, 1999), although this see more has not been demonstrated for the elicitin 6 precursor proteins identified by our blast analysis. However, SpHtp1 aligns only with the C-terminal region of the elicitin 6 precursor proteins identified by the blast analysis, containing an xPTx repeat region, and not with INF1, which is the highly expressed elicitin in mycelium stages from P. infestans (Kamoun et al., 1997) (Fig. S3). Moreover, blast of SpHtp1 against INF1 results in an E-value of 8.7. interpro analysis shows that the

xPTx repeat region is observed in a variety of proteins; however, it is not known whether they are homologous to each other and no specific function of this repeat region has been identified so far. In vitro transcript analysis showed that SpHtp1 is expressed in all life stages of S. parasitica, but compared with the transcript levels in mycelia, SpHtp1 transcripts are more abundant in zoospores/cysts and germinating cysts when normalized to transcript levels of the reference gene SpTub-b (Fig. 1c). In the RTG-2 model system, it was observed that SpHtp1 transcript

levels were very high at time point 0, representing the addition of the zoospores/cysts as an inoculum source. A decrease over time was observed, representing germination and subsequent mycelial growth (Fig. 1c). Similar results were obtained when other reference genes were used. However, the SpTub-b transcript levels showed the lowest variation between the life stages (Fig. S4). These results selleck chemicals indicate that SpHtp1 is predominantly expressed in the preinfection stages and in the very early stages of infection. To investigate whether SpHtp1 is secreted and where the protein is located during the infection of S. parasitica

on RTG-2 cells, a final bleed polyclonal antiserum was generated that was directed against a peptide of the SpHtp1 sequence (Fig. 1a). Western blot analysis showed that the Bcl-w antiserum recognized SpHtp1 synthesized in E. coli and a protein band of the same size in a protein fraction isolated from infected RTG-2 cells (Fig. S5). Several other bands in the protein samples isolated from uninfected and infected RTG-2 cells were recognized by the final bleed polyclonal antiserum, but these were also detected with the preimmune antiserum. Subsequent fluorescent immunolocalization studies on S. parasitica-infected RTG-2 cells resulted in SpHtp1 detection inside fish cells, surrounding the host nucleus, that are in close contact with the S. parasitica hyphae (Figs 2 and 3). This localization pattern was neither observed in infected RTG-2 cells treated with only preimmune antiserum nor in uninfected RTG-2 cells treated with preimmune or the final bleed polyclonal antiserum (Fig. 2), thereby demonstrating that the immunolocalization pattern in the infected cells of RTG-2 is only derived from translocated SpHtp1. Z-scans of fish cells that are in contact with hyphae from S.