gov/Blast.cgi), and the search for specific domains was performed using interproscan (http://www.ebi.ac.uk/Tools/InterProScan/). Alignments were generated using clustalx
1.81 or clustalw (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The sequence of the SpHtp1 has been deposited in GenBank under accession number GU345745. RTG-2 cells were grown as a confluent monolayer in 75 cm2 in cell culture flasks (Nunc) and challenged with 5 × 104 zoospore/cysts at 24 °C. At several time points, media were discarded, except for time point 0, and 5 mL of Qiazol (Qiagen) or Trizol reagent (Invitrogen) was added to each flask. Cells were scraped loose with a cell scraper (Fisher) and the suspension was aliquoted as 1 mL portions into 2-mL screw-cap tubes containing 10–35 glass beads of 1 mm diameter (Biospec). Samples this website Selleckchem Ibrutinib were frozen immediately in liquid N2. Frozen cells were homogenized in a Fastprep machine (ThermoSavant) and shaken several times at speed 5.0 for 45 s until defrosted and homogenized. RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s protocol, modified with an extra 1 : 1 (v/v) phenol : chloroform extraction after first chloroform addition. A similar approach was used for RNA isolation from zoospores/cysts and germinated cysts. RNA was isolated from mycelium and sporulating mycelium using the Qiagen RNeasy kit, according
to the manufacturer’s protocol for filamentous fungi. RNA was treated with Turbo DNA-free DNase (Ambion) according to the manufacturer’s protocol and checked for genomic DNA contamination by PCR with the primers used for quantitative RT-qPCR (Q-PCR). The concentration and purity of RNA were determined spectrophotometrically with Nanodrop at 260 and 260/280 nm ratios, respectively. Samples with a 260/280 nm ratio lower than 1.7 were discarded. Subsequent cDNA synthesis was performed using a First strand cDNA synthesis Dapagliflozin kit (GE Healthcare) with 3–5 μg of RNA per 33-μL sample using the pd(N)6 random hexamers according to the manufacturer’s protocol. Transcript levels of SpHtp1 were analysed with a LightCycler® 480 (Roche),
using the LightCycler® 480 SYBR Green I Master mix (Roche), with 1 μL of cDNA in a total of 10 μL and according to the manufacturer’s protocol. The reaction was performed with an initial incubation at 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 58 °C for 10 s and 72 °C for 5 s, respectively. A dissociation curve as described in the LightCycler® 480 SYBR Green I Master mix (Roche) was performed to check the specificity of the primers. The amplicon length and optimized concentrations of the primers were 104 bp and 250 nM for SpHtp1, respectively, and 129 bp and 400 nM for SpTub-b, respectively. To correct for differences in the template concentration, several reference genes suggested by Yan & Liou (2006) were tested initially (Supporting Information, Fig.