platani cp and 18S sequences (GenBank accession nos. EF017218 and U43777). The resulting 20× assay mix for the cp transcript analysis contained the primers cp-for 5′-GAAGTTCTCTATCCTACCCATGATTGC-3′, cp-rev 5′-TCAGGTCAGCGGCGTAGATA-3′, and the probe spanning the exon–exon boundary, 5′-CCGTCTCGATCTCTTATGAC-3′. The 20× assay mix for 18S transcript analysis contained the primers 18S-for 5′-GGAACAATTGGAGGGCAAGTCT-3′, 18S-rev 5′-CAACTACGAGCTTTTTAACCACAACA-3′, BI 2536 datasheet and the probe 5′-TTGGAGCTGGAATTAC-3′. The amplifications of the target gene and the endogenous control were run in triplicate on the same plate in separate tubes. Reactions (25 μL)
were carried out with 20 ng of cDNA, 1× TaqMan® Gene Expression Assay mix and 1× TaqMan® Gene Expression Master Mix following the manufacturer’s instructions. Amplifications were performed in an Applied Biosystems selleck chemicals 7300 Real-Time PCR System using the recommended thermocycling conditions. The size of the amplification products was verified on agarose gel. The relative amount of cp gene transcript in each sample was determined using the comparative CT method as described in the ABI PRISM 7700 Sequence Detection System User Bulletin #2 (Applied Biosystems). Before the quantification, a validation experiment was performed to ensure that the amplification efficiencies
of the target gene and the reference gene were approximately equal. The Universal GenomeWalker™ Kit (Clontech Laboratories Inc., Palo Alto, CA) was used to isolate the upstream region of the cp gene following the manufacturer’s instructions.
The genomic DNA of C. platani was digested with the blunt-end enzymes DraI, EcoRV, PvuII and StuI, respectively, and the resulting fragments were ligated to a GenomeWalker™ adaptor. Genome walking was then performed by two rounds of PCR with gene-specific primers: GW cp 1rev 5′-TCAGCGGCGTAGATAGGGTCATAAGAG-3′ for the first PCR and GW cp nest2rev 5′-GCGCTGGCAATCATGGGTAGGATAGAG-3′ for the nested PCR. Putative binding sites for transcription factors in the 5′-flanking region of the cp gene were investigated with the programmes Patch™ 1.0 (http://www.gene-regulation.com/pub/programs.html), based on the transfac® database release Uroporphyrinogen III synthase 7.0 and MatInspector 8.0.5 (Cartharius et al., 2005). Only binding sites with a high matrix similarity (≥ 0.85) were retained. The cp gene in C. platani is a single-copy gene as demonstrated by the Southern blot analysis after hybridization of the fungal genomic DNA digested with the enzymes HindIII and EcoRI with the cp probe (Fig. 1). The expression of cp was quantified by real-time PCR using the 18S gene as endogenous control (Bernardi et al., 2011). Before the analysis, the 18S gene was assessed for stability between samples (treated vs. control) under the growth conditions studied in the present work, and it did not show significant differences in the transcriptional level (data not shown).