As R428 a result the method was adapted such that different amounts of RNA (10, 20, 50, 100, and 150 ng of the normally used 200 ng RNA) were used in the reverse transcription reaction. Subsequently identical volumes of these reactions were used as template in real-time experiments. The standard curves for the three genes used (Uf-CON1, Uf-CON2, and Uf-TBB1) are depicted in Fig. 2a. The slopes of the three standard curves are almost identical. However, the standard curve for Uf-TBB1 is markedly shifted to higher Ct values, reflecting lower levels of transcript abundance of Uf-TBB1 compared with

the two other genes (Uf-CON1 and Uf-CON2). For the quantification of haustoria, three genes (Uf-HXT1, Uf-RTP1, and Uf-THI1) were used, which have been shown to be haustorium-specifically expressed (Hahn & Mendgen, 1997; Voegele et al., 2001). Again slopes of the standard curves are almost identical (Fig. 2b). The low CT numbers indicate high levels of transcript abundance. Indeed, all three genes have been shown to be among the most highly expressed genes in haustoria, representing between 0.7% and 2.8% of the total cDNA each (Hahn & Mendgen, 1997; Voegele et al., 2001). These standard curves were then used to perform an absolute quantification of U. fabae in planta. Figure 3a–c depicts the fraction of the constitutively expressed genes Uf-CON1 (a), Uf-CON2

(b), and Uf-TBB1(c) of the total RNA of samples from infected leaves as a function of

disease progression. These results mirror those obtained with dot plot analysis. It appears that there is a lag phase in PD0325901 ic50 the early days after inoculation, where hardly any fungus is detectable. Between 4 and 8 dpi, there is an exponential increase of the proportion of RNA made up by the fungus. Thereafter, the fungal fraction seemed to reach a steady-state level of around 50% of the total RNA. Results from these analyses correlated so well that data for the different genes could be integrated into a single graph (Fig. 4a). The fact that the proportion of fungal RNA does not seem to increase continuously might reflect the specific need of obligate biotrophic pathogens Methane monooxygenase to keep their host plants alive in order to assure propagation. Nine days post inoculation an equilibrium seems to be established enabling further pathogen development and proliferation without damaging the host plant to a point where it ceases growth. The proportion of about 50% fungal RNA is considerably higher than the amount of 20% fungal DNA reported for a compatible interaction of the poplar rust Melampsora medusae with its host (Boyle et al., 2005). This discrepancy might either be due to the problems associated with using DNA for quantification of rust fungi mentioned above, or to different levels of pathogen present in different host–parasite interactions. Jakupovic et al.

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