C. orbiculare Inoculations
C. orbiculare strain 104-T [41] was maintained on potato dextrose agar (Difco, MI, USA) at 24°C in the dark. Conidia were obtained by gently scraping 7 d cultures as described [42]. Mature leaves of 2.5 true leaf stage seedlings were used for virulence assays. Quantification of C. orbiculare was performed by spotting 5 µl of a conidial suspension (5×105 conidia ml−1 in distilled water) on the surface of detached cucumber leaves, and then five leaf-disks were cut using a cork borer (No. 3) at 6 dpi. Leave disks were frozen in liquid nitrogen and ground using an SH-48 grinding apparatus (Kurabo, Osaka, Japan). Preparation of total RNA and first-strand cDNA, qRT-PCR were performed according to a method described previously [34]. Nucleotide sequences of C. orbiculare actin (Cor-ACT) primers are listed in Table S1. QRT-PCR data for Cor-ACT and C. sativus EF1α (Cs-EF1α) expression from cucumber were collected as copy number obtained from a standard curve of cycle times. The abundance of Cor-ACT was normalized against Cs-EF1α in infected samples.