A31627) according to the manufacturer’s protocol. After washing three times with PBS, the immunostained coverslips were viewed using a Zeiss LSM 510 Meta

confocal microscope with a Plan Apochromat × 63/1.0 water-dipping objective lens. For the uptake experiment of recombinant SpHtp1 proteins, RTG-2 cells were washed three times with HBSS before a 20–30-min incubation with 20 μM recombinant SpHtp124-198(His)6 protein in L-15 medium containing 10% FCS. After washing cells three times with PBS, they were fixed as described above. Fixed cells were washed three times with PBS, permeabilized for 15 min with PBS containing 0.1% Triton-X 100 and washed again three times before incubation with the primary penta-His antibody at 37 °C for 1 h (Qiagen, No. 34660; titre 1 : 300). selleck compound Subsequently, the samples were washed three times with PBS, and incubated Trichostatin A chemical structure at 37 °C for 1 h with the secondary antibody [fluorescein

isothiocyanate (FITC) 488-conjugated goat-anti-mouse immunoglobulin G; Jackson ImmunoResearch] according to the manufacturer’s protocol. The immunostained coverslips were washed again three times with PBS and mounted onto microscope slides. Microscopy was carried out using a Zeiss LSM 510 META confocal microscope (Fluor 488/FITC: excitation is 488 nm, filter settings BP 505-530; detector gain 750. Iodide: excitation is 633 nm, filter settings LP 650; detector gain 540, all PI-1840 1 μm slices). In order to identify and investigate genes of S. parasitica that are expressed in the preinfection and early infection

stages, we set up a cDNA library from RNA isolated from zoospores, cysts and germinated cysts of S. parasitica and generated ESTs. End-sequencing of the cloned cDNA library and subsequent preliminary bioinformatic analysis resulted in the identification of a putative secreted protein with an RxLR motif located within the first 40 aa after the predicted signal peptide cleavage site. The ORF, SpHtp1 (S. parasitica host targeting protein 1), encodes a putative protein, SpHtp1, of 198 aa, of which the first 23 aa encode a signal peptide (Fig. 1a). The RxLR motif is located 22 aa downstream of the predicted signal peptide cleavage site, which is comparable to all known and characterized oomycete RxLR effector proteins (Fig. 1b, Fig. S1). Genome sequencing confirmed that the ORF is present in the genome and revealed an intron of 55 nt long, ranging from 74 nt up to 129 nt. Also, the oomycete conserved sequence motif in the promoter region of SpHtp1 was identified 35 nt upstream of the start codon (Pieterse et al., 1994; McCleod et al., 2004) (Fig. S2). blastp analysis of SpHtp1 yielded sequence similarity only to regions of low complexity in other proteins such as the elicitin 6 precursor proteins of Phytophthora medicaginis, Phytophthora ramorum and Phytophthora sojae (E-values 9e-19, 2e-16, 3e-16 and 52%, 68% and 51% identity, respectively).