fabae in planta. As a consequence, we focused on nucleic acid-based techniques. Most previous studies have used genomic DNA for quantification (Winton et al., 2003; Barnes & Szabo, 2007; Vincelli & Tisserat, 2008). However, different spore forms of rust fungi and the structures derived thereof show some distinct variations in DNA content.

Moreover, studies using for example Puccinia striiformis, U. fabae, and Uromyces appendiculatus have indicated multinucleate conditions in different differentiation stages of these rust fungi (Staples et al., 1984; Deising et al., 1991; Chong et al., 1992). From this evidence, it has to be concluded that the amount of genomic DNA cannot be used as a reliable marker for the quantitative determination GSK1120212 mouse of the fungus in planta. An alternative, which was used in this study, is the use of specific RNAs for quantification. In all organisms, some genes tend to be constitutively expressed, or to be more precise tend to exhibit constant levels of transcript abundance, regardless of the physiological condition or the differentiation stage. In U. fabae, a number of genes have been shown to be more or less constitutively expressed at a relatively high level.

Among those genes were Uf-TBB1 encoding β-tubulin, a major component of the cytoskeleton (Wirsel et al., 2004), and Uf-CON1 and Uf-CON2, two genes encoding hypothetical proteins of unknown function (Hahn & Mendgen, 1997). In

Trichostatin A initial experiments, we used dot-blot analysis for the quantification of the fungal fraction in mixed samples. Figure 1 shows such an analysis Cyclin-dependent kinase 3 with Uf-CON2 as example. Defined amounts of serial dilutions of RNA preparations from infected leaves were spotted onto Hybond-N+ membranes (GE-Healthcare, Munich, Germany) using a manifold (Gibco BRL, Gaithersburg, MD) to ensure equivalent dot sizes. As a reference, serial dilutions of RNA preparations from in vitro germinated spores (fungus only) were spotted. Quantification of spot intensity was performed using a Gel Doc 1000 System (BioRad, Munich, Germany) and the quantity one software (BioRad). The fraction of fungal RNA present in mixed samples was calculated and plotted against the time course of infection. Figure 1b shows that no fungal material could be detected before 5 dpi. Between 5 and 9 dpi, an almost exponential increase could be seen, which seemed to reach a steady-state level thereafter. While this setup yielded promising results, experiments were very labor-intensive and time-consuming. We therefore focused on real-time PCR analysis. mRNA was reverse transcribed in a separate reaction using the QuantiTect Reverse Transcription Kit (Qiagen). RNA prepared from germinated spores was again used to generate a standard for absolute quantification. Initially, dilutions of the reverse transcription reaction were used in real-time PCR assays to generate a standard curve.