77 cm2) were collected from the inoculated leaflets described above at each inoculation spot immediately https://www.selleckchem.com/products/AZD1152-HQPA.html after inoculation and then one, two, five and nine

days post-inoculation. The controls were fragments from leaves inoculated with water supplemented with 0.02 % Tween20. For each time point, three sets of inoculated fragments were analyzed independently (three biological replicates). Collected samples were lyophilized and stored at −20 °C. The total RNA was extracted from the samples using CTAB extraction buffer (Chang et al. 1993), treated with RNase-free RQ1 DNase (Promega), quantified by spectrophotometry and quality tested by electrophoresis on 1.2 % agarose gels. The first-strand cDNA was synthesized from 1 μg of total RNA using oligodT Compound C and SuperScript III (Invitrogen) according to the supplier’s protocol. Design of Cas-specific primers Several

pairs of primers were designed from the sequence of each Cas gene homologue, including at least one primer that overlapped an intron site. Their efficiency was tested on diluted cDNA pools of all time points for each isolate by cultivar set. The specificity of the amplification was analyzed using the melting temperature curves at the end of each run. The best primer pairs were selected for the real-time RT-PCR experiments. The primers selected to amplify the Cas1 transcripts were CasF12 and Cc-qCas1-R2. For Cas3 and Cas4 transcripts, the primers selected were Cc-qCas3,4-F1 and Cc-qCas3,4-R1. A

third primer pair (Cc-qCas1,3,4-F1/Cc-qCas1,3,4-R1) designed to amplify conserved regions of all Cas homologue cDNA sequences was used as a positive control. All of these primer pairs failed to amplify any product from cDNA derived from non-inoculated leaves. Primer sequences are listed in the Electronic Supplementary Material (ESM 2). Design of C. next cassiicola-specific reference gene primers Primers were designed based on conserved regions (framing one intron site) determined from the alignment of EF1α or actin gene sequences from various fungal species, most of which belonged to the order Pleosporales, like C. cassiicola. Primers designed from the EF1α sequences were Nc-EF1α-F2 and Cc-EF1α-R1. Primers designed from the actin sequences were Cc-Actin-F4 and Cc-Actin-R1. These primers were used to amplify partial genomic sequences from all of the C. cassiicola isolates from this study. The PCR products were sequenced as described above and compared by multiple sequence alignment. New primers were designed for real-time RT-PCR, with the forward primer overlapping the intron. For EF1α, two forward primers were designed depending on the isolate due to a one-nucleotide substitution in the primer binding site. Primer Cc-qEF1α-F1 was developed for isolates CCP, E78, and E70 and primer Cc-qEF1α-F3 was developed for isolates E79 and E139. The reverse primer, Cc-qEF1α-R1, was the same for all isolates. For the actin gene, the primers designed were Cc-qActin-F2 and Selonsertib mouse Cc-qActin-R2.