, 2000; Voegele et al.,
2001; Kemen et al., 2005). Primers were designed using the programs gene runner V3.05 (Hastings Software Inc., Westwood, NJ) and lasergene 7 (DNASTAR Inc., Madison, WI). Primer selection was based on a minimum formation of primer secondary structure (within a single primer and among primer pairs), similar annealing temperatures and an amplicon size of 100–200 bp. Primers finally chosen for this study are listed in Table 1. Generation of cDNA Selleckchem 3Methyladenine was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For samples to be quantified, 200 ng of total RNA were used. Incubation was for 30 min at 42 °C. In order to be able to perform an
absolute quantification of fungal RNA in a mixed sample, RNA from germinated spores and isolated haustoria was also used in amounts of 10, 20, 50, 100, and 150 ng, representing 5, 10, 25, 50, and 75% of the total RNA used for samples to be quantified. cDNA was quantified using a SmartCyclerII Real Time PCR device (Peqlab) and the QuantiTect SYBR Green PCR Kit (Qiagen). Reactions were carried out in a final volume of 25 μL. The reaction contained 1 μL cDNA (200 ng, or for standards fractions thereof), 12.5 μL 2 × QuantiTect SYBR Green PCR Master Mix, 1.25 μL of each primer, and 9 μL H2O. Amplification conditions consisted of an initial denaturation at 95 °C for 15 min followed by 45 three-step cycles of 94 °C for 15 s, 55 °C for 20 s, and Crizotinib 72 °C for 20 s. Cycle threshold was manually set to 20 RFU. Following the PCR, a melting curve analysis was performed by heating the samples from 60 to 90 °C at a rate of 0.2 °C s−1. All experiments included water instead of nucleic Selleck Nutlin3 acids as a negative control and all PCR assays were replicated at least three times. We set out to quantify the amount of pathogen present at any given time point in the obligate biotrophic interaction of the rust fungus U. fabae and its host plant V. faba. Traditionally, disease severity in this host–pathogen interaction is scored on the basis of macroscopically visible symptoms (Sillero & Rubiales, 2002). Histochemical analyses
may be used to complement such ratings (Sillero & Rubiales, 2002). However, this type of quantification is very labor-intensive and only semi-quantitative at best. Initially, we set out to use the Ergosterol content as a marker for fungal development in planta. However, using adapted extraction procedures according to established protocols (Newell et al., 1988; Martin et al., 1990) and subsequent HPLC analysis using a Nucleosil 100-5 C18 column (Macherey-Nagel, Düren, Germany) revealed that U. fabae has only a negligible Ergosterol content (data not shown). Controls using the addition of defined amounts of purified Ergosterol to diseased plant material before extraction indicated a detection limit in the range of 1 μg mL−1 extract. These results reflect similar findings by Weete et al.