubation for 2 days at 24 °C. To accomplish S.