, 2006; Kamoun, 2006; Dou et al., 2008b). Therefore, it is possible that the EER motif or the amino acids in the region located after the RxLR motif in Atr13 and SpHtp1 play an important role in the folding of these proteins and thereby targeting specific recognition sites in the host. Related to this, positive selection was found to have acted mostly on the C-terminal region of RxLR proteins, which ABT-263 purchase is consistent with the view that the N-terminal RxLR–EER region functions as a translocation signal and that it is not
required for effector activity (Morgan & Kamoun, 2007; Win et al., 2007). An exception to the recognition of the C-terminal part of oomycete effectors by cognate resistance genes in plants comes from the recently published P. sojae PsAvr4/6 effector, where the RxLR–EER region is recognized by Rps4 from soybean (Dou et al., 2010). Transcript analysis showed that SpHtp1 is mainly expressed in the zoospores/cysts and in the onset of the challenge of the RTG-2 cell line, corresponding to the zoospore/cysts stage. Transcript analysis of 38 predicted RxLR–EER effectors from P. infestans KU-60019 order showed various expression patterns: ‘predominantly upregulated in preinfection only; predominantly
in preinfection and biotrophy; preinfection and throughout infection; biotrophy only’ (Whisson et al., 2007). Also, seven genes encoding RxLR proteins lacking the EER motif conformed
to the profiles seen for the RxLR–EER effectors (Whisson et al., 2007). Although no time point 0 was included in their analysis and it is therefore not possible to compare that time point with our results, the expression profile of SpHtp1 would fit best in the group of preinfection only. Translocated SpHtp1 was detected inside the host cells, using an antiserum directed against a peptide of SpHtp1 (Figs 2 and S5), which has not been shown for any other oomycete RxLR effector thus far. In a P. infestans transformant expressing many recombinant Avr3a-mRFP, the RxLR protein localizes in the haustoria and the extrahaustorial matrix of infected potato leaves. However, translocation inside the infected plant cells was only observed in a recombinant Avr3a-GUS-expressing transformant (Whisson et al., 2007). Moreover, substitution of the RxLR and EER residues with alanine abolished Avr3a-GUS translocation inside the plant cells (Whisson et al., 2007). Here, we show that recombinant and exogenously applied SpHtp1 is also taken up into the fish cells (Fig. 4). Furthermore, Dou et al. (2008a) showed that recombinant PsAvr1b from P. sojae can also be translocated into nonhost onion cells, suggesting that the RxLR translocation mechanism is used by both plant and animal pathogenic oomycetes.